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

ABSTRACT: 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 IC(50)=16 microM (+/-1.6 microM) in our in vitro assay. This inhibitor (0831-1035) is highly water-soluble, and possesses an IC(50)=47 microM (+/-7.0 microM) in our primary cell culture assay (with virtually no cytotoxicity up to 500 microM), 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 K(I)=9 microM.

SUBMITTER: Cai S

PROVIDER: S-EPMC2876331 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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

Cai Shuowei S Lindo Paul P Park Jong-Beak JB Vasa Kruti K Singh Bal Ram BR

Toxicon : official journal of the International Society on Toxinology 20091207 4

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 IC(50)=16 microM (+/-1.6 microM) in our in vitro assay. This inhibitor (0831-1035) is ...[more]

PMID: 20003913

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Project description:Botulinum neurotoxin serotype A (BoNTA) causes a life-threatening neuroparalytic disease known as botulism. Current treatment for post exposure of BoNTA uses antibodies that are effective in neutralizing the extracellular toxin to prevent further intoxication but generally cannot rescue already intoxicated neurons. Effective small-molecule inhibitors of BoNTA endopeptidase (BoNTAe) are desirable because such inhibitors potentially can neutralize the intracellular BoNTA and offer complementary treatment for botulism. Previously we reported a serotype-selective, small-molecule BoNTAe inhibitor with a K(i) (app) value of 3.8+/-0.8 microM. This inhibitor was developed by lead identification using virtual screening followed by computer-aided optimization of a lead with an IC(50) value of 100 microM. However, it was difficult to further improve the lead from micromolar to even high nanomolar potency due to the unusually large enzyme-substrate interface of BoNTAe. The enzyme-substrate interface area of 4,840 A(2) for BoNTAe is about four times larger than the typical protein-protein interface area of 750-1,500 A(2). Inhibitors must carry several functional groups to block the unusually large interface of BoNTAe, and syntheses of such inhibitors are therefore time-consuming and expensive. Herein we report the development of a serotype-selective, small-molecule, and competitive inhibitor of BoNTAe with a K(i) value of 760+/-170 nM using synthesis-based computer-aided molecular design (SBCAMD). This new approach accounts the practicality and efficiency of inhibitor synthesis in addition to binding affinity and selectivity. We also report a three-dimensional model of BoNTAe in complex with the new inhibitor and the dynamics of the complex predicted by multiple molecular dynamics simulations, and discuss further structural optimization to achieve better in vivo efficacy in neutralizing BoNTA than those of our early micromolar leads. This work provides new insight into structural modification of known small-molecule BoNTAe inhibitors. It also demonstrates that SBCAMD is capable of improving potency of an inhibitor lead by nearly one order of magnitude, even for BoNTAe as one of the most challenging protein targets. The results are insightful for developing effective small-molecule inhibitors of protein targets with large active sites.

Dataset Information

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

Publications

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

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