Inhibitors to Clostridium perfrigens epsilon toxin

描述（由申请人提供）：产气荚膜梭菌epsilon毒素是B类选择性制剂，可导致严重的、通常是致命的肠毒血症，其特征是心脏、肺、肾和脑水肿。epsilon毒素起作用的机制尚不完全清楚。然而，人们认为毒素单体与敏感细胞上的特定受体结合，组装成寡聚复合物，并在细胞膜上形成孔。我们假设可以制备抑制剂来阻断产气荚膜梭菌毒素介导细胞毒性作用过程中的一个或多个关键步骤。本提案的具体目标是通过(1)鉴定显性阴性突变体和(2)鉴定小分子抑制剂来开发epsilon毒素活性抑制剂。在目标1中，氨基酸缺失、插入和替换将被引入编码epsilon毒素的基因，特别强调被认为插入目标细胞膜的蛋白质区域。重组突变蛋白将被检查缺乏细胞毒性和抑制野生型epsilon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epsilon毒素活性的结构-功能关系的理解，将提供对显性阴性突变毒素性质的见解，并确定对抗暴露于epsilon毒素的新治疗候选物。在目标2中，高通量筛选将用于鉴定抑制epsilon毒素细胞毒性活性的小分子。除了鉴定新的抑制剂外，该目标还将验证高通量分析，这些分析可能用于未来的研究中，以测试其他化合物抑制毒素活性或减轻毒素影响的能力。在R21探索性/发展提案中确定的抑制剂将为未来的研究提供基础，旨在优化抑制活性，检查抑制机制，并使用已建立的动物模型测试抑制剂的有效性。

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.