Research Article: Potent New Small-Molecule Inhibitor of Botulinum Neurotoxin Serotype A Endopeptidase Developed by Synthesis-Based Computer-Aided Molecular Design

Date Published: November 10, 2009

Publisher: Public Library of Science

Author(s): Yuan-Ping Pang, Anuradha Vummenthala, Rajesh K. Mishra, Jewn Giew Park, Shaohua Wang, Jon Davis, Charles B. Millard, James J. Schmidt, Narcis Fernandez-Fuentes.

Abstract: 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 Kiapp value of 3.8±0.8 µM. This inhibitor was developed by lead identification using virtual screening followed by computer-aided optimization of a lead with an IC50 value of 100 µM. 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 Å2 for BoNTAe is about four times larger than the typical protein-protein interface area of 750–1,500 Å2. 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 Ki 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.

Partial Text: Botulism is a life-threatening neuroparalytic disease that occurs in at least five forms–food-borne botulism, infant botulism, wound botulism, adult enteric infectious botulism, and inhalation botulism. The disease is caused by the specific action of botulinum neurotoxins. Seven antigenically distinct botulinum neurotoxins (serotypes A to G) are known, but primarily serotypes A, B, E, and F have been reported to cause botulism in humans.



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