Evaluation of Short Peptide Inhibitors to Counteract Botulinum Neurotoxin A (bont/a) Poisoning in Vitro

Botulinum neurotoxins (BoNTs) inhibit stimulated neuroexocytosis, resulting in flaccid paralysis. Among the seven serotypes (A–G), serotype A (BoNT/A) is the most toxic. BoNT/A consists of a 100 kD heavy chain and a 50 kD light chain (LC). The LC, a zinc endopeptidase, hydrolyzes the vesicle fusion protein SNAP‐25 and thus blocking neuroexocytosis. A prospective countermeasure 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 compared this peptide inhibitor and two other derivatives, which include an aldehyde or a fluoromethyl ketone (an irreversible inhibitor) side group, with respect to their inhibitory potencies against SNAP‐25 hydrolysis and blockade of neuroexocytosis induced by BoNT/A in a human neuronal model, M‐17. Using SNAP‐25 hydrolysis and high K+ (80 mM) stimulated (3H)‐glycine release assays, we observed that all three inhibitors significantly inhibited BoNT/A effects. We also observed using an Alexa555 fluorescent tag that the peptide inhibitors were permeable through neuronal cell membranes. These peptide inhibitors show promise as therapeutic intervention(s) against BoNT/A poisoning. Research supported by JSTO‐DTRA Award # T.T.0009_06_RC_B to R. Ray. The views represented are those of the authors and are not necessarily endorsed by the US Army.