Targeting Clostridioides difficile with microbiome-sparing, resistant-proof anti-toxins

使用保留微生物组、抗耐药性的抗毒素来靶向艰难梭菌

• 批准号: 10115406
• 负责人: Vern L. Schramm
• 金额: $ 66.87万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10115406
• 关键词: Affect; Affinity; Animals; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Apoptosis; Bacterial Toxins; Binding; Biological Assay; Catalytic Domain; Ceftriaxone; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Chemicals; Clinical; Clinical Trials; Clostridium difficile; Colitis; Complex; Crystallization; Crystallography; Cytoskeleton; Development; Disease; Drug Design; Drug resistance; Electrostatics; Environment; Epithelial Cells; Equilibrium; Evolution; FDA approved; Fatal Outcome; GTP-Binding Proteins; Glucose; Glucosyltransferase; Glucosyltransferases; Guidelines; Health Care Costs; Hospitals; Human; Hydrolysis; Hypotension; Ileus; Immunity; Infection; Infection prevention; Isotopes; Kinetics; Knowledge; Lactamase; Lead; Life; Maps; Medical; Megacolon; Methods; Microfilaments; Modeling; Molecular; Monoclonal Antibody Therapy; Morbidity - disease rate; North America; Oral; Organism; Pathology; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Population; Process; Production; Proliferating; Proteins; Pseudomembranous Colitis; Reaction; Recovery; Reporting; Reproduction spores; Resistance; Roentgen Rays; Shock; Structure; Technology; Testing; Thermodynamics; Threonine; Tissues; Toxic effect; Toxin; Transferase; United States National Institutes of Health; Uridine Diphosphate Sugars; Vaccines; Vancomycin; Virulence Factors; World Health Organization; analog; antitoxin; base; cdc42 GTP-Binding Protein; computational chemistry; cytotoxicity; design; fecal transplantation; gastrointestinal epithelium; glycosylation; glycosyltransferase; gut microbiome; gut microbiota; healthcare-associated infections; holotoxins; inhibitor/antagonist; innovation; insight; microbial; microbiome; mortality; mouse model; neutralizing antibody; novel; oral infection; pathogen; pathogenic bacteria; phase II trial; preservation; pressure; prevent; programs; quantum chemistry; recurrent infection; rho; screening; small molecule; symptom treatment

Abstract: Human gut infections by Clostridioides (Clostridium) difficile (here, C.diff.) are the most lethal urgent threat in the 2019 CDC Antibiotic Resistance Threats Report. Excess healthcare costs from these infections have been estimated to be over $5 billion annually. Antibiotic resistance has elicited an insightful RFA RA18-725, `Generating new insights and mechanistic understanding of antibiotic resistance'. C.diff. infections (CDI) typically arise following treatment of other clinical disorders with antibiotics. Antibiotic therapy disrupts normal gut microbiota, allowing C.diff. to proliferate and to repopulate the gut following treatment. Additional antibiotic therapy to treat CDI prevents return of normal gut microbiota, leading to recurrent infections in over 20% of patients. C.diff. has acquired resistance to several common antibiotics, compounding its therapy. Recent clinical guidelines (2018) for C.diff. infections are oral vancomycin for patients in shock, hypotension, ileus or megacolon. Fecal transplant is recommended for nonresponsive infections following vancomycin treatment. mAb therapies have been FDA- approved, but are not recommended. Despite these therapies, C. difficile causes an estimated 224,000 infections and 13,000 deaths per year (CDC in 2017). Gut epithelial cell cytotoxicity results from C.diff. production of secreted toxins, primarily TcdA and TcdB (Tcds). Tcds are processed in gut cells to form active UDP-glucosyl transferases that glucosylate cytoskeletal-regulating Rho, Rac and Cdc42 GTP-binding proteins on specific threonines. Loss of cytoskeletal integrity causes severe colitis and can have a fatal outcome. Anti-toxin immunity is a historic approach to prevent host damage from circulating bacterial toxins. We propose that small molecule, tight-binding inhibitors targeting C.diff. Tcds can prevent the morbidity and mortality from gut toxins in C.diff. infections. Our transition state analog approach uses kinetic isotope effects and quantum chemistry to solve transition state structures of Tcds. Solving the first transition state structures of G-protein glucosyltransferases, and developing the first transition state analog of any UDP-sugar transferase is innovative. Electrostatic potential models of Tcd transition states will guide the design and synthesis of transition state analogs. Lead transition state analog candidates will be elaborated by cycles of crystallography and chemical design. Candidate compounds and crystal structures of Tcd complexes have been obtained in preliminary studies. Inhibitors will be characterized against Tcds in human cells and in mouse models. Agents to prevent tissue damage from C.diff. infections, without disruption of the gut microbiome or pressure for microbial resistance have important medical relevance. Inhibition of Tcds in gut epithelial cells places no selective pressure for antibiotic or anti-toxin resistance on C.diff. or on the gut microbiome, while protecting the gut by neutralizing Tcds. Mechanistically, this approach is innovative in recapitulating vaccine-based antibody neutralization of toxins using the powerful approach of transition state analogs.

