Basic tetrapeptides as potent intracellular inhibitors of type A botulinum neurotoxin protease activity.
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 arginine-arginine-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.
Department Cell Biology and Biochemistry, Integrated Toxicology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702, USA.
Martha
M
Hale
George
G
Oyler
Subramanyam
S
Swaminathan
S Ashraf
SA
Ahmed
D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D019274:Botulinum Toxins, Type A / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002531:Cerebellum / Q000378:metabolism; D002645:Chickens; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D020125:Mutation, Missense; D009474:Neurons / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology*; D051381:Rats; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism
D004791:Enzyme Inhibitors; D009842:Oligopeptides; C494209:Snap25 protein, mouse; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D000255:Adenosine Triphosphate; D019274:Botulinum Toxins, Type A