Pre-clinical evaluation of Clostridium difficile toxin inhibitors

 描述（由申请人提供）： 艰难梭菌感染（CDI）是美国退伍军人发病率和死亡率的重要来源。主要毒力因子是TcdA和TcdB，它们是诱导腹泻、炎症和结肠内显著损伤的毒素。该提议是围绕以下假设设计的：抑制毒素活性代表了一种治疗方法，可以影响患有CDI的个体的临床治疗和结果。 TcdA和TcdB是修饰和修饰Rho家族GTP酶的同源葡糖基转移酶。毒素的葡糖基转移酶活性与活性细胞骨架的"致细胞病变"破坏有关，对小鼠中毒模型的全身毒性很重要。结合到TcdA和TcdB葡糖基转移酶结构域的小分子葡糖基转移酶抑制剂的X射线晶体结构（Aim 1）将为具有增强效力的分子的结构引导设计提供基础。 除了致细胞病变效应之外，TcdB是一种强效细胞毒素，其导致细胞和组织中的坏死性损伤。细胞毒性是由TcdB诱导的上皮细胞NADPH氧化酶（NOX）复合物的组装引起的。组装导致产生活性氧（ROS），其引起坏死。初步数据表明，Nox 1基因敲除小鼠受到保护，免受CDI组织损伤，并强调了抑制NOX 1通路将保护免受严重CDI病例中观察到的结肠组织损伤的假设。 一个高通量的筛选导致了176个小分子的鉴定，抑制TcdB诱导的坏死。目标2中的实验将根据其作用机制对这些化合物进行分类，并鉴定出先导化合物以供进一步分析。在目标3中，将在CDI小鼠模型中评价N-乙酰半胱氨酸（一种FDA批准的抗氧化剂）与目标1和2的先导化合物一起沿着的功效。用流行性M7404菌株和一组在一种或两种毒素中具有确定突变的变体进行孢子攻击将允许剖析每种化合物对TcdA和TcdB介导的事件的特异性作用。这些都是关键的研究需要推进小分子抑制剂的C。艰难梭菌毒素进入临床实践。

 DESCRIPTION (provided by applicant): Clostridium difficile infection (CDI) is an important source of morbidity and mortality among U.S. Military Veterans. The primary virulence factors are TcdA and TcdB, toxins that induce diarrhea, inflammation, and significant damage within the colon. This proposal is designed around the hypothesis that inhibition of toxin activity represents a therapeutic approach that can impact clinical treatment and outcome for individuals suffering from CDI. TcdA and TcdB are homologous glucosyltransferases that modify and inactivate Rho family GTPases. The glucosyltransferase activity of the toxins has been linked to a `cytopathic' disruption of the acti cytoskeleton and is important for systemic toxicity in a mouse intoxication model. The X-ray crystal structures of small molecule glucosyltransferase inhibitors bound to the TcdA and TcdB glucosyltransferase domains (Aim 1) will provide a foundation for structure-guided design of molecules with enhanced potency. In addition to the cytopathic effects, TcdB is a potent cytotoxin that causes necrotic damage in cells and tissue. The cytotoxicity results from TcdB-induced assembly of the epithelial cell NADPH oxidase (NOX) complex. The assembly results in the production of reactive oxygen species (ROS), which cause necrosis. Preliminary data indicate that a Nox1 knockout mouse is protected from CDI tissue damage and underscores the hypothesis that inhibition of the NOX1 pathway will protect against the colonic tissue damage observed in severe cases of CDI. A high-throughput screen has led to the identification of 176 small molecules that inhibit TcdB-induced necrosis. Experiments in Aim 2 will categorize these compounds according to their mechanism of action and result in the identification of lead compounds for further analysis. In Aim 3, the efficacy of N- acetylcysteine, an FDA-approved antioxidant, along with lead compounds from Aims 1 and 2 will be evaluated in a mouse model of CDI. Spore challenge with an epidemic M7404 strain and a panel of variants with defined mutations in one or both toxins will permit dissection of the specific effect each compound has on TcdA- and TcdB-mediated events. These are the key studies needed to advance small molecule inhibitors of the C. difficile toxins into clinical practice.