Covalent Modifiers of Botulinum Neurotoxin Counteract Toxin Persistence
Covalent Modifiers of Botulinum Neurotoxin Counteract Toxin Persistence
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DOI：
10.1021/acschembio.8b00937
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发表时间：
2019-01-01
影响因子：
4
通讯作者：
Bogyo, Matthew
中科院分区：
文献类型：
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作者：
Garland, Megan;Babin, Brett M.;Bogyo, Matthew
肉毒杆菌神经毒素（BoNTs）是人类已知的最有效的毒素，是生物恐怖主义武器的重大威胁。bont含有金属蛋白酶结构域，可阻断神经末梢的神经递质释放，导致下降性弛缓性瘫痪，死亡率为5-10%。现有的治疗方案无法在毒素内吞后获得或中和毒素，因此显然需要开发新的治疗方法。许多底物和锌螯合小分子抑制剂已被报道；然而，没有一个进展到临床。这可能是由于可逆抑制剂难以实现毒素的持续抑制，毒素在体内的半衰期为数月。减轻BoNT持久性的另一种策略是共价的、不可逆的毒素功能抑制。然而，共价BoNT抑制剂的例子很少被报道。在这里，我们描述了一种基于竞争的筛选方法，以鉴定BoNT/ a血清型中保守活性位点邻近半胱氨酸C165的共价修饰因子。我们发现含有半胱氨酸反应性亲电试剂的化合物不能结合C165，而硒化物化合物是半胱氨酸的有效共价结合物。重要的是，C165的共价修饰导致BoNT/A蛋白酶活性的持续、不可逆抑制。共价硒化物抑制剂在神经中毒试验中无毒且具有保护作用，使其成为研究BoNT/ a毒素以及设计新型治疗药物的有希望的新支架。
Botulinum neurotoxins (BoNTs) are the most potent toxins known to man and a significant threat as weapons of bioterrorism. BoNTs contain a metalloprotease domain that blocks neurotransmitter release in nerve terminals, resulting in a descending, flaccid paralysis with a 5-10% mortality rate. Existing treatment options cannot access or neutralize the toxin following its endocytosis, so there is a clear need to develop novel therapies. Numerous substrate-based and zinc-chelating small-molecule inhibitors have been reported; however, none have progressed to the clinic. This is likely due to the difficulty that reversible inhibitors have in achieving sustained inhibition of the toxin, which has a half-life of months in vivo. An alternative strategy for mitigating BoNT persistence is covalent, irreversible inhibition of toxin function. However, few examples of covalent BoNT inhibitors have been reported. Here, we describe a competition-based screen to identify covalent modifiers of the conserved active-site-adjacent cysteine C165 in the BoNT/A serotype. We found that compounds containing cysteine-reactive electrophiles designed to target cysteine proteases failed to bind C165 while selenide compounds were efficient covalent binders of this cysteine. Importantly, covalent modification at C165 resulted in sustained, irreversible inhibition of BoNT/A protease activity. Covalent selenide inhibitors were nontoxic and protective in a neuronal assay of intoxication, making them promising new scaffolds for the study of the BoNT/A toxin as well as for the design of novel therapy agents.