Proteopedia - User contributions [en]

User:Angelika Wackerl

2015-08-03T16:11:03Z

Angelika Wackerl:

* Full Real Name: Angelika Wackerl

* Position: Student

* Institution (NO ABBREVIATIONS): Albert-Ludwigs-Universität Freiburg

* City, State/Province, Country: Freiburg, Germany

* Field of Expertise or Study: Currently working to obtain degree 'Master of Science' in (Micro)Biology [August 2015]

Sandbox Reserved 712

2013-01-03T21:48:19Z

Angelika Wackerl: /* '''Phenotypic susceptibility and enzymatic analysis''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process gag & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. [[Darunavir]] is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

[[Darunavir]] (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large panel of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to [[darunavir]]. <ref> PMID:15917527 </ref>
One reason for the high potency of [[darunavir]] is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
[[Darunavir]] shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
[[Darunavir]] resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. Its most important residues are two aspartate amino acids.
Inhibitors occupy similar positions as the natural substrate-protein chains.
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.1 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.2 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.3 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.1, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.2 and Fig.3) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene>) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to [[amprenavir]], which is a structural related PI of [[darunavir]], it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to [[darunavir]]. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for [[darunavir]] in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.4 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more does the mutated PR support the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of [[darunavir]] and [[amprenavir]], because it has a different structure and resistance profile than the others.(Fig.4) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance.<ref name="Molecular" /> The knowledge we gain through that kind of experiments could help to develop new drugs for HIV-positive patients in the future.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-03T21:47:42Z

Angelika Wackerl: /* X-ray structure analysis of 3ggu */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process gag & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. [[Darunavir]] is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

[[Darunavir]] (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large panel of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to [[darunavir]]. <ref> PMID:15917527 </ref>
One reason for the high potency of [[darunavir]] is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
[[Darunavir]] shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
[[Darunavir]] resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. Its most important residues are two aspartate amino acids.
Inhibitors occupy similar positions as the natural substrate-protein chains.
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.1 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.2 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.3 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.1, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.2 and Fig.3) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene>) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to [[amprenavir]], which is a structural related PI of [[darunavir]], it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to [[darunavir]]. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for [[darunavir]] in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more does the mutated PR support the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of [[darunavir]] and [[amprenavir]], because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance.<ref name="Molecular" /> The knowledge we gain through that kind of experiments could help to develop new drugs for HIV-positive patients in the future.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-03T21:44:42Z

Angelika Wackerl: /* '''Applications''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process gag & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. [[Darunavir]] is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

[[Darunavir]] (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large panel of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to [[darunavir]]. <ref> PMID:15917527 </ref>
One reason for the high potency of [[darunavir]] is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
[[Darunavir]] shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
[[Darunavir]] resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. Its most important residues are two aspartate amino acids.
Inhibitors occupy similar positions as the natural substrate-protein chains.
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene>) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to [[amprenavir]], which is a structural related PI of [[darunavir]], it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to [[darunavir]]. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for [[darunavir]] in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more does the mutated PR support the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of [[darunavir]] and [[amprenavir]], because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance.<ref name="Molecular" /> The knowledge we gain through that kind of experiments could help to develop new drugs for HIV-positive patients in the future.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-03T21:43:00Z

Angelika Wackerl: /* '''Darunavir: HIV-Protease Inhibitor''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process gag & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. [[Darunavir]] is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

[[Darunavir]] (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large panel of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to [[darunavir]]. <ref> PMID:15917527 </ref>
One reason for the high potency of [[darunavir]] is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
[[Darunavir]] shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
[[Darunavir]] resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. Its most important residues are two aspartate amino acids.
Inhibitors occupy similar positions as the natural substrate-protein chains.
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene>) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to [[amprenavir]], which is a structural related PI of [[darunavir]], it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to [[darunavir]]. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for [[darunavir]] in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more does the mutated PR support the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of [[darunavir]] and [[amprenavir]], because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help to develop new drugs for HIV-positive patients in the future.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-03T21:29:11Z

Angelika Wackerl: /* '''Darunavir: HIV-Protease Inhibitor''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process gag & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. [[Darunavir]] is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

