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ANANBIOANAL - 2010
Botulinum Neurotoxin Protease Activity
S. Ashraf Ahmed
Department Cell Biology and Biochemistry, Integrated Toxicology Division, United States Army Medical Research Institute of Infectious
Diseases, Fort Detrick, MD, USA
B
otulinum neurotoxins (BoNT) are the most potent of all toxins. They 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
(RRGC) 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 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 cerebellar neurons for more than 40h, and inhibited BoNT/A
protease action inside the neurons in a dose- and time-dependent fashion. Our
results define a tetrapetide 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.
(This project received support from the DTRA-JSTO for Chemical and Biological
Defense grant #CBS.MEDBIO.01.10.RD.002. Opinions, interpretations, conclusions,
and recommendations are those of the author and are not necessarily endorsed by
the U.S. Army.)
Pharmaceutical R & D Summit
doi:10.4172/2155-9872.1000009
Basic Tetrapeptides as Potent Intracellular Inhibitors of Type A
ANALBIOANAL-2010
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