Pre-clinical evaluation of Clostridium difficile toxin inhibitors

艰难梭菌毒素抑制剂的临床前评价

• 批准号: 9487905
• 负责人: Dana Borden Lacy
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-10-01 至 2019-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9487905
• 关键词: Acetylcysteine; Actins; Antibiotics; Antioxidants; Apigenin; Categories; Cell model; Cells; Chemical Structure; Clinical Treatment; Clostridium difficile; Collection; Colon; Complex; Crystallization; Cytoskeleton; Cytotoxin; Data; Development; Diarrhea; Dihydropyridines; Disease; Disease Outbreaks; Dissection; Drug Design; Enzymes; Epidemic; Epithelial Cells; Evaluation; Event; FDA approved; Family; Foundations; Glucosyltransferase; Glucosyltransferases; Goals; Guanosine Triphosphate Phosphohydrolases; Human; Individual; Infection; Inflammation; Intoxication; Knockout Mice; Lead; Life; Link; Mediating; Military Personnel; Modeling; Morbidity - disease rate; Mus; Mutation; NADPH Oxidase; Necrosis; Nifedipine; Pathology; Pathway interactions; Perforation; Phloretin; Prevention; Process; Production; Property; Pseudomembranous Colitis; Reactive Oxygen Species; Recurrence; Relapse; Reproduction spores; Roentgen Rays; Sepsis; Source; Structure; Therapeutic; Tissues; Toxic Megacolon; Toxin; Translating; Treatment outcome; United States; Universities; Variant; Veterans; Virulence Factors; base; clinical practice; cytotoxicity; design; ebselen; efficacy testing; experimental study; high throughput screening; hypertension treatment; in vivo; inhibitor/antagonist; mortality; mouse model; novel; novel therapeutics; preclinical evaluation; prevent; protective efficacy; public health relevance; response; rho; small molecule; small molecule inhibitor; systemic toxicity; tissue culture; treatment strategy

 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.

 描述（由申请人提供）： 艰难梭菌感染 (CDI) 是美国退伍军人发病和死亡的重要来源，主要毒力因子是 TcdA 和 TcdB，它们是引起腹泻、炎症和结肠内严重损伤的毒素。该提案是围绕抑制的假设而设计的。毒素活性的研究代表了一种可以影响患有 CDI 的个体的临床治疗和结果的治疗方法，TcdA 和 TcdB 是同源葡萄糖基转移酶。修饰和灭活 Rho 家族 GTP 酶的毒素的葡萄糖基转移酶活性与 acti 细胞骨架的“细胞病变”破坏有关，并且对于小鼠中毒模型中的全身毒性很重要 小分子葡萄糖基转移酶抑制剂的 X 射线晶体结构。与 TcdA 和 TcdB 葡萄糖基转移酶结构域结合（目标 1）将为结构引导设计提供基础除了细胞病变效应外，TcdB 也是一种强效细胞毒素，可导致细胞和组织坏死。TcdB 诱导上皮细胞 NADPH 氧化酶 (NOX) 复合物的组装导致细胞毒性。活性氧 (ROS) 的产生会导致坏死，初步数据表明 Nox1 敲除小鼠可以免受 CDI 组织损伤，并强调了这一假设。抑制 NOX1 途径将防止在严重 CDI 病例中观察到的结肠组织损伤。高通量筛选已鉴定出 176 种抑制 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对这些化合物进行分类。在 Aim 3 中，N-乙酰半胱氨酸（一种 FDA 批准的抗氧化剂）以及来自 Aims 的先导化合物的功效。 1 和 2 将在流行性 M7404 菌株的 CDI 孢子攻击小鼠模型中进行评估，并且在一种或两种毒素中具有确定突变的一组变体将允许剖析每种化合物对 TcdA 和 TcdB 介导的特定作用。这些是将艰难梭菌毒素的小分子抑制剂推进临床实践所需的关键研究。