Inhibition of Clostridium perfringens epsilon toxin

描述(由申请人提供)：产气荚膜梭菌epsilon毒素是一种B类精选药物，可导致一种严重的、通常是致命的肠毒素血症，其特征是心脏、肺、肾脏和脑水肿。Epsilon毒素的作用机制尚不完全清楚。然而，人们认为毒素单体与敏感细胞上的特定受体结合，组装成低聚复合体，并在细胞膜上形成孔洞。我们推测，在产气荚膜梭菌epsilon毒素介导细胞毒作用的过程中，可以制备出阻断这些关键步骤中的一个或多个步骤的抑制剂。这项建议的具体目的是通过(1)鉴定和分析显性-负性突变蛋白，(2)鉴定epsilon毒素活性的小分子抑制物，以及(3)鉴定epsilon毒素活性所需的宿主细胞因子来开发epsilon毒素活性的抑制剂。在目标1中，编码epsilon毒素的基因将引入氨基酸的缺失、插入和替换，重点放在被认为插入到靶细胞膜的蛋白质区域。将检查重组突变蛋白是否缺乏细胞毒性，以及是否有能力抑制野生型epsilon毒素的细胞毒性活性。在这一目标中发现的突变将增加我们对支配epsilon毒素活性的结构-功能关系的理解，将提供对显性-负性突变毒素的性质的洞察，并找到新的治疗候选药物来对抗epsilon毒素的暴露。在目标2中，将使用高通量筛选来识别抑制epsilon毒素细胞毒活性的小分子。除了确定新的抑制剂外，这一目标还将验证高通量测试，这些测试可用于未来的研究，以测试更多化合物抑制毒素活性或减轻毒素影响的能力。在目标3中，对epsilon毒素敏感和耐药的MDCK细胞系进行蛋白质组学分析，以确定epsilon毒素活性所需的宿主细胞因子。这种方法有望确定毒素识别的细胞表面受体以及可能作为治疗干预目标的其他宿主因素。该提案中确定的抑制剂将为未来旨在优化抑制活性并使用已建立的动物模型测试抑制剂的有效性的研究提供基础。与公共卫生相关的产气荚膜梭菌epsilon毒素是最有效的细菌毒素之一。美国卫生与公众服务部和农业部已将epsilon毒素归类为选择性毒剂。目前，还没有被批准用于人类的对策，这项提案的具体目的是开发epsilon毒素活性的抑制剂。

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 C. perfringens epsilon toxin mediates cytotoxic effects. The specific aims of this proposal are designed to develop inhibitors of epsilon toxin activity by (1) identifying and analyzing dominant-negative mutant proteins, (2) identifying small molecule inhibitors of epsilon toxin activity, and (3) identifying host cell factors required for epsilon toxin activity. 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. In aim 3, proteomic analyses of epsilon toxin-sensitive and resistant MDCK cells lines will be used to identify host cell factors required for epsilon toxin activity. This approach is expected to identify the cell-surface receptor recognized by the toxin as well as other host factors that may be targeted for therapeutic intervention. The inhibitors identified in this proposal will provide the foundation for future studies aimed at optimizing the inhibitory activities and testing the effectiveness of the inhibitors using established animal models. PUBLIC HEALTH RELEVANCE The Clostridium perfringens epsilon toxin is one of the most potent bacterial toxins. The U.S. Department of Health and Human Services and the Department of Agriculture have classified the epsilon toxin as a select agent. Currently, there is no countermeasure approved for use in humans and the specific aims of this proposal are designed to develop inhibitors of epsilon toxin activity.