[[Darunavir]] (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to [[darunavir]]. <ref> PMID:15917527 </ref>
One reason for the high potency of [[darunavir]] is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
[[Darunavir]] shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
[[Darunavir]] resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. Its most important residues are two aspartate amino acids.
Inhibitors occupy similar positions as the natural substrate-protein chains.
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene>) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to [[amprenavir]], which is a structural related PI of [[darunavir]], it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to [[darunavir]]. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for [[darunavir]] in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more does the mutated PR support the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of [[darunavir]] and [[amprenavir]], because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help to develop new drugs for HIV-positive patients in the future.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-03T11:28:38Z

Angelika Wackerl: /* '''Phenotypic susceptibility and enzymatic analysis''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process gag & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. [[Darunavir]] is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

[[Darunavir]] (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to [[darunavir]]. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
[[Darunavir]] shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
[[Darunavir]] resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. Its most important residues are two aspartate amino acids.
Inhibitors occupy similar positions as the natural substrate-protein chains.
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene>) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to [[amprenavir]], which is a structural related PI of [[darunavir]], it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to [[darunavir]]. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for [[darunavir]] in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more does the mutated PR support the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of [[darunavir]] and [[amprenavir]], because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help to develop new drugs for HIV-positive patients in the future.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-03T10:46:02Z

Angelika Wackerl: /* '''Description''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process gag & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. [[Darunavir]] is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

[[Darunavir]] (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to [[darunavir]]. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
[[Darunavir]] shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
[[Darunavir]] resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. Its most important residues are two aspartate amino acids.
Inhibitors occupy similar positions as the natural substrate-protein chains.
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene>) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to [[amprenavir]], which is a structural related PI of [[darunavir]], it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to [[darunavir]]. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for [[darunavir]] in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of [[darunavir]] and [[amprenavir]], because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help to develop new drugs for HIV-positive patients in the future.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-02T19:48:31Z

Angelika Wackerl: /* '''Description''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process gag & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

[[Darunavir]] (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to [[darunavir]]. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
[[Darunavir]] shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
[[Darunavir]] resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. Its most important residues are two aspartate amino acids.
Inhibitors occupy similar positions as the natural substrate-protein chains.
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene>) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to [[amprenavir]], which is a structural related PI of [[darunavir]], it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to [[darunavir]]. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for [[darunavir]] in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of [[darunavir]] and [[amprenavir]], because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help to develop new drugs for HIV-positive patients in the future.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T19:32:34Z

Angelika Wackerl: /* General structure of HIV-1 proteases */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

[[Darunavir]] (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to [[darunavir]]. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
[[Darunavir]] shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
[[Darunavir]] resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. Its most important residues are two aspartate amino acids.
Inhibitors occupy similar positions as the natural substrate-protein chains.
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene>) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to [[amprenavir]], which is a structural related PI of [[darunavir]], it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to [[darunavir]]. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for [[darunavir]] in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of [[darunavir]] and [[amprenavir]], because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help to develop new drugs for HIV-positive patients in the future.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T18:35:18Z

Angelika Wackerl: /* '''Applications''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

[[Darunavir]] (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to [[darunavir]]. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
[[Darunavir]] shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
[[Darunavir]] resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. Its most important residues are two aspartate amino acids.

Inhibitors occupie similar positions as the natural substrate-protein chains.
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene>) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to [[amprenavir]], which is a structural related PI of [[darunavir]], it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to [[darunavir]]. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for [[darunavir]] in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of [[darunavir]] and [[amprenavir]], because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help to develop new drugs for HIV-positive patients in the future.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T18:31:44Z

Angelika Wackerl: /* '''Phenotypic susceptibility and enzymatic analysis''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

[[Darunavir]] (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to [[darunavir]]. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
[[Darunavir]] shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
[[Darunavir]] resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. Its most important residues are two aspartate amino acids.

Inhibitors occupie similar positions as the natural substrate-protein chains.
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene>) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to [[amprenavir]], which is a structural related PI of [[darunavir]], it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to [[darunavir]]. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for [[darunavir]] in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of [[darunavir]] and [[amprenavir]], because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T18:29:27Z

Angelika Wackerl: /* General structure of HIV-1 proteases */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

[[Darunavir]] (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to [[darunavir]]. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
[[Darunavir]] shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
[[Darunavir]] resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. Its most important residues are two aspartate amino acids.

