Development of LspA Inhibitors to Treat Gram-negative Bacterial Infections

开发治疗革兰氏阴性细菌感染的 LspA 抑制剂

• 批准号: 10508185
• 负责人: Holly Ann Sutterlin
• 金额: $ 17万
• 依托单位: PROKARYOTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10508185
• 关键词: Acinetobacter baumannii; Active Sites; American; Anti-Bacterial Agents; Antibiotics; Bacteremia; Binding; Binding Proteins; Biochemical; Biogenesis; Biological Assay; Cells; Centers for Disease Control and Prevention (U.S.); Chemosensitization; Clinical; Coculture Techniques; Collaborations; Combined Antibiotics; Combined Modality Therapy; Communities; Complement; Crystallization; Data; Development; Docking; Dose; Drug Design; Employment; Enterobacteriaceae; Enterobacteriaceae Infections; Escherichia coli; European; Extended-spectrum β-lactamase; Failure; Fright; Future; Genetic; Goals; Gram-Negative Bacterial Infections; HIV Protease Inhibitors; Health Care Costs; HepG2; Hospitals; Hour; Human; In Vitro; Infection; Innate Immune System; Intravenous; Klebsiella pneumoniae; Lead; Left; Length of Stay; Lipoproteins; Measures; Mediating; Membrane; Membrane Potentials; Minimum Inhibitory Concentration measurement; Modeling; Multi-Drug Resistance; Multidrug-resistant Acinetobacter; Mus; Natural Products; Nature; New Agents; Nosocomial pneumonia; Oral; Oral Administration; Orthologous Gene; Outcome; Penetration; Peptide Signal Sequences; Peptides; Permeability; Pharmaceutical Chemistry; Pharmaceutical Preparations; Plasma Proteins; Play; Polymyxin B; Polymyxins; Predisposition; Protease Inhibitor; Proteins; Research; Resistance; Roentgen Rays; Role; Septicemia; Series; Serum; Signal Transduction; Structure; Structure-Activity Relationship; Superbug; Testing; Therapeutic; Time; Work; analog; bactericide; base; carbapenem resistance; carbapenem-resistant Enterobacteriaceae; cell envelope; clinically relevant; cytotoxicity; dosage; in vivo; inhibitor; innovation; insight; interest; lead optimization; lead series; member; membrane biogenesis; mutant; novel; overexpression; pathogen; periplasm; programs; protein purification; resistance frequency; resistance mechanism; scale up; screening; signal peptidase; small molecule; small molecule inhibitor; standard of care; structural biology; synergism

Recently, WHO and CDC designated Carbapenem-resistant Enterobacteriaceae (CRE) a Priority 1 ‘critical superbug’ and an ‘Urgent Threat’, and warned that new treatments for superbugs, which kill nearly 50,000 Americans and Europeans a year, are unlikely to be developed in time if left to market forces alone. Few therapeutic options are left to treat CRE, and the fear of ‘pan-resistant’ CRE has emerged. Currently, most CRE infections occur in a hospital setting, but the potential spread of CRE in the community also exists. As current treatments are administered intravenously in a hospital setting, future agents offering oral administration would reduce hospital stay and overall healthcare costs. Entirely new agents with novel mechanisms of action languish; therefore, mechanistically novel antibiotics unaffected by clinically relevant resistance mechanisms and suitable for orally administered stepdown therapy are urgently needed. Our proposal aims to develop a mechanistically novel, IV and PO administered agent to treat infections caused by antibiotic susceptible and multidrug-resistant Enterobacteriaceae, including extended spectrum beta-lactamase producers and CRE. Using an innovative overexpression-based co-culture screen in Escherichia coli (Ec), we identified a small molecule inhibitor series targeting LspA, the essential and broadly conserved Gram-negative lipoprotein signal peptidase that plays a crucial role in outer membrane lipoprotein biogenesis. Preliminary work presented in this proposal establishes the great potential of this series and target. We seek to continue to develop this program through the following aims in a Hit-to-Lead campaign: Aim 1 - Develop toolset to prosecute LspAi Hit-to-Lead campaign. (1) Synthesize 25 mg of LspAi screening hits as well as key intermediates to interrogate nature of the warhead and facilitate analog synthesis in Aim 2. (2) Test LspAis in Ec LspA in vitro biochemical assay to establish in vitro potency. (3) Expand FOR analysis in Ec ΔtolC. (4) Perform FOR and mechanism of action (MOA) analysis in Klebsiella pneumoniae (Kp) ΔtolC to show MOA extends to Kp. Aim 2 – Hit-to-Lead campaign to identify 1 Lead series with WT activity and in vivo efficacy. (1) Hit-to- Lead med chem analog synthesis. (2) MIC determination against WT and matched-pair efflux and permeability modified Ec and Kp. (3) In vitro IC50 determination. (4) Assess plasma protein binding (ppb), mammalian HepG2 cytotoxicity, and hemolytic activity. (5) FOR determination, extended MOA analysis, kill curves and polymyxin synergy assays. (6) Measure PK (IV, SC, and PO). (7) Compound scale-up for in vivo studies. (8) Conduct dose- ranging mouse PK studies. (9) Demonstrate in vivo efficacy in murine septicemia model of WT Ec. Aim 3 - Obtain Ec LspAi-LspA X-ray co-crystal structure in collaboration with SSGCID to enable structure-based drug design approach. SSGCID-led Ec LspA heterologous expression, protein purification, and crystallization studies to enable SBDD strategy. See Research Strategy for associated Aim Milestones.

