Sandbox Reserved 817: Difference between revisions
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<ref name="first">PMID:18005427</ref> | <ref name="first">PMID:18005427</ref> | ||
<StructureSection load='4ivs' size='500' side='right' caption='Amino acid sequence of Beta secretase 1' scene=''> | |||
__NOTOC__ | |||
=== Post translational modification === | === Post translational modification === | ||
BACE1 is synthetised in the endoplasmamic reticulum as an inactive precursor, pro-BACE1. This precursor is then maturated in the Golgi apparatus. It undergoes glycosylation of 4 residues : <scene name='56/568015/Asn/1'>Asn(153), Asn(172), Asn(223) and Asn(354) </scene>. This has a role in the activity of BACE1. 3 cysteine residues of the cytosolic tail are also palmitoylated , it is has a role in the localization of the mature enzyme. The propeptide domain is then clived between Arg 45 and Glu 46 of pro-BACE1 by a proprotein convertase (protease). This clivage is known to increase the activity of the enzyme. <ref name="two">PMID:10887202</ref> | BACE1 is synthetised in the endoplasmamic reticulum as an inactive precursor, pro-BACE1. This precursor is then maturated in the Golgi apparatus. It undergoes glycosylation of 4 residues : <scene name='56/568015/Asn/1'>Asn(153), Asn(172), Asn(223) and Asn(354) </scene>. This has a role in the activity of BACE1. 3 cysteine residues of the cytosolic tail are also palmitoylated , it is has a role in the localization of the mature enzyme. The propeptide domain is then clived between Arg 45 and Glu 46 of pro-BACE1 by a proprotein convertase (protease). This clivage is known to increase the activity of the enzyme. <ref name="two">PMID:10887202</ref> | ||
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== Structures == | == Structures == | ||
Structurally, the 501 amino acid sequence of BACE1 belongs to the eukaryotic aspartic proteases of the pepsin family and contains a bilobal structure constituted of by an N- and a C-terminal domains. BACE is composed of 12 <scene name='56/568015/Helix/1'>alpha helix</scene> and 28 <scene name='56/568015/Beta_sheet/3'>beta sheets</scene>. Both N- and C-domains are formed by highly twisted β-sheet structures and each domain contributes with an aspartic acid to the catalytic module of the enzyme. The ligands containing a positive charged moiety might then be favorable to counteract the negative charged active site. BACE1 has two aspartic protease active site motifs, DTGS (<scene name='56/568015/93-96/2'>residues 93-96</scene>) and DSGT (<scene name='56/568015/289-292/2'>residues 289-292</scene>), and mutation of either aspartic acid renders the enzyme inactive. Like other aspartic proteases, BACE1 has an N-terminal signal sequence (residues 1–21) and a pro-peptide domain (residues 22–45) that are removed post-translationally, so the mature enzyme begins at residue Glu46. Importantly, BACE1 has a single transmembrane domain near its C-terminus (residues 455–480) and a palmitoylated cytoplasmic tail. Thus, BACE1 is a type I membrane protein with a luminal active site, features predicted for β-secretase. The position of the BACE1 active site within the lumen of intracellular compartments provides the correct topological orientation for cleavage of APP at the β-secretase site. As observed with other aspartic proteases, BACE1 has <scene name='56/568015/Six_cysteines/1'>six luminal cysteine residues</scene> that form three intramolecular disulfide bonds ('''yellow''') and several N-linked glycosylation sites.