A Novel Irreversible Peptide Inhibitor to Counteract Botulinum Neurotoxin A (BoNT/A) Poisoning In Vitro

Abstract

BoNT/A light chain (LC) hydrolyzes the vesicle fusion protein SNAP‐25, thus blocking neuroexocytosis and causing flaccid paralysis. A potential therapeutic approach against BoNT/A poisoning is to inhibit the LC‐induced hydrolysis of SNAP‐25 using a short peptide inhibitor. A high affinity synthetic peptide inhibitor, N‐acetyl‐CRATKML‐amide, has been reported. We studied a derivative of N‐Acetyl‐CRATKML, peptide inhibitor #3 (PI3), for its antagonism of SNAP‐25 hydrolysis and neuroexocytosis inhibition due to BoNT/A in the human neuronal M17 cell line. Using SNAP‐25 hydrolysis and high KCl (80 mM) stimulated [3H]‐glycine release assays, we observed that PI3 significantly inhibited BoNT/A effects. We also observed, by Alexa 488 labeling, that PI3 permeates through M17 cell membranes. Additionally, using dialysis and SNAPtide assays, we found that PI3 is an irreversible BoNT/A inhibitor. To our knowledge, this is the first irreversible BoNT/A inhibitor reported. Disclaimer: The opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army or the Department of Defense. This research was supported by the DTRA‐JSTO, Med. S&T Div.