Biological and Medicinal Chemistry

Irreversible Inhibition of BoNT/A Protease: Unique Warhead Reactivity and Function Contingent upon a Bifunctional Approach

Abstract

We describe a comprehensive screening campaign of warheads, linked to a hydroxamate chelating anchor, for the modification of Cys165 within the BoNT/A protease.
Engaging thorough enzyme kinetics, we detail a remarkable proximity-driven covalent bond with an epoxide warhead, a weak electrophile; yet, one that possessed superior irreversible inhibition, and pharmacological properties, when compared to intrinsically higher reactive warheads. This directed, selective covalent bond was contingent upon the crucial hydroxamate-Zn2+ chelating interaction as exemplified by examining non-chelating compounds.
We discuss previous approaches using non-target selective cysteine-reactive warheads to modify the BoNT/A protease of which none present any therapeutic potential – our bifunctional strategy allows the use of intrinsically less reactive warheads to intercept the cysteine, which will allow for less off-target modifications of such inhibitors. Moreover, we also broach that this bifunctional approach is not a one-off strategy that we believe can be broadly translated to other metalloproteases that possess non-catalytic, yet, nucleophilic residues within the enzymes catalytic sphere.

Content

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Supplementary material

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BoNT A Cov ToC
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ESI I
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ESI II

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