Proteopedia - User contributions [en]

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T18:22:01Z

Bryant Chen:

==''[http://en.wikipedia.org/wiki/Clostridium_botulinum Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions. [http://www.youtube.com/watch?v=6n3rrjbweO8 Mechanism of Botulinum Toxin]

==[http://www.sciencedaily.com/releases/2010/01/100120211009.htm Llama Antibody] Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for [http://www.nih.gov/researchmatters/march2007/03052007toxins.htm bio-terrorism]. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

===The New!===
The crystal structure of the substrate SNAP25 complexed to the BoNT/A Lc showed the extended nature of ligand recognition and identified potential exosites that are away from the catalytic active site. To display the range of bidning sites for potential BoNT/A Lc inhibitors, the authors of the paper generated and selected a non-immune camelid(llama) library of single-domain VHH antibodies for binding to the BoNT/A Lc. This single-domain antibody has been postulated to be more able to bind into enzymatic cavities. Furthermore VHH is able to withstand intracellular reducing environment.

==Uses of Botulinum Toxin==
===Cosmetic===
[http://www.youtube.com/watch?v=dkpohXE06pg&feature=related BOTOX®]

[[Image:Hilary.jpg|left]][[Image:Cox.jpg|190px]]

===[http://en.wikipedia.org/wiki/Botox Medicinal/Therapeutic]===
-Muscle Spasms,Upper motor neuron syndrome,Sweating, Cervical Dystonia, Migraines

==References==
J Mol Biol. 2010 Apr 9;397(4):1106-18. Epub 2010 Feb 6.
A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region.

Dong J, Thompson AA, Fan Y, Lou J, Conrad F, Ho M, Pires-Alves M, Wilson BA, Stevens RC, Marks JD.

Department of Anesthesia, University of California, San Francisco, San Francisco, CA 94110, USA.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T08:38:40Z

Bryant Chen:

==''[http://en.wikipedia.org/wiki/Clostridium_botulinum Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions. [http://www.youtube.com/watch?v=6n3rrjbweO8 Mechanism of Botulinum Toxin]

==[http://www.sciencedaily.com/releases/2010/01/100120211009.htm Llama Antibody] Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for [http://www.nih.gov/researchmatters/march2007/03052007toxins.htm bio-terrorism]. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

===The New!===
The crystal structure of the substrate SNAP25 complexed to the BoNT/A Lc showed the extended nature of ligand recognition and identified potential exosites that are away from the catalytic active site. To display the range of bidning sites for potential BoNT/A Lc inhibitors, the authors of the paper generated and selected a non-immune camelid(llama) library of single-domain VHH antibodies for binding to the BoNT/A Lc. This single-domain antibody has been postulated to be more able to bind into enzymatic cavities. Furthermore VHH is able to withstand intracellular reducing environment.

==Structure Complex of BoNT/A Lc and VHH==
'''All rights reserved by © Elsevier LTd. & Marks et. al'''
'''[[http://www.elsevier.com/wps/find/termsconditions.cws_home/termsconditions Terms and Conditions]]'''

VHH fragment consists of a single immunoglobulin domain with three CDR(complimentary-determining regions) defined by experimental electron density. Binding by the llama antibody fragment is driven by CDR1 and CDR3 interactions '''Figure 5B'''. The paratope(the antigen-binding site of an antibody.) formed by CDR1 and CDR3 is stabilized by several hydrogen bonds (VHH residues 37, 44, 117, and 124 interactions with BoNT/A Lc residues 113, 105, 102, and 345 respectively) and salt bridges (Lc Lys340 interactions with VHH Asp residues 113, 115 and 128) '''Figure 5C'''. Superimposed pictures of the BoNT/A Lc – VHH structure with the structure of SNAP25 bound to the Lc enzyme reveals that the paratope coincides with the an alpha-helical portion of the SNAP25 substrate. VHH paratope coincides with an alpha-helical portion of the SNAP25 substrate. '''Figure 5D'''. which is occupied by the heavy-chain belt of the neurotoxin '''Figure 5E'''. Both the CDR1 and CDR3 form small alpha-helices that appear to mimic the secondary structure of SNAP25 at the alpha-exosite( is a secondary binding site, remote from the active site. In order for an enzyme to be active, its exosite typically must be occupied). Although there is no primary homology sequence of SNAP25 and CDR1 and CDR3 alpha helices, the substrate mimicry is extended to tertiary structure because of a number of hydrophobic residues found on the VHH fragment coincide with similar residues found on SNAP25.'''Figure 5F'''. Residues include Met37 on CDR1 of the VHH fragment, which occupies the position of Met167 from SNAP25; Val116 from CDR3, which coincides with Leu160 of SNAP25; Val120 of CDR3, which occupies the space of Ile156 and Ile157 from SNAP25; and Val 123 of CDR3, which replaces Val154 of the natural substrate. Aa1 likely inhibits substrate cleavage by preventing efficient binding of SNAP25 and positioning of its scissile bond(bond of a substrate that is subject to enzymic cleavage).

[[Image:1.jpg|275px]][[Image:Lol.jpg|275px]]

[[Image:3.jpg|275px]][[Image:4.jpg|275px]]

[[Image:5.jpg|275px]][[Image:6.jpg|275px]]

==Uses of Botulinum Toxin==
===Cosmetic===
[http://www.youtube.com/watch?v=dkpohXE06pg&feature=related BOTOX®]

[[Image:Hilary.jpg|left]][[Image:Cox.jpg|190px]]

===[http://en.wikipedia.org/wiki/Botox Medicinal/Therapeutic]===
-Muscle Spasms,Upper motor neuron syndrome,Sweating, Cervical Dystonia, Migraines

==References==
J Mol Biol. 2010 Apr 9;397(4):1106-18. Epub 2010 Feb 6.
A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region.

Dong J, Thompson AA, Fan Y, Lou J, Conrad F, Ho M, Pires-Alves M, Wilson BA, Stevens RC, Marks JD.

Department of Anesthesia, University of California, San Francisco, San Francisco, CA 94110, USA.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T08:34:07Z

Bryant Chen:

==''[http://en.wikipedia.org/wiki/Clostridium_botulinum Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions. [http://www.youtube.com/watch?v=6n3rrjbweO8 Mechanism of Botulinum Toxin]

==[http://www.sciencedaily.com/releases/2010/01/100120211009.htm Lama Antibody] Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for [http://www.nih.gov/researchmatters/march2007/03052007toxins.htm bio-terrorism]. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

===The New!===
The crystal structure of the substrate SNAP25 complexed to the BoNT/A Lc showed the extended nature of ligand recognition and identified potential exosites that are away from the catalytic active site. To display the range of bidning sites for potential BoNT/A Lc inhibitors, the authors of the paper generated and selected a non-immune camelid(llama) library of single-domain VHH antibodies for binding to the BoNT/A Lc. This single-domain antibody has been postulated to be more able to bind into enzymatic cavities. Furthermore VHH is able to withstand intracellular reducing environment.