<ref name="first">PMID:18005427</ref> <ref name="three">PMID:23681056</ref> | Structurally, the 501 amino acid sequence of BACE1 belongs to the eukaryotic aspartic proteases of the pepsin family and contains a bilobal structure constituted of by an N- and a C-terminal domains. BACE is composed of 12 <scene name='56/568015/Helix/1'>alpha helix</scene> and 28 <scene name='56/568015/Beta_sheet/3'>beta sheets</scene>. Both N- and C-domains are formed by highly twisted β-sheet structures and each domain contributes with an aspartic acid to the catalytic module of the enzyme. The ligands containing a positive charged moiety might then be favorable to counteract the negative charged active site. BACE1 has two aspartic protease active site motifs, DTGS (<scene name='56/568015/93-96/2'>residues 93-96</scene>) and DSGT (<scene name='56/568015/289-292/2'>residues 289-292</scene>), and mutation of either aspartic acid renders the enzyme inactive. Like other aspartic proteases, BACE1 has an N-terminal signal sequence (residues 1–21) and a pro-peptide domain (residues 22–45) that are removed post-translationally, so the mature enzyme begins at residue Glu46. Importantly, BACE1 has a single transmembrane domain near its C-terminus (residues 455–480) and a palmitoylated cytoplasmic tail. Thus, BACE1 is a type I membrane protein with a luminal active site, features predicted for β-secretase. The position of the BACE1 active site within the lumen of intracellular compartments provides the correct topological orientation for cleavage of APP at the β-secretase site. As observed with other aspartic proteases, BACE1 has <scene name='56/568015/Six_cysteines/1'>six luminal cysteine residues</scene> that form three intramolecular disulfide bonds ('''yellow''') and several N-linked glycosylation sites.<ref name="first">PMID:18005427</ref> <ref name="three">PMID:23681056</ref> | ||
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=== Mechanism of inhibition === | === Mechanism of inhibition === | ||
Structural information about the interaction of substrate with the active site of BACE1 would greatly facilitate the rational design of small molecule BACE1 inhibitors. The molecular modeling identified several residues in BACE1 that potentially contribute to substrate specificity. In particular, <scene name='56/568015/Arg235/1'>Arg235</scene> forms a saltbridge with the P1' Asp+1 residue of the β-secretase cleavage site, thus explaining the unusual preference of BACE1 among aspartic proteases for substrates that are negatively charged at this position. In addition, several hydrophobic residues in BACE1 form a pocket for the hydrophobic P1 residue. The model also showed that the mutation LysMet→AsnLeu at P2-P1 interacts more favorably with Arg235 and the hydrophobic pocket of BACE1 than does wild-type substrate, providing an explanation for the enhanced cleavage of this mutation. Conversely, the substitution of Met→Val at P1 blocks the catalytic <scene name='56/568015/Arg32/1'>Asp32</scene> | Structural information about the interaction of substrate with the active site of BACE1 would greatly facilitate the rational design of small molecule BACE1 inhibitors. The molecular modeling identified several residues in BACE1 that potentially contribute to substrate specificity. In particular, <scene name='56/568015/Arg235/1'>Arg235</scene> forms a saltbridge with the P1' Asp+1 residue of the β-secretase cleavage site, thus explaining the unusual preference of BACE1 among aspartic proteases for substrates that are negatively charged at this position. In addition, several hydrophobic residues in BACE1 form a pocket for the hydrophobic P1 residue. The model also showed that the mutation LysMet→AsnLeu at P2-P1 interacts more favorably with <scene name='56/568015/Arg235/1'>Arg235</scene> and the hydrophobic pocket of BACE1 than does wild-type substrate, providing an explanation for the enhanced cleavage of this mutation. Conversely, the substitution of Met→Val at P1 blocks the catalytic <scene name='56/568015/Arg32/1'>Asp32</scene> residue, explaining the lack of cleavage of this mutation by BACE1. | ||
Four hydrogen bonds from the catalytic aspartic acid residues (Asp32 and Asp228) and ten additional hydrogen bonds from various residues in the active site are made with the inhibitor, most of which are conserved in other aspartic proteases. The X-ray structure indicates that <scene name='56/568015/Arg235/1'>Arg235</scene> and the hydrophobic pocket of the active site play an important role in substrate binding, confirming the results of the molecular modelling study. In addition, the bound inhibitor has an unusual kinked conformation from P2' to P4'. The BACE1 X-ray structure suggests that small molecules targeting <scene name='56/568015/Arg235/1'>Arg235</scene> and the hydrophobic pocket residues should inhibit β-secretase cleavage. Moreover, mimicking the unique P2'-P4' conformation of the bound inhibitor may increase the selectivity of inhibitors for BACE1 over BACE2 and the other aspartic proteases. <ref name="first">PMID:18005427</ref> | Four hydrogen bonds from the catalytic aspartic acid residues (<scene name='56/568015/Arg32/1'>Asp32</scene> and <scene name='56/568015/Asp228/1'>Asp228</scene>) and ten additional hydrogen bonds from various residues in the active site are made with the inhibitor, most of which are conserved in other aspartic proteases. The X-ray structure indicates that <scene name='56/568015/Arg235/1'>Arg235</scene> and the hydrophobic pocket of the active site play an important role in substrate binding, confirming the results of the molecular modelling study. In addition, the bound inhibitor has an unusual kinked conformation from P2' to P4'. The BACE1 X-ray structure suggests that small molecules targeting <scene name='56/568015/Arg235/1'>Arg235</scene> and the hydrophobic pocket residues should inhibit β-secretase cleavage. Moreover, mimicking the unique P2'-P4' conformation of the bound inhibitor may increase the selectivity of inhibitors for BACE1 over BACE2 and the other aspartic proteases. <ref name="first">PMID:18005427</ref> | ||
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In the past, major efforts in designing BACE1 inhibitors were focused on the transition state analogs such as hydroxyethylamines, hydroxyethylene, and tatine-based peptidomimetic inhibitors but their relatively large sizes and excessive number of hydrogen-bond donors and acceptors made it difficult for them to penetrate the blood brain barrier. Therefore the scientific community focused on other coompounds. | In the past, major efforts in designing BACE1 inhibitors were focused on the transition state analogs such as hydroxyethylamines, hydroxyethylene, and tatine-based peptidomimetic inhibitors but their relatively large sizes and excessive number of hydrogen-bond donors and acceptors made it difficult for them to penetrate the blood brain barrier. Therefore the scientific community focused on other coompounds. | ||
One of the discovered compound, 1-(2-(1H-indol-1-yl)ethyl)guanidine showed weak inhibition activity towards BACE1 (about 42% inhibition ratio at the ligand concentration of 100 μM in the fluorescence resonance energy transfer (FRET) assay system). These compound occupied the S1 pocket and the guanidine moiety formed key binding interactions with the two catalytic aspartic acids, <scene name='56/568015/32/1'>Asp32</scene> and <scene name='56/568015/Asp228/1'>Asp228</scene> (Figure 2). | One of the discovered compound, <scene name='56/568015/Inhibiteur/1'>1-(2-(1H-indol-1-yl)ethyl)guanidine</scene> showed weak inhibition activity towards BACE1 (about 42% inhibition ratio at the ligand concentration of 100 μM in the fluorescence resonance energy transfer (FRET) assay system). These compound occupied the S1 pocket and the guanidine moiety formed key binding interactions with the two catalytic aspartic acids, <scene name='56/568015/32/1'>Asp32</scene> and <scene name='56/568015/Asp228/1'>Asp228</scene> (Figure 2). | ||
Some known BACE1 inhibitors have an an acetylated guanidine group. Some indole acylguanidines were designed by introducing a carbonyl group into the α-position of the guanidine moiety to improve the inhibitor efficiency and the binding with BACE1 was studied. | Some known BACE1 inhibitors have an an acetylated guanidine group. Some indole acylguanidines were designed by introducing a carbonyl group into the α-position of the guanidine moiety to improve the inhibitor efficiency and the binding with BACE1 was studied. | ||
