Identification and Biochemical Characterization of Small-Molecule Inhibitors of Clostridium Botulinum Neurotoxin Serotype A

摘要

Botulinum neurotoxins (BoNTs) comprise a family of seven immunologically distinct proteins synthesized primarily by strains of Clostridium botulinum. BoNTs are the most lethal biological substances known, and are listed as category A agents by the Centers for Disease Control and Prevention. BoNTs are the etiological agents of botulism, an intoxication characterized by peripheral neuromascular blockade and flaccid paralysis. Currently, no effective therapeutics exists to counter botulism. Small-molecule inhibitors of BoNTs can serve as both prophylactics and post-exposure therapeutics. Methodology/Principal Findings: In this study, we report on five novel small-molecule BoNT/A inhibitors that have been discovered by using structure-based virtual screening (VS) in conjunction with tiered screening assays (protease activity, cell culture, and ex vivo mouse phrenic nerve hemidiaphragm assays). The National Cancer Institute (NCI) database that contains >250,000 small molecules was searched by VS against the crystal structure of BoNT/A light chain (LC; PDB code: 1E1H). Compounds identified as 'hits' and their analogs were subjected to HPLC-based assays against LC and BoNT/A (Hall). The top five candidates that effectively inhibited both the LC and the toxin were selected, and further advanced to cellular and tissue-based assays intended to mimic BoNT exposure. These five compounds (at 15 mM) protected N2a cells from BoNT/A-mediated cleavage of SNAP-25, and at low micromolar concentrations, delayed the toxin-induced paralytic time in mouse phrenic nerve hemidiaphragms by at least threefold. Conclusions/Significance: The inhibition of the protease activity of both LC and holotoxin, as well as the low micromolar protection of N2a cells and hemidiaphragms by these five leads are unique among the many recently reported small-molecule inhibitors of BoNT/A.