==Structure Complex of BoNT/A Lc and VHH==
'''All rights reserved by © Elsevier LTd. & Marks et. al'''
'''[[http://www.elsevier.com/wps/find/termsconditions.cws_home/termsconditions Terms and Conditions]]'''

VHH fragment consists of a single immunoglobulin domain with three CDR(complimentary-determining regions) defined by experimental electron density. Binding by the llama antibody fragment is driven by CDR1 and CDR3 interactions '''Figure 5B'''. The paratope(the antigen-binding site of an antibody.) formed by CDR1 and CDR3 is stabilized by several hydrogen bonds (VHH residues 37, 44, 117, and 124 interactions with BoNT/A Lc residues 113, 105, 102, and 345 respectively) and salt bridges (Lc Lys340 interactions with VHH Asp residues 113, 115 and 128) '''Figure 5C'''. Superimposed pictures of the BoNT/A Lc – VHH structure with the structure of SNAP25 bound to the Lc enzyme reveals that the paratope coincides with the an alpha-helical portion of the SNAP25 substrate. VHH paratope coincides with an alpha-helical portion of the SNAP25 substrate. '''Figure 5D'''. which is occupied by the heavy-chain belt of the neurotoxin '''Figure 5E'''. Both the CDR1 and CDR3 form small alpha-helices that appear to mimic the secondary structure of SNAP25 at the alpha-exosite( is a secondary binding site, remote from the active site. In order for an enzyme to be active, its exosite typically must be occupied). Although there is no primary homology sequence of SNAP25 and CDR1 and CDR3 alpha helices, the substrate mimicry is extended to tertiary structure because of a number of hydrophobic residues found on the VHH fragment coincide with similar residues found on SNAP25.'''Figure 5F'''. Residues include Met37 on CDR1 of the VHH fragment, which occupies the position of Met167 from SNAP25; Val116 from CDR3, which coincides with Leu160 of SNAP25; Val120 of CDR3, which occupies the space of Ile156 and Ile157 from SNAP25; and Val 123 of CDR3, which replaces Val154 of the natural substrate. Aa1 likely inhibits substrate cleavage by preventing efficient binding of SNAP25 and positioning of its scissile bond(bond of a substrate that is subject to enzymic cleavage).

[[Image:1.jpg|275px]][[Image:Lol.jpg|275px]]

[[Image:3.jpg|275px]][[Image:4.jpg|275px]]

[[Image:5.jpg|275px]][[Image:6.jpg|275px]]

==Uses of Botulinum Toxin==
===Cosmetic===
[http://www.youtube.com/watch?v=dkpohXE06pg&feature=related BOTOX®]

[[Image:Hilary.jpg|left]][[Image:Cox.jpg|190px]]

===[http://en.wikipedia.org/wiki/Botox Medicinal/Therapeutic]===
-Muscle Spasms,Upper motor neuron syndrome,Sweating, Cervical Dystonia, Migraines

==References==
J Mol Biol. 2010 Apr 9;397(4):1106-18. Epub 2010 Feb 6.
A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region.

Dong J, Thompson AA, Fan Y, Lou J, Conrad F, Ho M, Pires-Alves M, Wilson BA, Stevens RC, Marks JD.

Department of Anesthesia, University of California, San Francisco, San Francisco, CA 94110, USA.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T08:28:21Z

Bryant Chen:

==''[http://en.wikipedia.org/wiki/Clostridium_botulinum Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions. [http://www.youtube.com/watch?v=6n3rrjbweO8 Mechanism of Botulinum Toxin]

==[http://www.sciencedaily.com/releases/2010/01/100120211009.htm Lama Antibody] Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for [http://www.nih.gov/researchmatters/march2007/03052007toxins.htm bio-terrorism]. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

===The New!===
The crystal structure of the substrate SNAP25 complexed to the BoNT/A Lc showed the extended nature of ligand recognition and identified potential exosites that are away from the catalytic active site. To display the range of bidning sites for potential BoNT/A Lc inhibitors, the authors of the paper generated and selected a non-immune camelid(llama) library of single-domain VHH antibodies for binding to the BoNT/A Lc. This single-domain antibody has been postulated to be more able to bind into enzymatic cavities. Furthermore VHH is able to withstand intracellular reducing environment.

==Structure Complex of BoNT/A Lc and VHH==
'''All rights reserved by © Elsevier LTd. & Marks et. al'''
'''[[http://www.elsevier.com/wps/find/termsconditions.cws_home/termsconditions Terms and Conditions]]'''

VHH fragment consists of a single immunoglobulin domain with three CDR(complimentary-determining regions) defined by experimental electron density. Binding by the llama antibody fragment is driven by CDR1 and CDR3 interactions '''Figure 5B'''. The paratope(the antigen-binding site of an antibody.) formed by CDR1 and CDR3 is stabilized by several hydrogen bonds (VHH residues 37, 44, 117, and 124 interactions with BoNT/A Lc residues 113, 105, 102, and 345 respectively) and salt bridges (Lc Lys340 interactions with VHH Asp residues 113, 115 and 128) '''Figure 5C'''. Superimposed pictures of the BoNT/A Lc – VHH structure with the structure of SNAP25 bound to the Lc enzyme reveals that the paratope coincides with the an alpha-helical portion of the SNAP25 substrate. VHH paratope coincides with an alpha-helical portion of the SNAP25 substrate. '''Figure 5D'''. which is occupied by the heavy-chain belt of the neurotoxin '''Figure 5E'''. Both the CDR1 and CDR3 form small alpha-helices that appear to mimic the secondary structure of SNAP25 at the alpha-exosite( is a secondary binding site, remote from the active site. In order for an enzyme to be active, its exosite typically must be occupied). Although there is no primary homology sequence of SNAP25 and CDR1 and CDR3 alpha helices, the substrate mimicry is extended to tertiary structure because of a number of hydrophobic residues found on the VHH fragment coincide with similar residues found on SNAP25.'''Figure 5F'''. Residues include Met37 on CDR1 of the VHH fragment, which occupies the position of Met167 from SNAP25; Val116 from CDR3, which coincides with Leu160 of SNAP25; Val120 of CDR3, which occupies the space of Ile156 and Ile157 from SNAP25; and Val 123 of CDR3, which replaces Val154 of the natural substrate. Aa1 likely inhibits substrate cleavage by preventing efficient binding of SNAP25 and positioning of its scissile bond(bond of a substrate that is subject to enzymic cleavage).

[[Image:1.jpg|275px]][[Image:Lol.jpg|275px]]

[[Image:3.jpg|275px]][[Image:4.jpg|275px]]

[[Image:5.jpg|275px]][[Image:6.jpg|275px]]

==Uses of Botulinum Toxin==
[http://www.youtube.com/watch?v=dkpohXE06pg&feature=related BOTOX®]

[[Image:Hilary.jpg|left]][[Image:Cox.jpg|190px]]

==References==
J Mol Biol. 2010 Apr 9;397(4):1106-18. Epub 2010 Feb 6.
A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region.

Dong J, Thompson AA, Fan Y, Lou J, Conrad F, Ho M, Pires-Alves M, Wilson BA, Stevens RC, Marks JD.

