Inhibitors to Clostridium perfrigens epsilon toxin

描述（由申请方提供）：产气荚膜梭菌肠毒素是一种B类选择性制剂，可引起以心、肺、肾和脑水肿为特征的重度、通常致死性肠毒血症。但其作用机制尚不完全清楚。然而，据信毒素单体与敏感细胞上的特异性受体结合，组装成寡聚复合物，并在细胞膜中形成孔。我们假设可以制备抑制剂，其阻断产气荚膜梭菌毒素介导细胞毒性作用的过程中的一个或多个这些关键步骤。该提案的具体目的是通过（1）鉴定显性失活突变体和（2）鉴定小分子抑制剂来开发肉毒毒素活性抑制剂。在目标1中，将氨基酸缺失、插入和取代引入编码肉毒毒素的基因中，特别强调被认为插入靶细胞膜的蛋白质区域。将检查重组突变蛋白的细胞毒性缺乏和抑制野生型肉毒毒素的细胞毒性活性的能力。在这一目标中确定的突变体将增加我们对控制肉毒毒素活性的结构-功能关系的理解，将提供对显性负突变毒素性质的深入了解，并确定用于对抗肉毒毒素暴露的新的治疗候选物。在目标2中，将使用高通量筛选来鉴定抑制肉毒毒素的细胞毒性活性的小分子。除了鉴定新型抑制剂外，该目标还将验证可用于未来研究的高通量测定，以测试其他化合物抑制毒素活性或减轻毒素影响的能力。本R21探索性/开发提案中确定的抑制剂将为旨在优化抑制活性、检查抑制机制以及使用已建立的动物模型检测抑制剂有效性的未来研究提供基础。

DESCRIPTION (provided by applicant): The Clostridium perfringens epsilon toxin, a Category B Select Agent, is responsible for a severe, often fatal enterotoxemia characterized by cardiac, pulmonary, kidney, and brain edema. The mechanism by which epsilon toxin acts is incompletely understood. However, it is believed that toxin monomers bind to specific receptors on sensitive cells, assemble into oligomeric complexes, and form pores in the cell membrane. We hypothesize that inhibitors can be prepared that block one or more of these key steps in the process by which the Clostridium perfringens epsilon toxin mediates cytotoxic effects. The specific aims of this proposal are designed to develop inhibitors of epsilon toxin activity by (1) identifying dominant-negative mutants and (2) identifying small molecule inhibitors. In aim 1, amino acid deletions, insertions, and substitutions will be introduced into the gene encoding epsilon toxin, with special emphasis placed on a region of the protein believed to insert into the target cell membrane. Recombinant mutant proteins will be examined for both a lack of cytotoxicity and for the ability to inhibit the cytotoxic activity of wild-type epsilon toxin. Mutants identified in this aim will increase our understanding of the structure-function relationships that govern epsilon toxin activity, will provide insight into the nature of dominant-negative mutant toxins, and identify new therapeutic candidates for countering exposure to epsilon toxin. In aim 2, a high-throughput screen will be used to identify small molecules that inhibit the cytotoxic activity of epsilon toxin. In addition to identifying novel inhibitors, this aim will validate high-throughput assays that may be used in future studies to test additional compounds for the ability to either inhibit toxin activity or mitigate the effects of the toxin. The inhibitors identified in this R21 Exploratory/Developmental proposal will provide the foundation for future studies aimed at optimizing the inhibitory activities, examining the mechanisms of inhibition, and testing the effectiveness of the inhibitors using established animal models.