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，世卫组织和疾病预防控制中心将碳青霉烯类耐药肠杆菌科（CRE）列为优先级 1“关键” 超级细菌”和“紧急威胁”，并警告称，针对超级细菌的新疗法已导致近 50,000 人死亡 美国人和欧洲人一年，如果单靠市场力量，不太可能及时发展起来。 CRE 尚有治疗选择，而目前大多数 CRE 已出现“泛耐药”的恐惧。 感染发生在医院环境中，但目前 CRE 在社区中的潜在传播也存在。 治疗是在医院进行静脉注射，未来提供口服给药的药物将 减少住院时间和总体医疗费用。具有新颖作用机制的全新药物陷入困境； 因此，机械上新颖的抗生素不受临床相关耐药机制的影响，并且适合 我们的建议旨在开发一种机制上的口服降压疗法。 新型静脉注射和口服给药制剂，用于治疗抗生素敏感和多重耐药性引起的感染 肠杆菌科，包括超广谱 β-内酰胺酶生产者和 CRE。 基于大肠杆菌（Ec）过表达的共培养筛选，我们鉴定了一系列小分子抑制剂 靶向 LspA，一种重要且广泛保守的革兰氏阴性脂蛋白信号肽酶，在 该提案中提出的初步工作确立了外膜脂蛋白生物发生中的关键作用。 该系列的巨大潜力和目标，我们寻求通过以下方式继续开发该计划。 Hit-to-Lead 活动的目标： 目标 1 - 开发工具集来实施 LspAi Hit-to-Lead 活动 (1) 合成 25 毫克 LspAi 筛选。 命中以及关键中间体，以询问弹头的性质并促进目标 2 中的模拟合成。 (2) 在体外生化试验中测试 Ec LspA 中的 LspAis，以确定体外效力 (3) 扩展 FOR 分析。 Ec ΔtolC (4) 对肺炎克雷伯菌 (Kp) ΔtolC 进行 FOR 和作用机制 (MOA) 分析 显示 MOA 延伸至 Kp。 目标 2 – Hit-to-Lead 活动，以确定具有 WT 活性和体内功效的 1 个先导序列 (1) Hit-to-。 主要药物化学类似物合成 (2) 针对 WT 和匹配对流出和渗透性的 MIC 测定。 修改的 Ec 和 Kp。 (3) 体外 IC50 测定 (4) 评估血浆蛋白结合 (ppb)，哺乳动物 HepG2。 (5) FOR测定、扩展MOA分析、杀伤曲线和多粘菌素 (6) 测量 PK（IV、SC 和 PO）。 (7) 体内研究的化合物放大。 (9) 展示WT Ec在小鼠败血症模型中的体内功效。 目标 3 - 与 SSGCID 合作获得 Ec LspAi-LspA X 射线共晶结构，以实现 基于结构的药物设计方法，SSGCID 主导的 Ec LspA 异源表达、蛋白质纯化、 和结晶研究以实现 SBDD 策略，请参阅相关目标里程碑的研究策略。