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: 10656160
• 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/10656160
• 关键词: Affect; Affinity; Animal Experiments; 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; Clostridium difficile; Colitis; Complex; Crystallography; Cytoskeleton; Development; Disease; Drug Design; Drug resistance; Electrostatics; Environment; Epithelial Cells; Equilibrium; Evolution; Excretory function; 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; Recommendation; 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; cdc42 GTP-Binding Protein; computational chemistry; cytotoxicity; design; fecal transplantation; gastrointestinal epithelium; glycosylation; glycosyltransferase; gut microbiome; gut microbiota; healthcare-associated infections; holotoxins; inhibitor; 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：难辨梭状芽胞杆菌(Clostridioids，C.diff.)是最致命的紧急事件 2019年疾控中心抗生素耐药性威胁报告中的威胁。这些感染导致的额外医疗费用 据估计每年超过50亿美元。抗生素耐药性引发了一种有洞察力的RFA RA18-725， “对抗生素耐药性产生新的见解和机制理解”。C.diff.感染(CDI)通常 在用抗生素治疗其他临床疾病后出现。抗生素治疗扰乱正常肠道 微生物群，使C.diff.在治疗后增殖并重新填充肠道。额外的抗生素治疗 治疗CDI可以防止肠道微生物区系恢复正常，导致超过20%的患者再次感染。C.diff. 已经对几种常见的抗生素产生了抗药性，使其治疗复杂化。最新临床指南(2018) 对于C.diff。感染是口服万古霉素，用于休克、低血压、肠梗阻或巨结肠患者。粪便移植 推荐用于万古霉素治疗后无反应性感染。MAB疗法已经通过FDA- 已批准，但不推荐使用。尽管采取了这些治疗方法，艰难梭菌仍导致约22.4万人感染 每年有13,000人死亡(2017年疾控中心)。肠道上皮细胞毒性由C.diff引起。生产 分泌毒素，主要是TcdA和TcdB(TCDs)。TCD在肠道细胞中被加工成活性的UDP-葡萄糖 糖基化细胞骨架调节Rho、Rac和CDc42 GTP结合蛋白的转移酶 苏氨酸。细胞骨架完整性的丧失会导致严重的结肠炎，并可能导致致命的后果。 抗毒素免疫是防止细菌毒素循环对宿主造成损害的一种历史性的方法。我们 提出以C.diff为靶点的小分子、紧密结合的抑制剂。TCDS可以预防疾病的发生和死亡 来自C.diff的肠道毒素。感染。我们的过渡态模拟方法使用了动力学同位素效应和量子 解决TCDS过渡态结构的化学方法。G蛋白第一过渡态结构的求解 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过渡态的静电势模型将指导过渡态的设计和合成 类比。铅过渡态模拟候选物将通过结晶学和化学循环进行详细阐述 设计。初步获得了TCD配合物的候选化合物和晶体结构。 在人类细胞和小鼠模型中，抑制剂将针对TCDs进行表征。 防止C.diff对组织造成损害的药物。感染，而不破坏肠道微生物群或 压力对微生物的耐药性具有重要的医学意义。TCDs在肠上皮细胞部位的抑制作用 对C.diff的抗生素或抗毒素耐药性无选择性压力。或肠道微生物组，同时保护 通过中和TCDs来破坏肠道。从机理上讲，这种方法在重现疫苗抗体方面是创新的。 使用过渡态类似物的强大方法中和毒素。