Department of Anesthesia, University of California, San Francisco, San Francisco, CA 94110, USA.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T08:12:07Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions. [http://www.youtube.com/watch?v=6n3rrjbweO8 Mechanism of Botulinum Toxin]

==[http://www.sciencedaily.com/releases/2010/01/100120211009.htm Lama Antibody] Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for [http://www.nih.gov/researchmatters/march2007/03052007toxins.htm bio-terrorism]. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

===The New!===
The crystal structure of the substrate SNAP25 complexed to the BoNT/A Lc showed the extended nature of ligand recognition and identified potential exosites that are away from the catalytic active site. To display the range of bidning sites for potential BoNT/A Lc inhibitors, the authors of the paper generated and selected a non-immune camelid(llama) library of single-domain VHH antibodies for binding to the BoNT/A Lc. This single-domain antibody has been postulated to be more able to bind into enzymatic cavities. Furthermore VHH is able to withstand intracellular reducing environment.

==Structure Complex of BoNT/A Lc and VHH==
'''All rights reserved by © Elsevier LTd. & Marks et. al'''

[[Image:1.jpg|275px]][[Image:Lol.jpg|275px]]

[[Image:3.jpg|275px]][[Image:4.jpg|275px]]

[[Image:5.jpg|275px]][[Image:6.jpg|275px]]

==Uses of Botulinum Toxin==
[http://www.youtube.com/watch?v=dkpohXE06pg&feature=related BOTOX®]

[[Image:Hilary.jpg|left]][[Image:Cox.jpg|190px]]

==References==
J Mol Biol. 2010 Apr 9;397(4):1106-18. Epub 2010 Feb 6.
A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region.

Dong J, Thompson AA, Fan Y, Lou J, Conrad F, Ho M, Pires-Alves M, Wilson BA, Stevens RC, Marks JD.

Department of Anesthesia, University of California, San Francisco, San Francisco, CA 94110, USA.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T08:04:42Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions. [http://www.youtube.com/watch?v=6n3rrjbweO8 Mechanism of Botulinum Toxin]

==[http://www.sciencedaily.com/releases/2010/01/100120211009.htm Lama Antibody] Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for [http://www.nih.gov/researchmatters/march2007/03052007toxins.htm bio-terrorism]. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

===The New!===
The crystal structure of the substrate SNAP25 complexed to the BoNT/A Lc showed the extended nature of ligand recognition and identified potential exosites that are away from the catalytic active site. To display the range of bidning sites for potential BoNT/A Lc inhibitors, the authors of the paper generated and selected a non-immune camelid(llama) library of single-domain VHH antibodies for binding to the BoNT/A Lc. This single-domain antibody has been postulated to be more able to bind into enzymatic cavities. Furthermore VHH is able to withstand intracellular reducing environment.

==Structure Complex of BoNT/A Lc and VHH==
'''All rights reserved by © Elsevier LTd. & Marks et. al'''

[[Image:1.jpg]][[Image:2.jpg]]

[[Image:3.jpg]][[Image:4.jpg]]

[[Image:5.jpg]][[Image:6.jpg]]

==Uses of Botulinum Toxin==
[http://www.youtube.com/watch?v=dkpohXE06pg&feature=related BOTOX®]

[[Image:Hilary.jpg|left]][[Image:Cox.jpg|190px]]

==References==
J Mol Biol. 2010 Apr 9;397(4):1106-18. Epub 2010 Feb 6.
A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region.

Dong J, Thompson AA, Fan Y, Lou J, Conrad F, Ho M, Pires-Alves M, Wilson BA, Stevens RC, Marks JD.

Department of Anesthesia, University of California, San Francisco, San Francisco, CA 94110, USA.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T08:01:29Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions. [http://www.youtube.com/watch?v=6n3rrjbweO8 Mechanism of Botulinum Toxin]

==[http://www.sciencedaily.com/releases/2010/01/100120211009.htm Lama Antibody] Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for [http://www.nih.gov/researchmatters/march2007/03052007toxins.htm bio-terrorism]. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

===The New!===
The crystal structure of the substrate SNAP25 complexed to the BoNT/A Lc showed the extended nature of ligand recognition and identified potential exosites that are away from the catalytic active site. To display the range of bidning sites for potential BoNT/A Lc inhibitors, the authors of the paper generated and selected a non-immune camelid(llama) library of single-domain VHH antibodies for binding to the BoNT/A Lc. This single-domain antibody has been postulated to be more able to bind into enzymatic cavities. Furthermore VHH is able to withstand intracellular reducing environment.

==Structure Complex of BoNT/A Lc and VHH==
'''All rights reserved by © Elsevier LTd. & Marks et. al'''

[[Image:1.bmp]][[Image:2.bmp]]

[[Image:3.bmp]][[Image:4.bmp]]

[[Image:5.bmp]][[Image:6.bmp]]

==Uses of Botulinum Toxin==
[http://www.youtube.com/watch?v=dkpohXE06pg&feature=related BOTOX®]

[[Image:Hilary.jpg|left]][[Image:Cox.jpg|190px]]

==References==
J Mol Biol. 2010 Apr 9;397(4):1106-18. Epub 2010 Feb 6.
A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region.

Dong J, Thompson AA, Fan Y, Lou J, Conrad F, Ho M, Pires-Alves M, Wilson BA, Stevens RC, Marks JD.

Department of Anesthesia, University of California, San Francisco, San Francisco, CA 94110, USA.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T08:00:57Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions. [http://www.youtube.com/watch?v=6n3rrjbweO8 Mechanism of Botulinum Toxin]

==[http://www.sciencedaily.com/releases/2010/01/100120211009.htm Lama Antibody] Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for [http://www.nih.gov/researchmatters/march2007/03052007toxins.htm bio-terrorism]. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

===The New!===
The crystal structure of the substrate SNAP25 complexed to the BoNT/A Lc showed the extended nature of ligand recognition and identified potential exosites that are away from the catalytic active site. To display the range of bidning sites for potential BoNT/A Lc inhibitors, the authors of the paper generated and selected a non-immune camelid(llama) library of single-domain VHH antibodies for binding to the BoNT/A Lc. This single-domain antibody has been postulated to be more able to bind into enzymatic cavities. Furthermore VHH is able to withstand intracellular reducing environment.

==Structure Complex of BoNT/A Lc and VHH==
'''All rights reserved by © Elsevier LTd. & Marks et. al'''

[[Image:1.bmp]][[Image:2.bmp]]
[[Image:3.bmp]][[Image:4.bmp]]
[[Image:5.bmp]][[Image:6.bmp]]

==Uses of Botulinum Toxin==
[http://www.youtube.com/watch?v=dkpohXE06pg&feature=related BOTOX®]

[[Image:Hilary.jpg|left]][[Image:Cox.jpg|190px]]

==References==
J Mol Biol. 2010 Apr 9;397(4):1106-18. Epub 2010 Feb 6.
A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region.

Dong J, Thompson AA, Fan Y, Lou J, Conrad F, Ho M, Pires-Alves M, Wilson BA, Stevens RC, Marks JD.

