Novel Small Molecule Inhibitors of Botulinum Neurotoxin A

描述（由申请人提供）：项目总体目标是开发一种口服活性小分子BoNT/A抑制剂。BoNT/A LC的神经元底物是突触体相关蛋白25.000MW （SNAP-25）。我们的假设是，小分子BoNT/A抑制剂可以穿透神经元，防止BoNT LC切割SNAP-25，这将为毒素暴露前和暴露后的治疗提供潜力。我们的策略是通过化学文库筛选和3D子结构数据库挖掘相结合，确定“类药物”小分子BoNT/A LC抑制剂支架，适合在合理的药物设计程序中进一步进行化学细化。通过这一过程，我们在酶和细胞实验中发现了几种低uM效的BoNT/A抑制剂，它们可以阻止BoNT/A靶底物SNAP-25的细胞内蛋白水解。所确定的抑制剂类别是新颖的，缺乏非药物样的特征，如羟肟酸和肽骨架。这些经过验证的化学“命中”系列将构成开发有效、安全和口服生物可利用的抗BoNT/A药物的基础。我们将应用药物和组合化学的成熟技术；x射线晶体学和基于结构的药物设计（SBDD）来快速合成和评估这些新的经过验证的BoNT/A抑制剂支架的衍生物。在迭代过程中，我们将通过测量酶和细胞活性以及衍生物的特异性来探测重点化合物文库，以寻找有助于更紧密结合和更有效抑制BoNT/A的特征。我们将确定与BoNT活性位点结合的改进抑制剂的x射线结构，并使用这些数据开发完善的药效团模型，以指导进一步探索结构活性关系（SAR）。我们将评估化合物的最佳ADME（吸收、分布、代谢、消除）、药代动力学和生物利用度特性。具有足够的酶、细胞效力和ADME特性的化合物将在BoNT诱导的大鼠死亡模型中进行放大和有效性测试。成功拯救bont诱导死亡的大鼠将使化合物成为体内验证的先导物。我们将对这些先导药物进行毒性和安全性药理学评估，以便将其开发为IND前的临床候选药物，适用于人体临床试验。在这项拟议的研究和临床前开发计划结束时，我们的整体项目里程碑是为口服活性小分子BoNT/A抑制剂的临床人体安全性评估提交IND。

DESCRIPTION (provided by applicant): The overall project goal is to develop an orally active small molecule BoNT/A inhibitor. The neuronal substrate for BoNT/A LC is synaptosomal-associated protein of 25.000MW (SNAP-25). Our hypothesis is that small molecule BoNT/A inhibitors which can penetrate the neuron, preventing SNAP-25 cleavage by the BoNT LC, will provide the potential for treatment of both pre- and post-toxin exposure. Our strategy has been to identify "drug-like" small molecule BoNT/A LC inhibitor scaffolds, through a combination of chemical library screening and 3D sub-structure database mining, suitable for further chemical refinement in a rational drug design program. Through this process, we have identified several BoNT/A inhibitors with low uM potencies in enzymatic and cell based assays, which prevent BoNT/A intracellular proteolysis of its target substrate SNAP-25. The identified inhibitor classes are novel and devoid of non-drug like features such as hydroxamic acid and peptidic backbones. These validated chemical "hit" series will form the basis for development of efficacious, safe and orally bioavailable drugs against BoNT/A. We will apply proven techniques of medicinal and combinatorial chemistry; inhibitor-enzyme complex X-ray crystallography and structure-based drug design (SBDD) to rapidly synthesize and evaluate derivatives of these new validated BoNT/A inhibitor scaffolds. In an iterative process, we will probe focused compound libraries for features contributing to tighter binding and more potent inhibition of BoNT/A by measuring the enzymatic and cellular activity and specificity of derivatives. We will determine the X-ray structures of improved inhibitors bound to the BoNT active site, and use the data to develop refined pharmacophore models to guide further probing of the structure activity relationship (SAR). We will assess compounds for optimal ADME (Absorption, Distribution, Metabolism, Elimination), pharmacokinetic and bioavailability properties. Compounds with sufficient enzymatic, cellular potency and ADME properties will be scaled-up and tested for efficacy in a BoNT induced rat death model. Successful rescue of rats from BoNT-induced death will qualify compounds as in vivo-validated leads. We will evaluate these leads for investigational new drug (IND) enabling toxicity and safety pharmacology in order to develop them into pre-IND clinical candidates, suitable for human clinical trials. By the end of this proposed research and pre-clinical development plan, our overall project milestone is to file an IND for the clinical human safety evaluation of an orally active small molecule BoNT/A inhibitor.