Inhibitors occupie similar positions as the natural substrate-protein chains.
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to [[amprenavir]], which is a structural related PI of [[darunavir]], it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to [[darunavir]]. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for [[darunavir]] in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of [[darunavir]] and [[amprenavir]], because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T18:28:46Z

Angelika Wackerl: /* '''Phenotypic susceptibility and enzymatic analysis''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

[[Darunavir]] (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to [[darunavir]]. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
[[Darunavir]] shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
[[Darunavir]] resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

Inhibitors occupie similar positions as the natural substrate-protein chains.
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to [[amprenavir]], which is a structural related PI of [[darunavir]], it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to [[darunavir]]. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for [[darunavir]] in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of [[darunavir]] and [[amprenavir]], because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T18:26:51Z

Angelika Wackerl: /* '''Phenotypic susceptibility and enzymatic analysis''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

[[Darunavir]] (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to [[darunavir]]. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
[[Darunavir]] shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
[[Darunavir]] resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

Inhibitors occupie similar positions as the natural substrate-protein chains.
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to [[amprenavir]], which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of [[darunavir]] and [[amprenavir]], because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T18:24:53Z

Angelika Wackerl: /* '''Phenotypic susceptibility and enzymatic analysis''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

[[Darunavir]] (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to [[darunavir]]. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
[[Darunavir]] shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
[[Darunavir]] resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

Inhibitors occupie similar positions as the natural substrate-protein chains.
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to [[amprenavir]], which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T18:21:47Z

Angelika Wackerl: /* '''Darunavir: HIV-Protease Inhibitor''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

[[Darunavir]] (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to [[darunavir]]. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
[[Darunavir]] shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
[[Darunavir]] resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

Inhibitors occupie similar positions as the natural substrate-protein chains.
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T18:19:53Z

Angelika Wackerl:

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

Inhibitors occupie similar positions as the natural substrate-protein chains.
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T18:18:49Z

Angelika Wackerl: /* '''Description''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

Inhibitors occupie similar positions as the natural substrate-protein chains.
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T18:16:41Z

Angelika Wackerl: /* General structure of HIV-1 proteases */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

Inhibitors occupie similar positions as the natural substrate-protein chains.
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T18:14:50Z

Angelika Wackerl: /* '''Darunavir: HIV-Protease Inhibitor''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

(Inhibitors occupie similar positions like the natural substrate-protein chain.)
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T17:51:47Z

Angelika Wackerl:

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor ([[Molecular Playground/HIV Protease Inhibitor]]). The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

(Inhibitors occupie similar positions like the natural substrate-protein chain.)
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T17:49:51Z

Angelika Wackerl: /* '''Phenotypic susceptibility and enzymatic analysis''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor ([[Molecular Playground/HIV Protease Inhibitor]]). The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

(Inhibitors occupie similar positions like the natural substrate-protein chain.)
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T17:47:24Z

Angelika Wackerl: /* '''Applications''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor ([[Molecular Playground/HIV Protease Inhibitor]]). The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

(Inhibitors occupie similar positions like the natural substrate-protein chain.)
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T17:45:11Z

Angelika Wackerl: /* X-ray structure analysis of 3ggu */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor ([[Molecular Playground/HIV Protease Inhibitor]]). The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

(Inhibitors occupie similar positions like the natural substrate-protein chain.)
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T17:37:55Z

Angelika Wackerl: /* '''Description''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor ([[Molecular Playground/HIV Protease Inhibitor]]). The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

(Inhibitors occupie similar positions like the natural substrate-protein chain.)
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T17:37:10Z

Angelika Wackerl: /* '''Phenotypic susceptibility and enzymatic analysis''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

[[HIV-1 protease]]s (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor ([[Molecular Playground/HIV Protease Inhibitor]]). The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

(Inhibitors occupie similar positions like the natural substrate-protein chain.)
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T16:15:11Z

Angelika Wackerl: /* '''Description''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

[[HIV-1 protease]]s (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor ([[Molecular Playground/HIV Protease Inhibitor]]). The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to darunavir. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to darunavir. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has in comparison to the other samples a low vitality value for darunavir and the structural related amprenavir. The lopinavir pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