Department of Anesthesia, University of California, San Francisco, San Francisco, CA 94110, USA.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T07:59:06Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions. [http://www.youtube.com/watch?v=6n3rrjbweO8 Mechanism of Botulinum Toxin]

==[http://www.sciencedaily.com/releases/2010/01/100120211009.htm Lama Antibody] Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for [http://www.nih.gov/researchmatters/march2007/03052007toxins.htm bio-terrorism]. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

===The New!===
The crystal structure of the substrate SNAP25 complexed to the BoNT/A Lc showed the extended nature of ligand recognition and identified potential exosites that are away from the catalytic active site. To display the range of bidning sites for potential BoNT/A Lc inhibitors, the authors of the paper generated and selected a non-immune camelid(llama) library of single-domain VHH antibodies for binding to the BoNT/A Lc. This single-domain antibody has been postulated to be more able to bind into enzymatic cavities. Furthermore VHH is able to withstand intracellular reducing environment.

==Structure Complex of BoNT/A Lc and VHH==
'''All rights reserved by © Elsevier LTd. & Marks et. al'''

[[Image:1.jpg]][[Image:2.jpg]]
[[Image:3.jpg]][[Image:4.jpg]]
[[Image:5.jpg]][[Image:6.jpg]]

==Uses of Botulinum Toxin==
[http://www.youtube.com/watch?v=dkpohXE06pg&feature=related BOTOX®]

[[Image:Hilary.jpg|left]][[Image:Cox.jpg|190px]]

==References==
J Mol Biol. 2010 Apr 9;397(4):1106-18. Epub 2010 Feb 6.
A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region.

Dong J, Thompson AA, Fan Y, Lou J, Conrad F, Ho M, Pires-Alves M, Wilson BA, Stevens RC, Marks JD.

Department of Anesthesia, University of California, San Francisco, San Francisco, CA 94110, USA.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T07:45:43Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions. [http://www.youtube.com/watch?v=6n3rrjbweO8 Mechanism of Botulinum Toxin]

==[http://www.sciencedaily.com/releases/2010/01/100120211009.htm Lama Antibody] Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for [http://www.nih.gov/researchmatters/march2007/03052007toxins.htm bio-terrorism]. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

===The New!===
The crystal structure of the substrate SNAP25 complexed to the BoNT/A Lc showed the extended nature of ligand recognition and identified potential exosites that are away from the catalytic active site. To display the range of bidning sites for potential BoNT/A Lc inhibitors, the authors of the paper generated and selected a non-immune camelid(llama) library of single-domain VHH antibodies for binding to the BoNT/A Lc. This single-domain antibody has been postulated to be more able to bind into enzymatic cavities. Furthermore VHH is able to withstand intracellular reducing environment.

==Uses of Botulinum Toxin==
[http://www.youtube.com/watch?v=dkpohXE06pg&feature=related BOTOX®]

[[Image:Hilary.jpg|left]][[Image:Cox.jpg|190px]]

==References==
J Mol Biol. 2010 Apr 9;397(4):1106-18. Epub 2010 Feb 6.
A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region.

Dong J, Thompson AA, Fan Y, Lou J, Conrad F, Ho M, Pires-Alves M, Wilson BA, Stevens RC, Marks JD.

Department of Anesthesia, University of California, San Francisco, San Francisco, CA 94110, USA.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T07:38:57Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions. [http://www.youtube.com/watch?v=6n3rrjbweO8 Mechanism of Botulinum Toxin]

==[http://www.sciencedaily.com/releases/2010/01/100120211009.htm Lama Antibody] Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for [http://www.nih.gov/researchmatters/march2007/03052007toxins.htm bio-terrorism]. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

===The New!===
The crystal structure of the substrate SNAP25 complexed to the BoNT/A Lc showed the extended nature of ligand recognition and identified potential exosites that are away from the catalytic active site. To display the range of bidning sites for potential BoNT/A Lc inhibitors, the authors of the paper generated and selected a non-immune camelid(llama) library of single-domain VHH antibodies for binding to the BoNT/A Lc. This single-domain antibody has been postulated to be more able to bind into enzymatic cavities. Furthermore VHH is able to withstand intracellular reducing environment.

[[Image:1.jpg]] [[Image:2.jpg]]

[[Image:3.jpg]] [[Image:4.jpg]]

[[Image:5.jpg]] [[Image:6.jpg]]
==Uses of Botulinum Toxin==
[http://www.youtube.com/watch?v=dkpohXE06pg&feature=related BOTOX®]

[[Image:Hilary.jpg|left]][[Image:Cox.jpg|190px]]

==References==
J Mol Biol. 2010 Apr 9;397(4):1106-18. Epub 2010 Feb 6.
A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region.

Dong J, Thompson AA, Fan Y, Lou J, Conrad F, Ho M, Pires-Alves M, Wilson BA, Stevens RC, Marks JD.

Department of Anesthesia, University of California, San Francisco, San Francisco, CA 94110, USA.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T07:35:31Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions. [http://www.youtube.com/watch?v=6n3rrjbweO8 Mechanism of Botulinum Toxin]

==[http://www.sciencedaily.com/releases/2010/01/100120211009.htm Lama Antibody] Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for [http://www.nih.gov/researchmatters/march2007/03052007toxins.htm bio-terrorism]. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

===The New!===
The crystal structure of the substrate SNAP25 complexed to the BoNT/A Lc showed the extended nature of ligand recognition and identified potential exosites that are away from the catalytic active site. To display the range of bidning sites for potential BoNT/A Lc inhibitors, the authors of the paper generated and selected a non-immune camelid(llama) library of single-domain VHH antibodies for binding to the BoNT/A Lc. This single-domain antibody has been postulated to be more able to bind into enzymatic cavities. Furthermore VHH is able to withstand intracellular reducing environment.

==Uses of Botulinum Toxin==
[http://www.youtube.com/watch?v=dkpohXE06pg&feature=related BOTOX®]

[[Image:Hilary.jpg|left]][[Image:Cox.jpg|190px]]

==References==
J Mol Biol. 2010 Apr 9;397(4):1106-18. Epub 2010 Feb 6.
A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region.

Dong J, Thompson AA, Fan Y, Lou J, Conrad F, Ho M, Pires-Alves M, Wilson BA, Stevens RC, Marks JD.

Department of Anesthesia, University of California, San Francisco, San Francisco, CA 94110, USA.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T06:13:39Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions. [http://www.youtube.com/watch?v=6n3rrjbweO8 Mechanism of Botulinum Toxin]

==[http://www.sciencedaily.com/releases/2010/01/100120211009.htm Lama Antibody] Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for [http://www.nih.gov/researchmatters/march2007/03052007toxins.htm bio-terrorism]. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

===The New!===
The crystal structure of the substrate SNAP25 complexed to the BoNT/A Lc showed the extended nature of ligand recognition and identified potential exosites that are away from the catalytic active site. To display the range of bidning sites for potential BoNT/A Lc inhibitors, the authors of the paper generated and selected a non-immune camelid(llama) library of single-domain VHH antibodies for binding to the BoNT/A Lc. This single-domain antibody has been postulated to be more able to bind into enzymatic cavities. Furthermore VHH is able to withstand intracellular reducing environment.

