Covalent Inhibition as a Method to Counteract Botulinum Intoxication

项目摘要/摘要 A型肉毒神经毒素(BONT/A)是目前已知的效力最强的毒素，它会导致肉毒杆菌中毒。 对人类来说。BoNTs最常见的名称是BooxTM，它的使用越来越多，使其成为医源性的 肉毒杆菌中毒是一大令人担忧的问题。BoNTs是疾控中心指定为A类的仅有的六种病原体之一 生物恐惧剂因其毒性大、生产容易等优点而备受关注。此外，肉毒杆菌毒素在海洛因中的传播 用户是一个日益令人担忧的问题。 尽管存在潜在的威胁和疾病的严重性，但目前还没有治疗方法可以拯救 引起肉毒杆菌中毒的神经元中毒。最好的情况是，通过治疗可以减缓疾病的发展。 使用七价抗毒素，这仍然需要几个月的住院治疗。我们的长期目标是开发一种 临床上可行的治疗方法，能够逆转肉毒杆菌神经毒素的影响，除了阻止 进步。由于BONT中毒是一个孤立事件，我们假设不可逆的共价抑制剂能够 进入肌肉神经元可能永久性地损害其催化机制，为解决 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， 关于丝氨酸/半胱氨酸蛋白酶的不可逆转抑制剂的大量报道，不可逆转的抑制 金属蛋白酶是罕见的，这是催化机制不同的结果。因此，我们制定了一项战略 其中以变构反应残基为靶标的共价弹头连接到有效的活性部位抑制剂， 这样就产生了一种“双功能”抑制剂。这在本质上回避了酶的机制问题，现在允许共价 瞄准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)使用对接和结构活动关系(SAR)数据，我们将 使先前确定的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 可逆抑制物支架的存在，并解释由 可逆的抑制剂。3)我们将反复获得和分析晶体、细胞和药代动力学数据 改进我们的抑制剂，优先考虑效力、选择性和安全性，以最大限度地增加成功的机会 在活体研究中。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，我们 将评估我们开发的化合物的疗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.