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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 Lab. (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. https://doi.org/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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Works referenced in this record:

Botulinum neurotoxins: genetic, structural and mechanistic insights
journal, June 2014

  • Rossetto, Ornella; Pirazzini, Marco; Montecucco, Cesare
  • Nature Reviews Microbiology, Vol. 12, Issue 8
  • DOI: 10.1038/nrmicro3295

Tetanus and Botulinum Neurotoxins Are Zinc Proteases Specific for Components of the Neuroexocytosis Apparatus
journal, March 1994

  • Schiavo, Giampietro; Rossetto, Ornella; Benfenati, Fabio
  • Annals of the New York Academy of Sciences, Vol. 710, Issue 1 Toxins and Ex
  • DOI: 10.1111/j.1749-6632.1994.tb26614.x

Clostridial neurotoxins as tools to investigate the molecular events of neurotransmitter release
journal, August 1994

  • Schiavo, Giampietro; Rossetto, Ornella; Montecucco, Cesare
  • Seminars in Cell Biology, Vol. 5, Issue 4
  • DOI: 10.1006/scel.1994.1028

Amino acid composition of Clostridium botulinum type F neurotoxin
journal, January 1983


Purification and amino acid composition of type E botulinum neurotoxin
journal, January 1983


Separation, purification, partial characterization and comparison of the heavy and light chains of botulinum neurotoxin types A, B, and E.
journal, September 1985


Partial amino acid sequences of the heavy and light chains of botulinum neurotoxin type E
journal, March 1985

  • Sathyamoorthy, Venugopal; Dasgupta, Bibhuti R.
  • Biochemical and Biophysical Research Communications, Vol. 127, Issue 3
  • DOI: 10.1016/S0006-291X(85)80009-7

Mechanism of action of tetanus and botulinum neurotoxins
journal, July 1994


Structure and Function of Clostridium botulinum Toxins
journal, March 1995


Proteolysis of SNAP-25 by types E and A botulinal neurotoxins.
journal, January 1994


Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin
journal, October 1992

  • Schiavo, Giampietro G.; Benfenati, Fabio; Poulain, Bernard
  • Nature, Vol. 359, Issue 6398
  • DOI: 10.1038/359832a0

Botulinum neurotoxin serotype F is a zinc endopeptidase specific for VAMP/synaptobrevin
journal, June 1993


Identification of the nerve terminal targets of botulinum neurotoxin serotypes A, D, and E.
journal, November 1993


Botulinum G neurotoxin cleaves VAMP/synaptobrevin at a single Ala-Ala peptide bond.
journal, August 1994


Botulinum neurotoxin C1 blocks neurotransmitter release by means of cleaving HPC-1/syntaxin.
journal, December 1993


Strategies to Design Inhibitors of Clostridium Botulinum Neurotoxins
journal, March 2007


Botulism
journal, October 2005

  • Sobel, J.
  • Clinical Infectious Diseases, Vol. 41, Issue 8
  • DOI: 10.1086/444507

Iterative Structure-Based Peptide-Like Inhibitor Design against the Botulinum Neurotoxin Serotype A
journal, June 2010


A Potent Peptidomimetic Inhibitor of Botulinum Neurotoxin Serotype A Has a Very Different Conformation than SNAP-25 Substrate
journal, October 2008


Substrate Binding Mode and Its Implication on Drug Design for Botulinum Neurotoxin A
journal, September 2008


Identification of a Potent Botulinum Neurotoxin A Protease Inhibitor Using in Situ Lead Identification Chemistry
journal, April 2006

  • Boldt, Grant E.; Kennedy, Jack P.; Janda, Kim D.
  • Organic Letters, Vol. 8, Issue 8
  • DOI: 10.1021/ol0603211

Inhibition of Metalloprotease Botulinum Serotype A from a Pseudo-peptide Binding Mode to a Small Molecule That Is Active in Primary Neurons
journal, February 2007

  • Burnett, James C.; Ruthel, Gordon; Stegmann, Christian M.
  • Journal of Biological Chemistry, Vol. 282, Issue 7
  • DOI: 10.1074/jbc.M608166200

