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。主要病毒因子是TCDA和TCDB，诱导腹泻的毒素，感染和结肠内的重大损害。该提案的设计围绕以下假设：抑制毒素活性代表一种治疗方法，可以影响患有CDI的个体的临床治疗和结果。 TCDA和TCDB是​​同源的葡萄糖基转移酶，可修饰和失活Rho家族GTPase。毒素的葡萄糖基转移酶活性与Acti细胞骨架的“细胞性”破坏有关，对于小鼠中毒模型中的全身毒性很重要。与TCDA和TCDB葡萄糖基转移酶结构域结合的小分子葡萄糖基转移酶抑制剂的X射线晶体结构（AIM 1）将为结构制定的分子设计具有增强的效力。除了细胞疗法外，TCDB是​​一种潜在的细胞毒素，会在细胞和组织中导致坏蛋损伤。细胞毒性是由TCDB诱导的上皮细胞NADPH氧化物（NOX）复合物组装而产生的。组装导致活性氧（ROS）的产生，导致坏死。初步数据表明，NOX1基因敲除小鼠受到CDI组织损伤的保护，并强调了抑制NOX1途径的假设将预防在严重的CDI病例中观察到的殖民组织损伤。高通量屏幕导致鉴定了176个抑制TCDB诱导坏死的小分子。 AIM 2中的实验将根据这些化合物的作用机理对这些化合物进行分类，并导致识别铅化合物以进行进一步分析。在AIM 3中，将在CDI的小鼠模型中评估N-乙酰半胱氨酸（FDA批准的抗氧化剂）以及AIMS 1和2的铅化合物的效率。孢子质量M7404菌株和一个具有定义突变的变体的孢子挑战将允许对每种化合物对TCDA和TCDB介导的事件的特定效果进行解剖。这些是将艰难梭菌毒素的小分子抑制剂推向临床实践所需的关键研究。