摘要：艰难梭菌 (Clostridium) difficile（此处为 C.diff.）引起的人类肠道感染是最致命的紧急疾病 2019 年 CDC 抗生素耐药性威胁报告中的威胁。这些感染造成的额外医疗费用已经 据估计每年超过 50 亿美元。抗生素耐药性引发了富有洞察力的 RFA RA18-725， “产生对抗生素耐药性的新见解和机制理解”。 C.差异。感染（CDI）通常 用抗生素治疗其他临床疾病后出现。抗生素治疗会破坏正常肠道 微生物群，允许艰难梭菌。治疗后增殖并重新填充肠道。额外的抗生素治疗 治疗 CDI 会阻碍正常肠道微生物群的恢复，导致超过 20% 的患者出现复发性感染。 C.差异。 对几种常见抗生素产生了耐药性，使其治疗更加复杂。最近的临床指南（2018） 对于 C.diff。感染休克、低血压、肠梗阻或巨结肠的患者口服万古霉素。粪便移植 建议用于万古霉素治疗后无反应的感染。 mAb 疗法已获得 FDA 批准 批准，但不推荐。尽管有这些疗法，艰难梭菌仍导致估计 224,000 例感染 每年有 13,000 人死亡（CDC，2017 年）。艰难梭菌对肠上皮细胞的细胞毒性作用。生产 分泌毒素，主要是 TcdA 和 TcdB (Tcds)。 Tcd 在肠道细胞中加工形成活性 UDP-葡萄糖基 转移酶可对特定的细胞骨架调节 Rho、Rac 和 Cdc42 GTP 结合蛋白进行糖基化 苏氨酸。细胞骨架完整性的丧失会导致严重的结肠炎，并可能产生致命的结果。 抗毒素免疫是防止宿主因循环细菌毒素而受到损害的一种历史方法。我们 提出针对艰难梭菌的小分子紧密结合抑制剂。 TCD 可以预防发病率和死亡率 来自艰难梭菌的肠道毒素。感染。我们的过渡态模拟方法使用动力学同位素效应和量子 化学来解决 Tcd 的过渡态结构。解析 G 蛋白的第一个过渡态结构 葡萄糖基转移酶，并开发任何 UDP 糖转移酶的第一个过渡态类似物是创新的。 Tcd过渡态静电势模型将指导过渡态的设计与合成 类似物。先导过渡态类似物候选物将通过晶体学和化学循环来详细阐述 设计。初步研究已获得Tcd配合物的候选化合物和晶体结构。 抑制剂将在人类细胞和小鼠模型中针对 Tcd 进行表征。 防止艰难梭菌组织损伤的药物。感染，不会破坏肠道微生物组或 微生物耐药性的压力具有重要的医学意义。 Tcds 在肠道上皮细胞中的抑制作用 对艰难梭菌没有抗生素或抗毒素抗性的选择压力。或肠道微生物组，同时保护 通过中和 Tcd 来肠道。从机制上讲，这种方法在重现基于疫苗的抗体方面是创新的 使用过渡态类似物的强大方法中和毒素。