最近，世界卫生组织和疾病预防控制中心将耐碳青霉烯类肠杆菌科(CRE)列为优先1‘关键 并警告说，针对导致近5万人死亡的超级细菌的新疗法 美国人和欧洲人一年来，如果单靠市场力量，不太可能及时发展起来。一些 治疗CRE的选择剩下了，人们已经出现了对“泛耐药”CRE的恐惧。目前，大多数CRE 感染发生在医院环境中，但社区中也存在CRE的潜在传播。当前状态 治疗是在医院环境下静脉注射的，未来提供口服给药的代理人将 减少住院时间和整体医疗成本。具有新的作用机制的全新的代理人正在衰败； 因此，新型抗菌药物不受临床相关耐药机制的影响，适用于 对于口服给药，迫切需要阶梯疗法。我们的建议旨在开发一种机械的 新型静脉给药和PO给药治疗抗生素敏感和多药耐药感染 肠杆菌科，包括超广谱β-内酰胺酶产生菌和Cre。使用创新的 基于超表达的共培养筛选，我们鉴定了一个小分子抑制物系列 靶向LspA是一种基本的、广泛保守的革兰氏阴性脂蛋白信号肽酶，它发挥着 在外膜脂蛋白生物发生中的关键作用。本提案中提出的初步工作确定 这个系列的巨大潜力和目标。我们寻求通过以下方式继续发展这一计划 目标是一场打到领先的竞选活动： 目标1-开发工具集来起诉LspAi Hit-to-Lead运动。(1)合成25 mg LspAi筛选 命中以及关键中间体，以审问弹头的性质，并促进目标2中的模拟合成。 (2)在EC LspA体外生化试验中检测LspAis，以确定其体外效力。(3)展开以供分析 ECΔTolC.(4)肺炎克雷伯氏菌(Kp)ΔTolC的分析及其作用机制 SHOW MOA延伸到KP。 目的开展2-Hit-to-Lead运动，以确定1个具有WT活性和体内疗效的铅系列。(1)击打到- 主导化学模拟合成。(2)对WT和配对流出和渗透率的MIC测定 改进的EC和Kp。(3)体外IC50测定。(4)测定血浆蛋白结合率(Ppb)，哺乳动物HepG2 细胞毒性和溶血活性。(5)用于测定、扩展的MOA分析、杀伤曲线和多粘菌素 协作性分析。(6)测量PK(IV、SC、PO)。(7)用于体内研究的化合物放大。(8)进行剂量- 射程小鼠PK研究。(9)在小鼠败血症模型上验证WT EC的体内药效。 目标3-与SSGCID合作获得EC LspAI-LspA X射线共晶结构，以使 基于结构的药物设计方法。SSGCID引导的EC LspA异源表达，蛋白纯化， 和结晶研究，以实现SBDD战略。有关相关的目标里程碑，请参阅研究战略。