Novel Small Molecule Inhibitors of Botulinum Neurotoxin A

描述（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25.000MW的突触体相关蛋白（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SNAP-25被BoNT LC切割，将提供治疗毒素暴露前和毒素暴露后的潜力。我们的策略是通过化学文库筛选和3D亚结构数据库挖掘相结合，鉴定“药物样”小分子BoNT/A LC抑制剂支架，适合于在合理的药物设计方案中进一步化学细化。通过该过程，我们已经在酶和基于细胞的测定中鉴定了几种具有低uM效力的BoNT/A抑制剂，其防止BoNT/A的靶底物SNAP-25的细胞内蛋白水解。所鉴定的抑制剂类别是新颖的并且缺乏非药物样特征，例如异羟肟酸和肽骨架。这些经过验证的化学“命中”系列将形成开发有效、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;酶-酶复合物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，我们将通过测量衍生物的酶活性和细胞活性以及特异性来探测有助于更紧密结合和更有效抑制BoNT/A的特征的聚焦化合物库。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精细的药效团模型，以指导进一步探索构效关系（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。将放大具有足够的酶、细胞效力和ADME性质的化合物，并在BoNT诱导的大鼠死亡模型中测试其功效。从BoNT诱导的死亡中成功拯救大鼠将使化合物有资格作为体内验证的先导物。我们将评估这些先导药物的研究性新药（IND），使其具有毒性和安全性药理学，以便将其开发为适合人体临床试验的IND前临床候选药物。在拟定的研究和临床前开发计划结束时，我们的总体项目里程碑是提交IND，用于口服活性小分子BoNT/A抑制剂的临床人体安全性评价。

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.