Structures of Clostridium botulinum Neurotoxin Serotype A Light Chain Complexed with Small-Molecule Inhibitors Highlight Active-Site Flexibility
journal, May 2007


Catalytic Features of the Botulinum Neurotoxin A Light Chain Revealed by High Resolution Structure of an Inhibitory Peptide Complex
journal, May 2008

  • Silvaggi, Nicholas R.; Wilson, David; Tzipori, Saul
  • Biochemistry, Vol. 47, Issue 21
  • DOI: 10.1021/bi8001067

Serotype-selective, small-molecule inhibitors of the zinc endopeptidase of botulinum neurotoxin serotype A
journal, January 2006

  • Park, Jewn Giew; Sill, Peter C.; Makiyi, Edward F.
  • Bioorganic & Medicinal Chemistry, Vol. 14, Issue 2
  • DOI: 10.1016/j.bmc.2005.08.018

Structure- and Substrate-based Inhibitor Design for Clostridium botulinum Neurotoxin Serotype A
journal, July 2008

  • Kumaran, Desigan; Rawat, Richa; Ludivico, Matthew L.
  • Journal of Biological Chemistry, Vol. 283, Issue 27
  • DOI: 10.1074/jbc.M801240200

Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters
journal, April 2011

  • Ruge, Daniel R.; Dunning, F. Mark; Piazza, Timothy M.
  • Analytical Biochemistry, Vol. 411, Issue 2
  • DOI: 10.1016/j.ab.2011.01.002

[20] Processing of X-ray diffraction data collected in oscillation mode
book, January 1997


Overview of the CCP 4 suite and current developments
journal, March 2011

  • Winn, Martyn D.; Ballard, Charles C.; Cowtan, Kevin D.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4
  • DOI: 10.1107/S0907444910045749

REFMAC 5 for the refinement of macromolecular crystal structures
journal, March 2011

  • Murshudov, Garib N.; Skubák, Pavol; Lebedev, Andrey A.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4
  • DOI: 10.1107/S0907444911001314

Features and development of Coot
journal, March 2010

  • Emsley, P.; Lohkamp, B.; Scott, W. G.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
  • DOI: 10.1107/S0907444910007493

PROCHECK: a program to check the stereochemical quality of protein structures
journal, April 1993

  • Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.
  • Journal of Applied Crystallography, Vol. 26, Issue 2
  • DOI: 10.1107/S0021889892009944

Identification of Residues Surrounding the Active Site of Type A Botulinum Neurotoxin Important for Substrate Recognition and Catalytic Activity
journal, January 2008

  • Ahmed, S. Ashraf; Olson, Mark A.; Ludivico, Matthew L.
  • The Protein Journal, Vol. 27, Issue 3
  • DOI: 10.1007/s10930-007-9118-8

Arg 362 and Tyr 365 of the Botulinum Neurotoxin Type A Light Chain Are Involved in Transition State Stabilization
journal, February 2002

  • Binz, Thomas; Bade, Steffen; Rummel, Andreas
  • Biochemistry, Vol. 41, Issue 6
  • DOI: 10.1021/bi0157969

Peptide inhibitors of botulinum neurotoxin serotype A: design, inhibition, cocrystal structures, structure–activity relationship and pharmacophore modeling
journal, April 2012

  • Kumar, Gyanendra; Kumaran, Desigan; Ahmed, S. Ashraf
  • Acta Crystallographica Section D Biological Crystallography, Vol. 68, Issue 5
  • DOI: 10.1107/S0907444912003551

Substrate recognition strategy for botulinum neurotoxin serotype A
journal, December 2004


A high-affinity competitive inhibitor of type A botulinum neurotoxin protease activity
journal, December 2002


Presenting your structures: the CCP 4 mg molecular-graphics software
journal, March 2011

  • McNicholas, S.; Potterton, E.; Wilson, K. S.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4
  • DOI: 10.1107/S0907444911007281

Synthesis of all-hydrocarbon stapled α-helical peptides by ring-closing olefin metathesis
journal, May 2011

  • Kim, Young-Woo; Grossmann, Tom N.; Verdine, Gregory L.
  • Nature Protocols, Vol. 6, Issue 6
  • DOI: 10.1038/nprot.2011.324

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