Covalent Inhibition as a Method to Counteract Botulinum Intoxication

项目概要/摘要 A型肉毒神经毒素（BoNT/A）是目前已知的毒性最强的毒素，可引起肉毒中毒 BoNTs最常见的是BotoxTM，其使用的增加使得医源性 肉毒杆菌中毒是一个主要问题。BoNT是CDC指定为A类的六种病原体之一 由于其毒性和易于生产，它是生物恐怖主义制剂。此外，肉毒杆菌中毒在海洛因中的传播 用户越来越关注。 尽管存在潜在的威胁和疾病的严重性，但没有治疗方法可用于挽救 导致肉毒杆菌中毒的神经元中毒最好的情况是，通过治疗缓解疾病的进展 注射了一种七价抗毒素但仍需要住院数月我们的长期目标是发展一个 除了抑制肉毒杆菌神经毒素的作用外， 中求进工作总由于BoNT中毒是一个孤立的事件，我们认为一种不可逆的共价抑制剂能够 进入肌肉神经元可能会永久地损害其催化机制， 神经元中小分子的寿命和神经毒素的持久性之间的差异。相比之下 对于丝氨酸/半胱氨酸蛋白酶的不可逆抑制剂的许多报道， 金属蛋白酶是罕见的，催化机制的差异的结果。因此我们设计了一个策略 其中靶向变构反应性残基的共价弹头与有效的活性位点抑制剂连接， 从而产生“双功能”抑制剂。这实质上回避了酶的机械问题，现在允许共价键 靶向BoNT/LC。 基于有希望的初步数据，我们提出了四个具体目标，将导致识别 有效的、反应性的和选择性的分子。1)使用对接和结构活性关系（SAR）数据，我们将 使先前鉴定的BoNT/A轻链的可逆抑制剂适应双功能共价策略。2)我们 将在存在可逆抑制剂的情况下筛选共价片段，以选择耐受 可逆抑制剂支架的存在，并解释活性位点构象变化诱导的 可逆抑制剂。3)我们将获得并分析晶体学、细胞学和药代动力学数据， 改进我们的抑制剂，优先考虑效力，选择性和安全性，以最大限度地提高成功的机会 在体内研究中。4)最后，通过在FDA金标准小鼠致死模型中测试我们的化合物， 将评估我们开发的化合物的功效及其对临床前和临床研究的适用性。

PROJECT SUMMARY/ABSTRACT Botulinum neurotoxin serotype A (BoNT/A), which causes the disease botulism, is the most potent toxin known to man. BoNTs are most commonly encountered as BotoxTM, the increasing use of which has made iatrogenic botulism a major concern. BoNTs are one of only six pathogens designated by the CDC as a category A bioterrorism agent due to its toxicity and ease of production. Furthermore, the spread of botulism among heroin users is a growing concern. Despite the potential threat and the severity of the disease, there is no therapeutic available for rescuing the neuronal intoxication that causes botulism. At best, the progression of the disease is mitigated by treatment with a heptavalent antitoxin, which still requires months of hospitalization. Our long-term goal is to develop a clinically viable therapeutic capable of reversing the effects of botulinum neurotoxin, in addition to arresting progress. As BoNT intoxication is a solitary event, we posit that an irreversible covalent inhibitor capable of entering muscle neurons could permanently compromise its catalytic machinery, providing a solution to the discrepancy between the lifetime of a small molecule in neurons and the persistence of the neurotoxin. In contrast to the numerous reports of irreversible inhibitors of serine/cysteine proteases, irreversible inhibition of metalloproteinases is rare, a result of differences in catalytic mechanisms. As such we have devised a strategy wherein a covalent warhead that targets an allosteric reactive residue is tethered to a potent active site inhibitor, thus creating a “bifunctional” inhibitor. This in essence skirts enzyme mechanistic issues and now allows covalent targeting of the BoNT/LC. Based on promising preliminary data, we propose four specific aims that will lead to the identification of potent, reactive, and selective molecules. 1) Using docking and structure activity relationship (SAR) data we will adapt previously identified reversible inhibitors of BoNT/A light chain to the bifunctional covalent strategy. 2) We will screen covalent fragments in the presence of reversible inhibitors to select for warheads that tolerate the presence of the reversible inhibitor scaffold, and to account for active site conformational changes induced by the reversible inhibitor. 3) We will obtain and analyze crystallographic, cell and pharmacokinetic data to iteratively improve our inhibitors, prioritizing potency, selectivity, and safety in order to maximize the chance for success during in vivo studies. 4) Finally, by testing our compounds in the FDA gold standard mouse lethality model, we will assess the efficacy of our developed compounds and their suitability for pre-clinical and clinical studies.