==Uses of Botulinum Toxin==
[http://www.youtube.com/watch?v=dkpohXE06pg&feature=related BOTOX®]

[[Image:Hilary.jpg|left]][[Image:Cox.jpg|190px]]

==References==
J Mol Biol. 2010 Apr 9;397(4):1106-18. Epub 2010 Feb 6.
A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region.

Dong J, Thompson AA, Fan Y, Lou J, Conrad F, Ho M, Pires-Alves M, Wilson BA, Stevens RC, Marks JD.

Department of Anesthesia, University of California, San Francisco, San Francisco, CA 94110, USA.

File:Cox.jpg

2010-12-02T06:10:20Z

Bryant Chen:

File:Hilary.jpg

2010-12-02T06:09:29Z

Bryant Chen:

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T06:09:10Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions. [http://www.youtube.com/watch?v=6n3rrjbweO8 Mechanism of Botulinum Toxin]

==[http://www.sciencedaily.com/releases/2010/01/100120211009.htm Lama Antibody] Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for [http://www.nih.gov/researchmatters/march2007/03052007toxins.htm bio-terrorism]. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

===The New!===
The crystal structure of the substrate SNAP25 complexed to the BoNT/A Lc showed the extended nature of ligand recognition and identified potential exosites that are away from the catalytic active site. To display the range of bidning sites for potential BoNT/A Lc inhibitors, the authors of the paper generated and selected a non-immune camelid(llama) library of single-domain VHH antibodies for binding to the BoNT/A Lc. This single-domain antibody has been postulated to be more able to bind into enzymatic cavities. Furthermore VHH is able to withstand intracellular reducing environment.

==Uses of Botulinum Toxin==
[http://www.youtube.com/watch?v=dkpohXE06pg&feature=related BOTOX®]

[[Image:Hilary.jpg|left]][[Image:Cox.jpg|right]]
==References==
J Mol Biol. 2010 Apr 9;397(4):1106-18. Epub 2010 Feb 6.
A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region.

Dong J, Thompson AA, Fan Y, Lou J, Conrad F, Ho M, Pires-Alves M, Wilson BA, Stevens RC, Marks JD.

Department of Anesthesia, University of California, San Francisco, San Francisco, CA 94110, USA.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T06:04:05Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions. [http://www.youtube.com/watch?v=6n3rrjbweO8 Mechanism of Botulinum Toxin]

==[http://www.sciencedaily.com/releases/2010/01/100120211009.htm Lama Antibody] Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for [http://www.nih.gov/researchmatters/march2007/03052007toxins.htm bio-terrorism]. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

===The New!===
The crystal structure of the substrate SNAP25 complexed to the BoNT/A Lc showed the extended nature of ligand recognition and identified potential exosites that are away from the catalytic active site. To display the range of bidning sites for potential BoNT/A Lc inhibitors, the authors of the paper generated and selected a non-immune camelid(llama) library of single-domain VHH antibodies for binding to the BoNT/A Lc. This single-domain antibody has been postulated to be more able to bind into enzymatic cavities. Furthermore VHH is able to withstand intracellular reducing environment.

==Uses of Botulinum Toxin==
[http://www.youtube.com/watch?v=dkpohXE06pg&feature=related BOTOX®]

==References==
J Mol Biol. 2010 Apr 9;397(4):1106-18. Epub 2010 Feb 6.
A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region.

Dong J, Thompson AA, Fan Y, Lou J, Conrad F, Ho M, Pires-Alves M, Wilson BA, Stevens RC, Marks JD.

Department of Anesthesia, University of California, San Francisco, San Francisco, CA 94110, USA.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T05:57:05Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions. [http://www.youtube.com/watch?v=6n3rrjbweO8 Mechanism of Botulinum Toxin]

==[http://www.sciencedaily.com/releases/2010/01/100120211009.htm Lama Antibody] Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for [http://www.nih.gov/researchmatters/march2007/03052007toxins.htm bio-terrorism]. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

===The New!===
The crystal structure of the substrate SNAP25 complexed to the BoNT/A Lc showed the extended nature of ligand recognition and identified potential exosites that are away from the catalytic active site. To display the range of bidning sites for potential BoNT/A Lc inhibitors, the authors of the paper generated and selected a non-immune camelid(llama) library of single-domain VHH antibodies for binding to the BoNT/A Lc. This single-domain antibody has been postulated to be more able to bind into enzymatic cavities. Furthermore VHH is able to withstand intracellular reducing environment.

==References==
J Mol Biol. 2010 Apr 9;397(4):1106-18. Epub 2010 Feb 6.
A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region.

Dong J, Thompson AA, Fan Y, Lou J, Conrad F, Ho M, Pires-Alves M, Wilson BA, Stevens RC, Marks JD.

Department of Anesthesia, University of California, San Francisco, San Francisco, CA 94110, USA.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T05:50:39Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions. [http://www.youtube.com/watch?v=6n3rrjbweO8 Mechanism of Botulinum Toxin]

==[http://www.sciencedaily.com/releases/2010/01/100120211009.htm Lama Antibody] Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for bio-terrorism. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

===The New!===
The crystal structure of the substrate SNAP25 complexed to the BoNT/A Lc showed the extended nature of ligand recognition and identified potential exosites that are away from the catalytic active site. To display the range of bidning sites for potential BoNT/A Lc inhibitors, the authors of the paper generated and selected a non-immune camelid(llama) library of single-domain VHH antibodies for binding to the BoNT/A Lc. This single-domain antibody has been postulated to be more able to bind into enzymatic cavities. Furthermore VHH is able to withstand intracellular reducing environment.

==References==
J Mol Biol. 2010 Apr 9;397(4):1106-18. Epub 2010 Feb 6.
A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region.

Dong J, Thompson AA, Fan Y, Lou J, Conrad F, Ho M, Pires-Alves M, Wilson BA, Stevens RC, Marks JD.

Department of Anesthesia, University of California, San Francisco, San Francisco, CA 94110, USA.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T05:42:37Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions. [http://www.youtube.com/watch?v=6n3rrjbweO8 Mechanism of Botulinum Toxin]

==Llama Antibody Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for bio-terrorism. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

===The New!===
The crystal structure of the substrate SNAP25 complexed to the BoNT/A Lc showed the extended nature of ligand recognition and identified potential exosites that are away from the catalytic active site. To display the range of bidning sites for potential BoNT/A Lc inhibitors, the authors of the paper generated and selected a non-immune camelid(llama) library of single-domain VHH antibodies for binding to the BoNT/A Lc. This single-domain antibody has been postulated to be more able to bind into enzymatic cavities. Furthermore VHH is able to withstand intracellular reducing environment.

==References==
J Mol Biol. 2010 Apr 9;397(4):1106-18. Epub 2010 Feb 6.
A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region.

Dong J, Thompson AA, Fan Y, Lou J, Conrad F, Ho M, Pires-Alves M, Wilson BA, Stevens RC, Marks JD.

