Substrate-based inhibitors exhibiting excellent protective and therapeutic effects against Botulinum Neurotoxin A intoxication

Abstract

Potent inhibitors to reverse Botulinum neurotoxins (BoNTs) activity in neuronal cells are currently not available. A better understanding of the substrate recognition mechanism of BoNTs enabled us to design a novel class of peptide inhibitors which were derivatives of the BoNT/A substrate, SNAP25. Through a combination of in vitro, cellular based, and in vivo mouse assays, several potent inhibitors of approximately one nanomolar inhibitory strength both in vitro and in vivo have been identified. These compounds represent the first set of inhibitors that exhibited full protection against BoNT/A intoxication in mice model with undetectable toxicity. Our findings validated the hypothesis that a peptide inhibitor targeting the two BoNT structural regions which were responsible for substrate recognition and cleavage respectively could exhibit excellent inhibitory effect, thereby providing insight on future development of more potent inhibitors against BoNTs.

A novel role of C-terminus in introducing a functionally flexible structure critical for the biological activity of botulinum neurotoxin

“…The optimum activity of both LCAs was observed at 37 °C, yet tLCA has consistently been observed to have less activity than fLCA 8 – 10 , 32 . The more compact secondary and tertiary structure of tLCA at both 25 °C and 37 °C could provide steric hindrances and occlude the active site from optimum interaction with a peptide substrate, or possibly causing a buildup of cleaved product in the active site 9 , whereas a more loosely packed fLCA more readily facilitates substrate interaction and dissociation at the active site. The active site of LCA-424 has been previously shown to be a flexible region 19 , however, this flexibility in the active site region could be further increased through the fluctuations of the unstructured C-terminus inducing a global loosening of the overall structure.…”

Structural Biology and Molecular Modeling to Analyze the Entry of Bacterial Toxins and Virulence Factors into Host Cells

“…The AntiBotABE program has started the development of an oligoclonal antibody cocktail [43,44,45]. Virtual screening of BoNTs target Lc producing numerous compounds preventing the cleavage of SNARE proteins by binding to the active site of the zinc metalloprotease [46,47,48,49,50,51,52,53]. But, by contrast and thanks to their characteristic paralysis effects, BoNTs are also used to treat an increasing number of medical disorders [42] including human neuromuscular disorders characterized by involuntary muscle contractions (strabismus, blepharospasm, and hemifacial spasm), as well as cosmetic applications.…”