Quinoline-Based Inhibitors of Botulinum Neurotoxin A

描述（由申请人提供）：肉毒杆菌神经毒素（BoNTs）代表一组已知的毒性最强的生物物质（1- 3）。7种不同血清型BoNT （BoNT/A至BoNT/G）由厌氧孢子形成细菌肉毒梭菌、丁酸梭菌和巴拉氏梭菌分泌（4-7）。与由破伤风梭菌产生的破伤风神经毒素（TeNT）一起，bont构成了梭菌神经毒素（CNT）家族(8)。血清A型BoNT （BoNT/A）人静脉注射致死剂量为1-5ng/kg（9,10）。如果意外暴露于BoNT（例如，从受污染的食品中），可能会发生生命丧失或危及生命的瘫痪（11）。最重要的是，bont已经被“武器化”为剧毒气溶胶形式，因此对平民和军队人口构成重大威胁（9,12）。bont一旦被吸入肺部，就会被血液吸收，以周围胆碱能神经末梢为靶点，并通过中断自主神经功能而导致死亡。BoNTs的锌依赖性内肽酶轻链（LC）部分通过对神经传递中必需的SNARE（可溶性nsf -乙基酰亚胺敏感因子附着蛋白受体）成分的蛋白质水解而损害神经元胞外分泌。本项目的总体目标是从BoNT/A轻链金属蛋白酶活性的多个支架中开发小分子抑制剂来治疗肉毒杆菌中毒。通过筛选NCI化学多样性集，已经确定了BoNT /A LC的小分子喹啉类抑制剂（13,14）。这些经过验证的命中化合物是BoNT/A抑制剂药物发现的合适起点，通过基于结构的药物设计（SBDD）来改善和优化它们的效力和“类药物”性质。在第一阶段，我们将使用经过验证的药物和平行合成化学技术来生产“类药物”分子。我们将使用分子模拟方法和x射线晶体学来探索酶结合抑制剂复合物的结构特征，并使用平行合成来制备与理想抑制剂相关的化合物的重点文库。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测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金属蛋白酶。我们将使用不断增长的数据集来开发精细的药效团模型，以指导结构活性关系（sar）的发展。此外，为了加快药物开发过程，并大大减少该项目二期所需的动物实验数量，我们将常规评估所有目标化合物的最佳体外ADME-T（吸收、分布、代谢、消除和细胞毒性）特性。我们将从不同的支架中制造出优化的先导化合物和备用化合物。在II期，我们将进一步优化这些先导物的体内疗效、药代动力学特性、毒性（在两个物种中）和安全性药理学，以将其开发为ind前临床候选药物（III期）。“肉毒杆菌神经毒素是已知最毒的生物物质之一。从受污染的食物或生物恐怖主义行为中接触这些神经毒素后，可能会发生生命丧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发展。

DESCRIPTION (provided by applicant): The botulinum neurotoxins (BoNTs) represent a group of the most poisonous biological substances known (1- 3). Seven different serotypes of BoNTs (BoNT/A through BoNT/G) are secreted by the anaerobic spore- forming bacteria Clostridium botulinum, Clostridium butyricum and Clostridium baratii (4-7). Together with the tetanus neurotoxin (TeNT), which is produced by Clostridium tetani, the BoNTs comprise the clostridial neurotoxin (CNT) family (8). The lethal intravenous dose of BoNT serotype A (BoNT/A) in humans is 1-5ng/kg (9,10). If accidental exposure to BoNT occurs (e.g., from contaminated foodstuffs), loss of life or life-threatening paralysis can occur (11). Most importantly, the BoNTs have already been "weaponized" in a highly toxic aerosol form, and the BoNTs consequently pose a significant threat to both civilian and military populations (9, 12). Once inhaled into the lung, BoNTs are taken up by the blood stream, target the peripheral cholinergic nerve endings, and cause death by interrupting autonomic nerve function. The zinc-dependent endopeptidase light chain (LC) portion of BoNTs impair neuronal exocytosis through proteolysis of essential SNARE (soluble NSF-ethylmaleimide-sensitive factor attachment protein receptor) components of neurotransmission. The overall goal or this project is to develop small molecule inhibitors from multiple scaffolds of the BoNT/A light chain (LC) metalloprotease activity to treat botulinum poisoning. Small molecule quinoline-based inhibitors of BoNT /A LC have been identified by screening the NCI chemical diversity set (13, 14). These validated hit compounds are suitable starting points for BoNT/A inhibitor drug discovery by using structure-based drug design (SBDD) to improve and optimize their potencies and "drug-like" properties. In Phase I we will use proven techniques of medicinal and parallel synthetic chemistry, to produce "drug-like" molecules. We will use molecular modeling approaches and X-ray crystallography to explore the structural features of enzyme bound inhibitor complexes, and use parallel synthesis to prepare focused libraries of compounds related to idealized inhibitors. 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 metalloprotase by measuring the enzymatic and cellular activities, and specificity of the enzyme inhibitors. We will use the growing data set to develop refined pharmacophore models that will guide the development of the structure activity relationships (SARs). Also, to accelerate the drug development process, and dramatically reduce the number of animal experiments needed in Phase II of this project, we will routinely assess all of our target compounds for optimal in vitro ADME-T (Absorption, Distribution, Metabolism, and Elimination and cyto- Toxicity) properties. We will produce an optimized lead compound and a back-up compound from a different scaffold. In Phase II, we will further optimize these leads for in vivo efficacy, pharmacokinetic properties, toxicity (in two species) and safety pharmacology to develop them as pre-IND clinical candidates (Phase III). "The botulinum neurotoxins are some of the most poisonous biological 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."