Department of Anesthesia, University of California, San Francisco, San Francisco, CA 94110, USA.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T05:28:13Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack. Dr. Kim Janda of The Scripps Research Institute and his colleagues, with funding from NIH's National Institute of Allergy and Infectious Diseases (NIAID) and The Skaggs Institute for Chemical Biology, set out to find other compounds that could act against botulinum toxin A.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions. [http://www.youtube.com/watch?v=6n3rrjbweO8 Mechanism of Botulinum Toxin]

==Llama Antibody Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for bio-terrorism. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

===The New!===
The crystal structure of the substrate SNAP25 complexed to the BoNT/A Lc showed the extended nature of ligand recognition and identified potential exosites that are away from the catalytic active site. To display the range of bidning sites for potential BoNT/A Lc inhibitors, the authors of the paper generated and selected a non-immune camelid(llama) library of single-domain VHH antibodies for binding to the BoNT/A Lc. This single-domain antibody has been postulated to be more able to bind into enzymatic cavities. Furthermore VHH is able to withstand intracellular reducing environment.

==References==
J Mol Biol. 2010 Apr 9;397(4):1106-18. Epub 2010 Feb 6.
A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region.

Dong J, Thompson AA, Fan Y, Lou J, Conrad F, Ho M, Pires-Alves M, Wilson BA, Stevens RC, Marks JD.

Department of Anesthesia, University of California, San Francisco, San Francisco, CA 94110, USA.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T05:25:25Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack. Dr. Kim Janda of The Scripps Research Institute and his colleagues, with funding from NIH's National Institute of Allergy and Infectious Diseases (NIAID) and The Skaggs Institute for Chemical Biology, set out to find other compounds that could act against botulinum toxin A.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions. [http://www.youtube.com/watch?v=6n3rrjbweO8 Mechanism of Botulinum Toxin]

==Llama Antibody Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for bio-terrorism. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

===The New!===
The crystal structure of the substrate SNAP25 complexed to the BoNT/A Lc showed the extended nature of ligand recognition and identified potential exosites that are away from the catalytic active site. To display the range of bidning sites for potential BoNT/A Lc inhibitors, the authors of the paper generated and selected a non-immune camelid(llama) library of single-domain VHH antibodies for binding to the BoNT/A Lc. This single-domain antibody has been postulated to be more able to bind into enzymatic cavities. Furthermore VHH is able to withstand intracellular reducing environment.

==References==

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T05:20:01Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack. Dr. Kim Janda of The Scripps Research Institute and his colleagues, with funding from NIH's National Institute of Allergy and Infectious Diseases (NIAID) and The Skaggs Institute for Chemical Biology, set out to find other compounds that could act against botulinum toxin A.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions.

==Llama Antibody Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for bio-terrorism. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

===The New!===
The crystal structure of the substrate SNAP25 complexed to the BoNT/A Lc showed the extended nature of ligand recognition and identified potential exosites that are away from the catalytic active site. To display the range of bidning sites for potential BoNT/A Lc inhibitors, the authors of the paper generated and selected a non-immune camelid(llama) library of single-domain VHH antibodies for binding to the BoNT/A Lc. This single-domain antibody has been postulated to be more able to bind into enzymatic cavities. Furthermore VHH is able to withstand intracellular reducing environment.

==Structure of BoNT/A Lc-Aa1 VHH Complex==
[[Image:1.jpg]][[Image:2.jpg]]
[[Image:3.jpg]][[Image:4.jpg]]
[[Image:5.jpg]][[Image:6.jpg]]

[[Image:7.jpg]]

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T04:58:48Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack. Dr. Kim Janda of The Scripps Research Institute and his colleagues, with funding from NIH's National Institute of Allergy and Infectious Diseases (NIAID) and The Skaggs Institute for Chemical Biology, set out to find other compounds that could act against botulinum toxin A.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions.

==Llama Antibody Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for bio-terrorism. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

===The New!===
The crystal structure of the substrate SNAP25 complexed to the BoNT/A Lc showed the extended nature of ligand recognition and identified potential exosites that are away from the catalytic active site. To display the range of bidning sites for potential BoNT/A Lc inhibitors, the authors of the paper generated and selected a non-immune camelid(llama) library of single-domain VHH antibodies for binding to the BoNT/A Lc. This single-domain antibody has been postulated to be more able to bind into enzymatic cavities. Furthermore VHH is able to withstand intracellular reducing environment.

==Structure of BoNT/A Lc-Aa1 VHH Complex==

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T04:40:51Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack. Dr. Kim Janda of The Scripps Research Institute and his colleagues, with funding from NIH's National Institute of Allergy and Infectious Diseases (NIAID) and The Skaggs Institute for Chemical Biology, set out to find other compounds that could act against botulinum toxin A.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions.

==Llama Antibody Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for bio-terrorism. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT. The problem with this antitoxin is that they do not work once the toxin has entered the neuron and therefore can not reverse paralysis. Therefore, there is a huge interest in developing inhibitors of the translocation and catalytic domains.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T04:36:26Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack. Dr. Kim Janda of The Scripps Research Institute and his colleagues, with funding from NIH's National Institute of Allergy and Infectious Diseases (NIAID) and The Skaggs Institute for Chemical Biology, set out to find other compounds that could act against botulinum toxin A.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions.

==Llama Antibody Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for bio-terrorism. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

===The Old?===
The mainstream treatment for botulism is an antitoxin. An antitoxin is an antibody that has the ability to specifically neutralize a toxin. The antitoxin would be able to clearing toxins from the circulation, prevent BoNT entering neurons, and also could block uptake of BoNT.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T04:21:14Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack. Dr. Kim Janda of The Scripps Research Institute and his colleagues, with funding from NIH's National Institute of Allergy and Infectious Diseases (NIAID) and The Skaggs Institute for Chemical Biology, set out to find other compounds that could act against botulinum toxin A.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions.

==Llama Antibody Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for bio-terrorism. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis. The translocation domain at the N-terminal portion of the heavy chain mediates escape of the toxin light chain from the endosome. The light chain is responsible for cleaving SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) which is responsible for docking the synaptic vesicle, resulting in an inhibition of releasing acetylcholine.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T04:18:14Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px|alt Clostridium botulinum bacteria up close. Image courtesy of CDC.]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack. Dr. Kim Janda of The Scripps Research Institute and his colleagues, with funding from NIH's National Institute of Allergy and Infectious Diseases (NIAID) and The Skaggs Institute for Chemical Biology, set out to find other compounds that could act against botulinum toxin A.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]
Botulinum Toxin is very deadly in a sense that it prevents muscle contraction. Botulinum toxin prevents the release of [http://en.wikipedia.org/wiki/Acetylcholine#Function Acetylcholine]into the synaptic cleft to relay the signaling to another neuron. Acetylcholine has functions both in the peripheral nervous system (PNS) and in the central nervous system (CNS) as a neuromodulator.In the peripheral nervous system, acetylcholine activates muscles, and is a major neurotransmitter in the autonomic nervous system.In the central nervous system, acetylcholine and the associated neurons form a neurotransmitter system, the cholinergic system, which tends to cause anti-excitatory actions.