There is a large hydrophobic sub-site at the top of the guanidine moiety. The acylguanidine formed crucial interactions with two catalytic aspartic acids (<scene name='56/568015/Asp228/1'>Asp228</scene> and <scene name='56/568015/32/1'>Asp32</scene>) through three hydrogen bonds. The carboxyl oxygen atoms of the acylguanidine formed water-bridged hydrogen bonds with the side chains of <scene name='56/568015/Gln73/1'> | There is a large hydrophobic sub-site at the top of the guanidine moiety. The acylguanidine formed crucial interactions with two catalytic aspartic acids (<scene name='56/568015/Asp228/1'>Asp228</scene> and <scene name='56/568015/32/1'>Asp32</scene>) through three hydrogen bonds. The carboxyl oxygen atoms of the acylguanidine formed water-bridged hydrogen bonds with the side chains of <scene name='56/568015/Gln73/1'>Gln73</scene> and <scene name='56/568015/Thr72/1'>Thr72</scene>, and a direct hydrogen bond with <scene name='56/568015/Gln73/1'>Gln73</scene> as well. | ||
A benzyl group extending from the terminus of the guanidine moiety could fill this sub-pocket and thereby potentially increase the binding affinity. The introduction of the simple benzyl group did not enhance the inhibitory activity. However, when a 3,5-dichlorobenyl group was introduced, the potency was improved.The substituted benzyl group occupied the S1 subsite of the substrate binding pocket of BACE1. The carboxyl oxygen atom of acetamide also forms a water-bridged hydrogen bond with the nitrogen atom of the amide group of <scene name='56/568015/Gln73/1'> | A benzyl group extending from the terminus of the guanidine moiety could fill this sub-pocket and thereby potentially increase the binding affinity. The introduction of the simple benzyl group did not enhance the inhibitory activity. However, when a 3,5-dichlorobenyl group was introduced, the potency was improved. The substituted benzyl group occupied the S1 subsite of the substrate binding pocket of BACE1. The carboxyl oxygen atom of acetamide also forms a water-bridged hydrogen bond with the nitrogen atom of the amide group of <scene name='56/568015/Gln73/1'>Gln73</scene>. | ||
Surprisingly, a compound which has an acetamide group between two chlorine atoms showed a more substantial increase in potency. If two chlorine atoms are remplaced with two hydrogen atoms or reduced the amide to an amine, the resulting compounds displayed much weaker activities against BACE1. The acetamide nitrogen atom forms another hydrogen bond directly with the main chain carboxyl oxygen of <scene name='56/568015/Phe108/1'>Phe108</scene>. The two chlorine atoms may force the acetamide group adopting a perpendicular angle with respect to the benzyl group, which plays an important role in binding interactions between acetamide and BACE1.Besides, the hydrogen bonds of the inhibitor with residues <scene name='56/568015/Gln73/1'> | Surprisingly, a compound which has an acetamide group between two chlorine atoms showed a more substantial increase in potency. If two chlorine atoms are remplaced with two hydrogen atoms or reduced the amide to an amine, the resulting compounds displayed much weaker activities against BACE1. The acetamide nitrogen atom forms another hydrogen bond directly with the main chain carboxyl oxygen of <scene name='56/568015/Phe108/1'>Phe108</scene>. The two chlorine atoms may force the acetamide group adopting a perpendicular angle with respect to the benzyl group, which plays an important role in binding interactions between acetamide and BACE1. Besides, the hydrogen bonds of the inhibitor with residues <scene name='56/568015/Gln73/1'>Gln73</scene> and <scene name='56/568015/Thr72/1'>Thr72</scene> induce a semi-closed conformation. Such a conformation of the flap further strengthens the ligand binding to the enzyme. | ||
Analogs were also synthesized based on indole. The indole group pointed toward the back of the S1’ pocket forming a cation-πinteraction with <scene name='56/568015/Arg235/1'>Arg235</scene>, which appears to contribute further interactions to improve the potency of the inhibitor. <ref name="three">PMID:23681056</ref> | Analogs were also synthesized based on indole. The indole group pointed toward the back of the S1’ pocket forming a cation-πinteraction with <scene name='56/568015/Arg235/1'>Arg235</scene>, which appears to contribute further interactions to improve the potency of the inhibitor. <ref name="three">PMID:23681056</ref> | ||
</StructureSection> | |||