(Inhibitors occupie similar positions like the natural substrate-protein chain.)
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T16:14:31Z

Angelika Wackerl: /* '''Description''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

[[HIV-1 protease]]s (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, HIV-PRs are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor ([[Molecular Playground/HIV Protease Inhibitor]]). The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to darunavir. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to darunavir. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has in comparison to the other samples a low vitality value for darunavir and the structural related amprenavir. The lopinavir pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

(Inhibitors occupie similar positions like the natural substrate-protein chain.)
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T16:13:43Z

Angelika Wackerl: /* '''Description''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

[[HIV-1 protease]] (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
[[HIV-1 protease]] contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, HIV-PRs are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor ([[Molecular Playground/HIV Protease Inhibitor]]). The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to darunavir. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to darunavir. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has in comparison to the other samples a low vitality value for darunavir and the structural related amprenavir. The lopinavir pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

(Inhibitors occupie similar positions like the natural substrate-protein chain.)
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T16:11:47Z

Angelika Wackerl: /* '''Description''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant HIV protease. Shown is a patient's variant in complex with [[darunavir]].

[[HIV-1 protease]] (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
HIV proteases contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, HIV-PRs are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor ([[Molecular Playground/HIV Protease Inhibitor]]). The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to darunavir. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to darunavir. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has in comparison to the other samples a low vitality value for darunavir and the structural related amprenavir. The lopinavir pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

(Inhibitors occupie similar positions like the natural substrate-protein chain.)
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T16:10:48Z

Angelika Wackerl: /* '''Description''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant HIV protease. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
HIV proteases contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, HIV-PRs are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor ([[Molecular Playground/HIV Protease Inhibitor]]). The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to darunavir. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to darunavir. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has in comparison to the other samples a low vitality value for darunavir and the structural related amprenavir. The lopinavir pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

(Inhibitors occupie similar positions like the natural substrate-protein chain.)
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T16:08:58Z

Angelika Wackerl: /* General structure of HIV-1 proteases */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant HIV protease. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
HIV proteases contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/1'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, HIV-PRs are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor ([[Molecular Playground/HIV Protease Inhibitor]]). The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to darunavir. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to darunavir. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has in comparison to the other samples a low vitality value for darunavir and the structural related amprenavir. The lopinavir pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

(Inhibitors occupie similar positions like the natural substrate-protein chain.)
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T15:53:21Z

Angelika Wackerl: /* X-ray structure analysis of 3ggu */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant HIV protease. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
HIV proteases contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/1'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, HIV-PRs are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor ([[Molecular Playground/HIV Protease Inhibitor]]). The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to darunavir. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to darunavir. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has in comparison to the other samples a low vitality value for darunavir and the structural related amprenavir. The lopinavir pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
In the tunnel the acitve site is located. The acitve site uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

(Inhibitors occupie similar positions like the natural substrate-protein chain.)
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type, PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T15:30:58Z

Angelika Wackerl: /* '''Darunavir: HIV-Protease Inhibitor''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant HIV protease. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
HIV proteases contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/1'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, HIV-PRs are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor ([[Molecular Playground/HIV Protease Inhibitor]]). The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to darunavir. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to darunavir. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has in comparison to the other samples a low vitality value for darunavir and the structural related amprenavir. The lopinavir pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
In the tunnel the acitve site is located. The acitve site uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

(Inhibitors occupie similar positions like the natural substrate-protein chain.)
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T15:01:33Z

Angelika Wackerl: /* '''Description''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant HIV protease. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process [[Hiv-1 gag]] & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
HIV proteases contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/1'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, HIV-PRs are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor ([[Molecular Playground/HIV Protease Inhibitor]]). The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>. Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to darunavir. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to darunavir. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has in comparison to the other samples a low vitality value for darunavir and the structural related amprenavir. The lopinavir pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
In the tunnel the acitve site is located. The acitve site uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

(Inhibitors occupie similar positions like the natural substrate-protein chain.)
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T14:45:45Z

Angelika Wackerl: /* '''Darunavir: HIV-Protease Inhibitor''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant HIV protease. Shown is a patient's variant in complex with darunavir.

