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。主要的毒力因子是TcdA和TcdB，它们是导致腹泻、炎症和结肠内重大损害的毒素。这一建议是围绕这样一种假设设计的，即抑制毒素活性代表着一种治疗方法，可以影响CDI患者的临床治疗和结果。TcdA和TcdB是修饰和失活Rho家族GTP酶的同源糖基转移酶。毒素的葡萄糖转移酶活性与肌动细胞骨架的“细胞病态”破坏有关，对小鼠中毒模型的全身毒性很重要。结合TcdA和TcdB糖基转移酶结构域的小分子糖基转移酶抑制剂(Aim 1)的X射线晶体结构将为结构导向设计具有增强效力的分子提供基础。除了细胞病变作用外，TcdB还是一种强大的细胞毒素，可导致细胞和组织的坏死性损害。这种细胞毒性是由于TcdB诱导上皮细胞NADPH氧化酶(NOX)复合体的组装造成的。这种组装会导致产生活性氧物种(ROS)，从而导致坏死。初步数据表明，NOX1基因敲除小鼠可以免受CDI组织损伤，并强调了这样一个假设，即抑制NOX1通路将保护结肠组织免受严重CDI病例的损伤。高通量筛选已经鉴定出176个抑制TcdB诱导的坏死的小分子。目标2中的实验将根据这些化合物的作用机制对它们进行分类，并鉴定出先导化合物以供进一步分析。在目标3中，FDA批准的抗氧化剂N-乙酰半胱氨酸以及来自目标1和目标2的先导化合物的有效性将在CDI小鼠模型中进行评估。用流行的M7404菌株和一组在一种或两种毒素中都有明确突变的变异体挑战孢子，将允许剖析每种化合物对TcdA和TcdB介导的事件的特定影响。这些都是将艰难梭菌毒素的小分子抑制剂推向临床所需的关键研究。

 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.