Dynamic Drug Design Targeting Botulinum Neurotoxins

描述（由申请人提供）：肉毒杆菌神经毒素（BoNTs）是一种危险的生物恐怖主义威胁，因为它们具有极强的效力和致命性，并且易于生产和运输。如果不及时治疗，BoNTs中毒可发展为弛缓性麻痹，并因呼吸衰竭而死亡。然而，及时的接触后干预可以限制循环毒素的影响。我们的总体、长期研究目标是产生一种新的治疗方法，可以用于治疗被BoNT中毒的个体。每个BoNT由催化轻链组成，其进入神经元是由重链介导的。我们的策略是基于这样一个模型，即肉毒中毒相关的弛缓性麻痹是由BoNT轻链阐述的锌依赖性内肽酶活性的下游结果。使BoNT轻链内肽酶功能失活的最有效方法之一是合理设计靶向活性位点的抑制剂。为了实现这一目标，我们将结合计算和实验方法来开发铅抑制剂模板。在具体目标1中，我们将使用一种称为动态药效团建模的强大计算方法来确定阻断bont内肽酶活性的计算导联。在这种方法中，通过分子动力学模拟和使用分子动力学生成的蛋白质构象集合生成共识或动态药效团模型来考虑蛋白质和活性位点的构象灵活性。然后，动态药效团模型用于搜索商业上可用的小分子数据库，以生成计算先导化合物。在特异性目标2中，我们将使用酶测定和体外细胞测定来测试每个计算铅的抑制活性。这项工作的一个里程碑将是鉴定一个或多个铅抑制剂模板，阻断野生型毒素的作用，使用体外模型系统。这项研究的结果将为未来开发和使用已建立的动物模型进行这些新型抑制剂的体内测试奠定基础和理由。

DESCRIPTION (provided by applicant): Botulinum neurotoxins (BoNTs) are a dangerous bioterrorism threat due to their extreme potency and lethality, as well as their ease of production and transport. If untreated, poisoning by the BoNTs can progress to flaccid paralysis and death due to respiratory failure. However, timely post-exposure intervention can limit the effects of the circulating toxin. Our overall, long-term research objective is to generate a novel class of therapeutics that can be administered to individuals who have been poisoned by BoNT. Each BoNT is composed of a catalytic light chain whose entry into neurons is mediated by the heavy chain. Our strategy is based on the model that botulism-related flaccid paralysis is a downstream consequence of the zinc-dependent endopeptidase activity elaborated by the BoNT light chain. One of the most powerful approaches to inactivate the endopeptidase function of the BoNT light chains is rational design of inhibitors targeting the active site. To achieve this, we wilt combine computational and experimental approaches to develop lead inhibitor templates. In Specific Aim 1, we will use a powerful computational approach called dynamic pharmacophore modeling to identify computational leads to block the endopeptidase activities of the BoNTs. In this approach, the conformational flexibility of the protein and active site are taken into account through molecular dynamics simulations and the generation of a consensus, or dynamic, pharmacophore model using an ensemble of molecular dynamics-generated protein conformations. The dynamic pharmacophore model is then used to search databases of commercially available small molecules to generate computational lead compounds. In Specific Aim 2, we will test each computational lead for inhibitory activity using enzyme assays and in vitro cellular assays. A milestone of this work will be the identification of one or more lead inhibitor templates that block the action of wild type toxin using in vitro model systems. The results from this research will establish the groundwork and justification for future development and in vivo testing of these novel inhibitors using established animal models.