The identification and biochemical characterization of drug-like compounds that inhibit botulinum neurotoxin serotype A endopeptidase activity

A robust, high-throughput, two-tiered assay for screening small molecule inhibitors against botulinum neurotoxin serotype A was developed and employed to screen 16,544 compounds. Thirty-four compounds were identified as potent hits employing the first-tier assay. Subsequently, nine were confirmed as actives by our second-tier confirmatory assay. Of these, one displayed potent inhibitory efficacy, possessing an IC50 = 16 μM (±1.6 μM) in our in vitro assay. This inhibitor (0831-1035) is highly water-soluble, and possesses an IC50 = 47 μM (±7.0 μM) in our primary cell culture assay (with virtually no cytotoxicity up to 500 μM), suggesting that this inhibitor is a good candidate for further development as a therapeutic countermeasure to treat botulism resulting from botulinum neurotoxin serotype A intoxication. An enzyme kinetics study indicated that this inhibitor exhibits mixed non-competitive inhibition, with a KI = 9 μM.