==Llama Antibody Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==Introduction==
Human Botulism is caused by ''Clostridium botulinum''neurotoxin(BoNT) that is characterized by flaccid muscle paralysis. Without immediate care could result in death. In addition, BoNT is also classified as one of the top six highest risk threat for bio-terrorism. BoNT has 3 domains. It is composed of a light-chain segment and two heavy-chain segments. The C-terminal portion of the heavy chain is the cell binding domain which docks the toxin to ganglioside receptors and a protein receptor on presynaptic neurons resulting in toxin endocytosis.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T04:07:36Z

Bryant Chen:

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T01:39:05Z

Bryant Chen:

==''[http://www.nih.gov/researchmatters/march2007/03052007toxins.htm Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px]]
C. botulinum bacteria, which are commonly found in soil, can form spores that allow them to survive in a dormant state until they're exposed to conditions that support their growth. Infant botulism, the most common type of botulism, occurs when infants consume these spores, which then grow in their intestines and release toxin. In adults, most outbreaks are caused by contaminated home-canned foods. You can also get botulism through contaminated wounds.

If diagnosed early, botulism can be treated with an antitoxin, which blocks the action of the toxin in the blood. But recovery still takes many months. The antitoxin can also cause serious side effects, has only a very short window of application and is expensive to produce in large enough quantities to combat a bioterrorism attack. Dr. Kim Janda of The Scripps Research Institute and his colleagues, with funding from NIH's National Institute of Allergy and Infectious Diseases (NIAID) and The Skaggs Institute for Chemical Biology, set out to find other compounds that could act against botulinum toxin A.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]

==Llama Antibody Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T01:36:00Z

Bryant Chen:

==''[http://en.wikipedia.org/wiki/Clostridium_botulinum#Pathology Clostridium Botulinum]''==
[[Image:toxins_l.gif|right|180px]]
Clostridium botulinum is a gram-positive, rod shaped bacterium that produces neurotoxins known as botulinum neurotoxins types A-G, which causes flaccid muscular paralysis seen in botulism, and is also the main paralytic agent in botox. It is an anaerobic spore-former, which produces oval, subterminal endospores and is commonly found in soil.Botulism poisoning can occur due to improperly preserved or home canned low-acid food that was not processed using correct preservation times and/or pressure.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]

==Llama Antibody Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

File:Toxins l.gif

2010-12-02T01:33:57Z

Bryant Chen:

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T01:33:37Z

Bryant Chen:

==''[http://en.wikipedia.org/wiki/Clostridium_botulinum#Pathology Clostridium Botulinum]''==
[[Image:toxins_l|right|300px]]
Clostridium botulinum is a gram-positive, rod shaped bacterium that produces neurotoxins known as botulinum neurotoxins types A-G, which causes flaccid muscular paralysis seen in botulism, and is also the main paralytic agent in botox. It is an anaerobic spore-former, which produces oval, subterminal endospores and is commonly found in soil.Botulism poisoning can occur due to improperly preserved or home canned low-acid food that was not processed using correct preservation times and/or pressure.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]

==Llama Antibody Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T01:31:32Z

Bryant Chen:

==''[http://en.wikipedia.org/wiki/Clostridium_botulinum#Pathology Clostridium Botulinum]''==
[[Image:C.Botulinum.jpg|right|300px]]
Clostridium botulinum is a gram-positive, rod shaped bacterium that produces neurotoxins known as botulinum neurotoxins types A-G, which causes flaccid muscular paralysis seen in botulism, and is also the main paralytic agent in botox. It is an anaerobic spore-former, which produces oval, subterminal endospores and is commonly found in soil.Botulism poisoning can occur due to improperly preserved or home canned low-acid food that was not processed using correct preservation times and/or pressure.

==Botulinum Toxin==
[[Image:Figure1.png|left|300px]]

==Llama Antibody Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T01:30:57Z

Bryant Chen:

File:Figure1.png

2010-12-02T01:25:39Z

Bryant Chen: uploaded a new version of "Image:Figure1.png"

File:Figure1.png

2010-12-02T01:24:38Z

Bryant Chen:

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T01:23:05Z

Bryant Chen:

==''[http://en.wikipedia.org/wiki/Clostridium_botulinum#Pathology Clostridium Botulinum]''==
[[Image:C.Botulinum.jpg|right]]
Clostridium botulinum is a gram-positive, rod shaped bacterium that produces neurotoxins known as botulinum neurotoxins types A-G, which causes flaccid muscular paralysis seen in botulism, and is also the main paralytic agent in botox. It is an anaerobic spore-former, which produces oval, subterminal endospores and is commonly found in soil.

==Botulinum Toxin==
[[Image:Figure1.jpg|left]]
==Llama Antibody Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T01:20:55Z

Bryant Chen:

==''[http://en.wikipedia.org/wiki/Clostridium_botulinum#Pathology Clostridium Botulinum]''==
[[Image:C.Botulinum.jpg|right]]
Clostridium botulinum is a gram-positive, rod shaped bacterium that produces neurotoxins known as botulinum neurotoxins types A-G, which causes flaccid muscular paralysis seen in botulism, and is also the main paralytic agent in botox. It is an anaerobic spore-former, which produces oval, subterminal endospores and is commonly found in soil.

==Llama Antibody Inhibits Botulinum Neruotoxin==
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T01:07:33Z

Bryant Chen:

==''[http://en.wikipedia.org/wiki/Clostridium_botulinum#Pathology Clostridium Botulinum]''==
Clostridium botulinum is a gram-positive, rod shaped bacterium that produces neurotoxins known as botulinum neurotoxins types A-G, which causes flaccid muscular paralysis seen in botulism, and is also the main paralytic agent in botox. It is an anaerobic spore-former, which produces oval, subterminal endospores and is commonly found in soil.
[[Image:C.Botulinum.jpg] align=right]

{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T01:05:17Z

Bryant Chen:

==''[http://en.wikipedia.org/wiki/Clostridium_botulinum#Pathology Clostridium Botulinum]''==
Clostridium botulinum is a gram-positive, rod shaped bacterium that produces neurotoxins known as botulinum neurotoxins types A-G, which causes flaccid muscular paralysis seen in botulism, and is also the main paralytic agent in botox. It is an anaerobic spore-former, which produces oval, subterminal endospores and is commonly found in soil.
[[Image:C.Botulinum.jpg]]

{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T01:02:21Z

Bryant Chen:

==''[http://en.wikipedia.org/wiki/Clostridium_botulinum#Pathology Clostridium Botulinum]''==
Clostridium botulinum is a rod-shaped microorganism. It is an obligate anaerobe, meaning that oxygen is poisonous to the cells. However, C. botulinum tolerates traces of oxygen due to the enzyme called superoxide dismutase (SOD) which is an important antioxidant defense in nearly all cells exposed to oxygen. [1] C. botulinum does not form endospores as a way to protect the viability of the organism, but rather as a mechanism to produce the neurotoxin. C. botulinum is only able to produce the neurotoxin during sporulation, which can only happen in an anaerobic environment. Other bacterial species produce spores in an unfavorable growth environment to preserve the organism's viability and permit survival in a dormant state until the spores are exposed to favorable conditions.
{{Taxobox
| color = white
| name = ''Clostridium botulinum''
| image = Clostridium botulinum_01.png
| image_width = 240px
| image_caption = ''Clostridium botulinum'' stained with [[gentian violet]].
| domain = [[Bacteria]]
| divisio = [[Firmicutes]]
| classis = [[Clostridia]]
| ordo = [[Clostridia]]les
| familia = [[Clostridiaceae]]
| genus = ''[[Clostridium]]''
| species = '''''C. botulinum'''''
| binomial = ''Clostridium botulinum''
| binomial_authority = van Ermengem, 1896
}}

