Quinoline-based Inhibitors of BoNT/A LC

描述(申请人提供)：肉毒杆菌神经毒素(BoNTs)是已知的最有毒的生物物质。人静脉注射A型BONT的致死剂量为1-5 ng/kg。如果发生意外接触BONT(例如，由于受污染的食品)，可能会发生生命损失或危及生命的瘫痪。最重要的是，核爆弹药已经以剧毒的气雾剂形式“武器化”，因此对平民和军人都构成重大威胁。一旦被摄取，BoNTs就会靶向外周胆碱能神经末梢，通过干扰自主神经功能而导致死亡。BNTs的锌依赖内肽酶轻链(LC)部分通过蛋白分解神经传递的基本成分SNARE(可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体)来损害神经元的胞吐功能。该项目的总体目标是开发BoNT/A轻链(LC)金属蛋白酶活性的小分子抑制剂来治疗肉毒杆菌中毒。我们的策略是优化一种新的化学类型的BONT/A LC抑制剂，以提高效力、选择性和类药物性能。在初步研究中，我们改进了结构，为药物化学提供了一个新的起点，其化学核心具有比原始化合物系列更多的类似药物的性质，并具有十倍于原来的化合物系列的效力。在BONT/A中毒的雏鸡神经细胞模型中也观察到了显著的活动。这些经过验证的早期先导化合物比最初的系列更适合用于BONT/A抑制剂药物开发，它们构成了这一优化计划的基础，以产生先进的先导化合物。我们在第一阶段的方法是使用经过验证的药物和平行合成化学技术来优化先导系列化合物的效力和选择性，并生产具有推进临床前开发潜力的“类药物”分子。在一个迭代的过程中，我们将通过测量酶和细胞活性以及酶抑制剂的特异性来探索这些重点化合物文库的结构特征，这些结构特征有助于更紧密地结合和更有效地抑制BONT/A金属蛋白酶。我们将把化合物对BONT/A LC的体外活性与BONT/A作用的细胞模型中的活性相关联，并使用该系列中最有希望的成员在体内BoNT/A中毒的小鼠模型中验证这些抑制剂。在第一阶段结束时，我们将定义一种先进的先导化合物，它将从体内验证的抑制剂组中挑选出来，显示出适合进一步临床前开发的体外药代动力学特性。在第二阶段，我们将进一步优化和评估两个物种的体内疗效、药代动力学特性、毒性和安全药理学，并将其开发为适合人类临床试验的IND前临床候选药物(第三阶段)。 与公共卫生相关：肉毒杆菌神经毒素是已知的一些最有毒的生物物质。接触受污染食物中的这些神经毒素或生物恐怖主义行为后，可能会发生生命损失或危及生命的瘫痪。本提案描述了治疗肉毒杆菌中毒的新药的制备和开发。

DESCRIPTION (provided by applicant): The botulinum neurotoxins (BoNTs) are the most poisonous biological substances known. The lethal intravenous dose of BoNT serotype A (BoNT/A) in humans is 1-5 ng/kg. If accidental exposure to BoNT occurs (e.g., from contaminated foodstuffs), loss of life or life-threatening paralysis can occur. Most importantly, the BoNTs have already been "weaponized" in a highly toxic aerosol form, and they consequently pose a significant threat to both civilian and military populations. Once ingested, BoNTs target the peripheral cholinergic nerve endings and cause death by interrupting autonomic nerve function. The zinc-dependent endopeptidase light chain (LC) portion of BoNTs impairs neuronal exocytosis through proteolysis of essential SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) components of neurotransmission. The overall goal or this project is to develop small molecule inhibitors of the BoNT/A light chain (LC) metalloprotease activity to treat botulinum poisoning. Our strategy is to optimize a novel chemotype of BoNT/A LC inhibitors to improve potency, selectivity, and drug-like properties. In preliminary studies, we refined the structures to provide a new starting point for medicinal chemistry based on a chemical core with more drug-like properties than the original compound series and with ten-fold better potency. Significant activity was also observed in a chick neuronal cell model of BoNT/A intoxication. These validated early lead compounds are more suitable lead compounds for BoNT/A inhibitor drug discovery than were the original series, and they form the basis for this optimization program to generate an advanced lead compound. Our approach in Phase I is to use proven techniques of medicinal and parallel synthetic chemistry to optimize the potency and selectivity of the lead series of compounds and produce "drug-like" molecules with the potential to advance to pre-clinical development. In an iterative process, we will probe these focused compound libraries for structural features that contribute to tighter binding and more potent inhibition of the BoNT/A metalloprotease by measuring the enzymatic and cellular activities, as well as the specificity of the enzyme inhibitors. We will correlate the in vitro activity of the compounds against BoNT/A LC with activity in a cellular model of BoNT/A action, and use the most promising members of the series to validate the inhibitors in an in vivo mouse model of BoNT/A intoxication. At the end of Phase I we will define an advanced lead compound, which will be selected from the group of in vivo-validated inhibitors that displays suitable in vitro pharmacokinetic properties for further preclinical development. In Phase II, we will further optimize and evaluate the lead for in vivo efficacy, pharmacokinetic properties, toxicity and safety pharmacology in two species, and develop it into a pre-IND clinical candidate, suitable for human clinical trials (Phase III). PUBLIC HEALTH RELEVANCE: The botulinum neurotoxins are some of the most poisonous biologic substances known. Loss of life or life-threatening paralysis can occur following exposure to these neurotoxins from contaminated foodstuffs or acts of bioterrorism. This proposal describes the preparation and development of novel drugs to treat botulinum poisoning.