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Title: Interactions of a potent cyclic peptide inhibitor with the light chain of botulinum neurotoxin A: insights from x-ray crystallography

Abstract

The seven antigenically distinct serotypes (A to G) of botulinum neurotoxin (BoNT) are responsible for the deadly disease botulism. BoNT serotype A (BoNT/A) exerts its lethal action by cleaving the SNARE protein SNAP-25, leading to inhibition of neurotransmitter release, flaccid paralysis and autonomic dysfunction. BoNTs are dichain proteins: the heavy chain is responsible for neurospecific binding, internalization and translocation, and the light chain is responsible for substrate cleavage. Because of their extreme toxicity and prior history of weaponization, the BoNTs are considered to be potential bioterrorism agents. No post-symptomatic therapeutic interventions are available for BoNT intoxication other than critical care; therefore it is imperative to develop specific antidotes against this neurotoxin. To this end, a cyclic peptide inhibitor (CPI-1) was synthesized and found to inhibit BoNT/A light chain (Balc) with high affinity. When tested in a cell-free Förster resonance excitation transfer (FRET) assay, CPI-1 was found to have a Ki of 13.9 nM using full-length Balc448 and 42.1 nM using a truncated crystallizable form of light chain (Balc424). Co-crystallization of CPI-1 with Balc424 revealed that in the Balc-CPI-1 complex, the inhibitor adopts a helical conformation, occupies a high percentage of the active site cavity and interacts in an amphipathic mannermore » with critical active site residues. The data suggest that CPI-1 prevents SNAP-25 from accessing the Balc active site by blocking both the substrate binding path at the surface and the Zn2+ binding region involved in catalysis. This is in contrast to linear peptide inhibitors described to date which block only the latter« less

Authors:
 [1];  [2];  [2];  [2];  [3];  [1]
  1. Brookhaven National Lab. (BNL), Upton, NY (United States)
  2. U.S. Army Medical Research Inst. of Chemical Defense (USAMRICD), Aberdeen Proving Ground, MD (United States). Analytical Toxicology Division
  3. U.S. Army Medical Research Inst. of Chemical Defense (USAMRICD), Aberdeen Proving Ground, MD (United States). Research Division
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL), Upton, NY (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1232693
Alternate Identifier(s):
OSTI ID: 1250934
Report Number(s):
BNL-108605-2015-JA
Journal ID: ISSN 0968-0896
Grant/Contract Number:  
SC00112704; DEAC02-98CH10886; CB4080
Resource Type:
Accepted Manuscript
Journal Name:
Bioorganic and Medicinal Chemistry
Additional Journal Information:
Journal Volume: 23; Journal Issue: 22; Journal ID: ISSN 0968-0896
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Botulinum toxin; Botulinum neurotoxin; Cyclic peptide; Inhibition mechanism

Citation Formats

Kumaran, D., Adler, M., Levit, M., Krebs, M., Sweeney, R., and Swaminathan, S. Interactions of a potent cyclic peptide inhibitor with the light chain of botulinum neurotoxin A: insights from x-ray crystallography. United States: N. p., 2015. Web. doi:10.1016/j.bmc.2015.10.024.
Kumaran, D., Adler, M., Levit, M., Krebs, M., Sweeney, R., & Swaminathan, S. Interactions of a potent cyclic peptide inhibitor with the light chain of botulinum neurotoxin A: insights from x-ray crystallography. United States. https://doi.org/10.1016/j.bmc.2015.10.024
Kumaran, D., Adler, M., Levit, M., Krebs, M., Sweeney, R., and Swaminathan, S. Sat . "Interactions of a potent cyclic peptide inhibitor with the light chain of botulinum neurotoxin A: insights from x-ray crystallography". United States. https://doi.org/10.1016/j.bmc.2015.10.024. https://www.osti.gov/servlets/purl/1232693.
@article{osti_1232693,
title = {Interactions of a potent cyclic peptide inhibitor with the light chain of botulinum neurotoxin A: insights from x-ray crystallography},
author = {Kumaran, D. and Adler, M. and Levit, M. and Krebs, M. and Sweeney, R. and Swaminathan, S.},
abstractNote = {The seven antigenically distinct serotypes (A to G) of botulinum neurotoxin (BoNT) are responsible for the deadly disease botulism. BoNT serotype A (BoNT/A) exerts its lethal action by cleaving the SNARE protein SNAP-25, leading to inhibition of neurotransmitter release, flaccid paralysis and autonomic dysfunction. BoNTs are dichain proteins: the heavy chain is responsible for neurospecific binding, internalization and translocation, and the light chain is responsible for substrate cleavage. Because of their extreme toxicity and prior history of weaponization, the BoNTs are considered to be potential bioterrorism agents. No post-symptomatic therapeutic interventions are available for BoNT intoxication other than critical care; therefore it is imperative to develop specific antidotes against this neurotoxin. To this end, a cyclic peptide inhibitor (CPI-1) was synthesized and found to inhibit BoNT/A light chain (Balc) with high affinity. When tested in a cell-free Förster resonance excitation transfer (FRET) assay, CPI-1 was found to have a Ki of 13.9 nM using full-length Balc448 and 42.1 nM using a truncated crystallizable form of light chain (Balc424). Co-crystallization of CPI-1 with Balc424 revealed that in the Balc-CPI-1 complex, the inhibitor adopts a helical conformation, occupies a high percentage of the active site cavity and interacts in an amphipathic manner with critical active site residues. The data suggest that CPI-1 prevents SNAP-25 from accessing the Balc active site by blocking both the substrate binding path at the surface and the Zn2+ binding region involved in catalysis. This is in contrast to linear peptide inhibitors described to date which block only the latter},
doi = {10.1016/j.bmc.2015.10.024},
journal = {Bioorganic and Medicinal Chemistry},
number = 22,
volume = 23,
place = {United States},
year = {2015},
month = {10}
}

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