Inhibition of Clostridium perfringens 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，阻断C。产气荚膜杆菌毒素介导细胞毒性作用。该提案的具体目的是通过（1）鉴定和分析显性阴性突变蛋白，（2）鉴定抗真菌毒素活性的小分子抑制剂，和（3）鉴定抗真菌毒素活性所需的宿主细胞因子来开发抗真菌毒素活性的抑制剂。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。在目标3中，将使用对肉毒毒素敏感和抗性MDCK细胞系的蛋白质组学分析来鉴定肉毒毒素活性所需的宿主细胞因子。这种方法预计将确定由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子。本提案中确定的抑制剂将为未来的研究提供基础，旨在优化抑制活性并使用已建立的动物模型测试抑制剂的有效性。产气荚膜梭菌毒素是最强的细菌毒素之一。美国卫生与公众服务部和农业部已将这种毒素列为一种选择剂。目前，没有批准用于人类的对策，本提案的具体目的是开发肉毒毒素活性的抑制剂。

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.