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process gag & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
HIV proteases contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/1'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, HIV-PRs are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor ([[Molecular Playground/HIV Protease Inhibitor]]). The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>. Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to darunavir. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to darunavir. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has in comparison to the other samples a low vitality value for darunavir and the structural related amprenavir. The lopinavir pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
In the tunnel the acitve site is located. The acitve site uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

(Inhibitors occupie similar positions like the natural substrate-protein chain.)
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T14:31:55Z

Angelika Wackerl:

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant HIV protease. Shown is a patient's variant in complex with darunavir.

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process gag & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
HIV proteases contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/1'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, HIV-PRs are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>. Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to darunavir. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to darunavir. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has in comparison to the other samples a low vitality value for darunavir and the structural related amprenavir. The lopinavir pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
In the tunnel the acitve site is located. The acitve site uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

(Inhibitors occupie similar positions like the natural substrate-protein chain.)
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T14:29:03Z

Angelika Wackerl: /* '''Phenotypic susceptibility and enzymatic analysis''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant HIV protease. Shown is a patient's variant in complex with darunavir.

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process gag & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
HIV proteases contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/1'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, HIV-PRs are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>. Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to darunavir. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to darunavir. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has in comparison to the other samples a low vitality value for darunavir and the structural related amprenavir. The lopinavir pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
In the tunnel the acitve site is located. The acitve site uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

(Inhibitors occupie similar positions like the natural substrate-protein chain.)
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T14:27:09Z

Angelika Wackerl: /* General structure of HIV-1 proteases */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant HIV protease. Shown is a patient's variant in complex with darunavir.

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process gag & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
HIV proteases contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/1'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, HIV-PRs are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>. Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to darunavir. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to darunavir. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has in comparison to the other samples a low vitality value for darunavir and the structural related amprenavir. The lopinavir pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
In the tunnel the acitve site is located. The acitve site uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

(Inhibitors occupie similar positions like the natural substrate-protein chain.)
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2013-01-01T13:24:24Z

Angelika Wackerl: /* '''Structure''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant HIV protease. Shown is a patient's variant in complex with darunavir.

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process gag & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
HIV proteases contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/1'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, HIV-PRs are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>. Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to darunavir. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to darunavir. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has in comparison to the other samples a low vitality value for darunavir and the structural related amprenavir. The lopinavir pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

HIV-1 proteases are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
In the tunnel the acitve site is located. The acitve site uses water molecules to break the subrate-protein chain. It's most important residues are two aspartate amino acids.

(Inhibitors occupie similar positions like the natural substrate-protein chain.)
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2012-12-31T18:12:05Z

Angelika Wackerl: /* '''Description''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant HIV protease. Shown is a patient's variant in complex with darunavir.

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process gag & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
HIV proteases contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/1'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, HIV-PRs are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>. Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to darunavir. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to darunavir. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has in comparison to the other samples a low vitality value for darunavir and the structural related amprenavir. The lopinavir pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2012-12-31T17:49:30Z

Angelika Wackerl: /* X-ray structure analysis of 3ggu */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
3ggu is a drug resistant HIV protease. Shown is a patient's variant in complex with darunavir.

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process gag & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
HIV proteases contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/1'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, HIV-PRs are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>. Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to darunavir. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to darunavir. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has in comparison to the other samples a low vitality value for darunavir and the structural related amprenavir. The lopinavir pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2012-12-31T17:40:22Z

Angelika Wackerl: /* '''Phenotypic susceptibility and enzymatic analysis''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
3ggu is a drug resistant HIV protease. Shown is a patient's variant in complex with darunavir.

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process gag & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
HIV proteases contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/1'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, HIV-PRs are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>. Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to darunavir. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to darunavir. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has in comparison to the other samples a low vitality value for darunavir and the structural related amprenavir. The lopinavir pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values. <ref name="Molecular"/>]]

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

Mutations <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2012-12-31T17:39:54Z

Angelika Wackerl: /* X-ray structure analysis of 3ggu */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
3ggu is a drug resistant HIV protease. Shown is a patient's variant in complex with darunavir.

