Basic Tetrapeptides as Potent Intracellular Inhibitors of Type A Botulinum Neurotoxin Protease Activity

Abstract

Botulinum neurotoxins (BoNT) are the most potent of all toxins that cause flaccid muscle paralysis leading to death. They are also potential biothreat agents. A systematic investigation of various short peptide inhibitors of the BoNT protease domain with a 17-residue peptide substrate led to argininearginine-glycine-cysteine having a basic tetrapeptide structure as the most potent inhibitor. When assayed in the presence of dithiothreitol (DTT), the inhibitory effect was drastically reduced. Replacing the terminal cysteine with one hydrophobic residue eliminated the DTT effect but with two hydrophobic residues made the pentapeptide a poor inhibitor. Replacing the first arginine with cysteine or adding an additional cysteine at the N terminus did not improve inhibition. When assessed using mouse brain lysates, the tetrapeptides also inhibited BoNT/A cleavage of the endogenous SNAP-25. The peptides penetrated the neuronal cell lines, N2A and BE(2)-M17, without adversely affecting metabolic functions as measured by ATP production and P-38 phosphorylation. Biological activity of the peptides persisted within cultured chick motor neurons and rat and mouse cerebellar neurons for more than 40 h and inhibited BoNT/A protease action inside the neurons in a dose-and time-dependent fashion. Our results define a tetrapeptide as the smallest peptide inhibitor in the backdrop of a large substrate protein of 200؉ amino acids having multiple interaction regions with its cognate enzyme. The inhibitors should also be valuable candidates for drug development. Botulinum neurotoxins (BoNT)2 produced by strains of Clostridium botulinum, are the deadliest of all toxins (1). There are seven distinct serotypes, A-F, of which BoNT/A afflicts humans most frequently. The latter is also the most stable in neurons causing long term paralysis. The toxins are 150-kDa proteins comprising a 50-kDa protease light chain (LC) and a 100-kDa binding-translocating heavy chain. Upon entry to animal body, the toxins travel to peripheral neurons where heavy chain binds and translocates LC into the neurons. After dissociating from the heavy chain (2), LC cleaves at specific sites on one of three SNARE (soluble N-ethylmaleimide factor attachment protein receptor) proteins, thereby blocking exocytosis of acetylcholine into the neuromuscular junction and causing muscle paralysis. The paralysis prevents normal respiration and eventually leads to death of the animal. One of the hallmark features of BoNT/A intoxication is the extreme persistence of symptoms with neuroparalysis often lasting in excess of 3-4 months. Currently, there is no effective postexposure therapeutic available against botulism. Because of extreme toxicity, BoNT is a potential biowarfare and bioterrorism threat, and both Centers for Disease Control and Prevention and the United States Department of Agriculture have placed it as a category A threat agent.The substrate for BoNT/A is an ϳ210-residue SNAP-25 depending on species. Schmidt and Bostian (3) reported that unlike most other proteases,...