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.

中文翻译：

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BoNT / A蛋白酶的不可逆抑制：独特的战斗部反应性和功能取决于双功能方法。

我们描述了一个弹头的全面筛选运动，链接到异羟肟酸酯螯合锚，用于BoNT / A蛋白酶内Cys165的修饰。结合透彻的酶动力学，我们详细介绍了与环氧化物战斗部（弱亲电试剂）的显着邻近驱动共价键；然而，与本质上更高的反应弹头相比，它具有不可逆的抑制作用和药理特性。这种有针对性的，选择性的共价键取决于关键的异羟肟酸酯-Zn2 +螯合相互作用，这通过检查非螯合化合物来说明。我们讨论了以前使用非目标选择性半胱氨酸反应性战斗部来修饰BoNT / A蛋白酶的方法，而BoNT / A蛋白酶则没有任何治疗潜力-我们的双功能策略允许使用本质上反应性较小的战斗部来拦截半胱氨酸，从而减少脱落抑制剂的靶向修饰。此外，我们还提出，这种双功能方法不是一种一次性策略，我们认为可以将其广泛转化为在酶催化领域内具有非催化性但亲核性残基的其他金属蛋白酶。