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process gag & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
HIV proteases contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/1'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, HIV-PRs are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>. Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to darunavir. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to darunavir. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has in comparison to the other samples a low vitality value for darunavir and the structural related amprenavir. The lopinavir pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values <ref name="Molecular"/>]]

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

Mutations <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2012-12-31T17:38:42Z

Angelika Wackerl: /* '''Applications''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
3ggu is a drug resistant HIV protease. Shown is a patient's variant in complex with darunavir.

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process gag & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
HIV proteases contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/1'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, HIV-PRs are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>. Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to darunavir. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to darunavir. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has in comparison to the other samples a low vitality value for darunavir and the structural related amprenavir. The lopinavir pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values <ref name="Molecular"/>]]

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

Mutations <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes.<ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2012-12-30T19:29:12Z

Angelika Wackerl: /* '''Applications''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
3ggu is a drug resistant HIV protease. Shown is a patient's variant in complex with darunavir.

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process gag & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
HIV proteases contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/1'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, HIV-PRs are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>. Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to darunavir. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to darunavir. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has in comparison to the other samples a low vitality value for darunavir and the structural related amprenavir. The lopinavir pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values <ref name="Molecular"/>]]

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

Mutations <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes.<ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS(Acquired immunodeficiency syndrome)is one of the most threating viruses today.
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

Sandbox Reserved 712

2012-12-30T19:27:39Z

Angelika Wackerl: /* '''Applications''' */

{{Sandbox_ESBS_2012}}

{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}

== '''Description''' ==
3ggu is a drug resistant HIV protease. Shown is a patient's variant in complex with darunavir.

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process gag & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes.
HIV proteases contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/1'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, HIV-PRs are the major target for anti-HIV treatment.
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PRDRV5) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

Darunavir (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. <ref> PMID:15917527 </ref>
One reason for the high potency of darunavir is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>. Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Phenotypic susceptibility and enzymatic analysis''' ==

Samples PRDRV1 to PRDRV6 have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PRDRV5 (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.

These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to darunavir. In comparison to ampenavir, which is a structural related PI of darunavir, it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PRDRV5 which explains the smaller phenotypic changes in the susceptibility to darunavir. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. PRDRV5 - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ Ki values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>

The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more supports the mutated PR the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to darunavir. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PRDRV4 > PRDRV1 > PRDRV2 > PRDRV6). Due to the fact that PRDRV5 does not have the key mutations, it has in comparison to the other samples a low vitality value for darunavir and the structural related amprenavir. The lopinavir pattern looks different than the overall pattern of darunavir and amprenavir, because it has a different structure and resistance profile than the others.(Fig1) <ref name="Molecular"> PMID:19535439 </ref>

[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | left | Fig.1 Relative vitality values <ref name="Molecular"/>]]

Despite the many mutations the kcat values were still between 30% and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" />

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.2 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.3 Structural changes in PRDRV5 mutant relative to wild-type PR <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.4 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with darunavir with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PRDRV5 contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.2, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

Mutations <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>,
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.3 and Fig.4) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PRDRV1 and comparative structure of wild-type PRDRV1 and PRDRV5 follow the links:
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PRDRV mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes.<ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS(Acquired immunodeficiency syndrome)is one of the most threating viruses today.
The high turnover of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offers a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help in the future to develop new drugs for HIV-positive patients.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl

File:Detailed view of darunavir-enzyme interactions.jpg

2012-12-30T19:00:41Z

Angelika Wackerl: /* Summary */

== Summary ==
Detailed view of the darunavir-enzyme interactions. The wild-type structure is yellow and PRDRV5 is blue. Residues 32, 82, and 84 are represented by their solvent-accessible surfaces (solid yellow for the wild type, mesh for the mutant variant). Dashed lines indicate hydrogen bond interactions, and the water molecule involved in water-mediated hydrogen contacts in the wild-type complex is shown as a yellow sphere. Interactions are shown for one darunavir orientation; however, they are conserved for both alternative conformations.

== Licensing ==
{{subst:Permission from license selector}}

File:Structural changes in PRdrv5 mutant.jpg

2012-12-30T18:59:14Z

Angelika Wackerl: /* Summary */

== Summary ==
Structural changes in PRDRV5 mutant relative to wild-type PR indicated in blue

== Licensing ==
{{subst:Permission from license selector}}