'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T01:00:52Z

Bryant Chen:

==''[http://en.wikipedia.org/wiki/Clostridium_botulinum#Pathology Clostridium Botulinum]''==
Clostridium botulinum is a rod-shaped microorganism. It is an obligate anaerobe, meaning that oxygen is poisonous to the cells. However, C. botulinum tolerates traces of oxygen due to the enzyme called superoxide dismutase (SOD) which is an important antioxidant defense in nearly all cells exposed to oxygen. [1] C. botulinum does not form endospores as a way to protect the viability of the organism, but rather as a mechanism to produce the neurotoxin. C. botulinum is only able to produce the neurotoxin during sporulation, which can only happen in an anaerobic environment. Other bacterial species produce spores in an unfavorable growth environment to preserve the organism's viability and permit survival in a dormant state until the spores are exposed to favorable conditions. In the laboratory Clostridium botulinum is usually isolated in tryptose sulfite cycloserine (TSC) growth media in an anaerobic environment with less than 2% of oxygen. This can be achieved by several commercial kits that use a chemical reaction to replace O2 with CO2 (E.J. GasPak System). C. botulinum is a lipase negative microorganism that grows between pH of 4.8 and 7 and it can't use lactose as a primary carbon source, characteristics important during a biochemical identification.[2]

'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-12-02T00:59:48Z

Bryant Chen:

==''[http://en.wikipedia.org/wiki/Clostridium_botulinum#Pathology Clostridium Botulinum]''==
Clostridium botulinum is a rod-shaped microorganism. It is an obligate anaerobe, meaning that oxygen is poisonous to the cells. However, C. botulinum tolerates traces of oxygen due to the enzyme called superoxide dismutase (SOD) which is an important antioxidant defense in nearly all cells exposed to oxygen. [1] C. botulinum does not form endospores as a way to protect the viability of the organism, but rather as a mechanism to produce the neurotoxin. C. botulinum is only able to produce the neurotoxin during sporulation, which can only happen in an anaerobic environment. Other bacterial species produce spores in an unfavorable growth environment to preserve the organism's viability and permit survival in a dormant state until the spores are exposed to favorable conditions. In the laboratory Clostridium botulinum is usually isolated in tryptose sulfite cycloserine (TSC) growth media in an anaerobic environment with less than 2% of oxygen. This can be achieved by several commercial kits that use a chemical reaction to replace O2 with CO2 (E.J. GasPak System). C. botulinum is a lipase negative microorganism that grows between pH of 4.8 and 7 and it can't use lactose as a primary carbon source, characteristics important during a biochemical identification.[2]
'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''

{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

Llama Antibody Inhibits Botulinum Neruotoxin

2010-11-16T22:57:44Z

Bryant Chen:

'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==''[http://en.wikipedia.org/wiki/Clostridium_botulinum#Pathology Clostridium Botulinum]''==
Clostridium botulinum is a rod-shaped microorganism. It is an obligate anaerobe, meaning that oxygen is poisonous to the cells. However, C. botulinum tolerates traces of oxygen due to the enzyme called superoxide dismutase (SOD) which is an important antioxidant defense in nearly all cells exposed to oxygen. [1] C. botulinum does not form endospores as a way to protect the viability of the organism, but rather as a mechanism to produce the neurotoxin. C. botulinum is only able to produce the neurotoxin during sporulation, which can only happen in an anaerobic environment. Other bacterial species produce spores in an unfavorable growth environment to preserve the organism's viability and permit survival in a dormant state until the spores are exposed to favorable conditions. In the laboratory Clostridium botulinum is usually isolated in tryptose sulfite cycloserine (TSC) growth media in an anaerobic environment with less than 2% of oxygen. This can be achieved by several commercial kits that use a chemical reaction to replace O2 with CO2 (E.J. GasPak System). C. botulinum is a lipase negative microorganism that grows between pH of 4.8 and 7 and it can't use lactose as a primary carbon source, characteristics important during a biochemical identification.[2]

Llama Antibody Inhibits Botulinum Neruotoxin

2010-11-16T22:57:10Z

Bryant Chen:

Llama Antibody Inhibits Botulinum Neruotoxin

2010-11-16T22:55:10Z

Bryant Chen:

Llama Antibody Inhibits Botulinum Neruotoxin

2010-11-16T22:34:40Z

Bryant Chen:

'''[http://www.rcsb.org/pdb/explore/explore.do?structureId=3K3Q PubMed Abstract]:'''
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==''[http://en.wikipedia.org/wiki/Clostridium_botulinum#Pathology Clostridium Botulinum]''==
''C. Botulinum'' is a Gram Positive bacteria belonging to the group of Firmicutes. They are obligate anaerobes capable of producing endospores. ''C. Botulinum'' is capable of producing a neurotoxin that causes muscle paralysis. The toxin can enter the body in many different ways: colonization of the digestive tract in both adults (Adult intestinal toxemia) and infants (infant botulism), ingestion of toxin from foods (Foodborne Botulism), and entry of a wound (Wound botulism).

Llama Antibody Inhibits Botulinum Neruotoxin

2010-11-16T22:32:50Z

Bryant Chen:

'''PubMed Abstract:'''
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==''[http://en.wikipedia.org/wiki/Clostridium_botulinum#Pathology Clostridium Botulinum]''==
''C. Botulinum'' is a Gram Positive bacteria belonging to the group of Firmicutes. They are obligate anaerobes capable of producing endospores. ''C. Botulinum'' is capable of producing a neurotoxin that causes muscle paralysis. The toxin can enter the body in many different ways: colonization of the digestive tract in both adults (Adult intestinal toxemia) and infants (infant botulism), ingestion of toxin from foods (Foodborne Botulism), and entry of a wound (Wound botulism).

Llama Antibody Inhibits Botulinum Neruotoxin

2010-11-16T22:31:34Z

Bryant Chen:

'''PubMed Abstract:'''
{{STRUCTURE_3k3q| PDB=3k3q | SCENE= }}
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest... [ Read More & Search PubMed Abstracts ]
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

==''[http://en.wikipedia.org/wiki/Clostridium_botulinum#Pathology Clostridium]''==
''C. Botulinum'' is a Gram Positive bacteria belonging to the group of Firmicutes. They are obligate anaerobes capable of producing endospores. ''C. Botulinum'' is capable of producing a neurotoxin that causes muscle paralysis. The toxin can enter the body in many different ways: colonization of the digestive tract in both adults (Adult intestinal toxemia) and infants (infant botulism), ingestion of toxin from foods (Foodborne Botulism), and entry of a wound (Wound botulism).