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: 10654841
• 负责人: Holly Ann Sutterlin
• 金额: $ 20.4万
• 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/10654841
• 关键词: Acinetobacter baumannii; Active Sites; American; Anti-Bacterial Agents; Antibiotic susceptibility; Antibiotics; Bacteremia; Binding; Binding Proteins; Biochemical; Biogenesis; Biological Assay; Cell Death; Cells; 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; Klebsiella pneumoniae; Lead; Left; Length of Stay; Lipoproteins; Marketing; Measures; Mediating; Membrane; Minimum Inhibitory Concentration measurement; Modeling; Multi-Drug Resistance; Multidrug-resistant Acinetobacter; Mus; Natural Products; Nature; New Agents; Nosocomial pneumonia; 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; Structure; Structure-Activity Relationship; Superbug; Testing; Therapeutic; Time; Work; analog; bactericide; carbapenem resistance; carbapenem-resistant Enterobacteriaceae; cell envelope; clinically relevant; cytotoxicity; dosage; improved; in vivo; inhibitor; innovation; insight; interest; intravenous administration; 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，谁和疾病预防控制中心设计了耐碳青霉烯的肠杆菌科（CRE）优先级1' Superbug’和“紧急威胁”，并警告说，对超级细菌的新疗法杀死了近50,000 如果只留给市场力量，那么美国和欧洲人每年就不太可能及时发展。很少 治疗选择要治疗CRE，并且出现了对“耐泛力” CRE的恐惧。目前，大多数Cre 感染发生在医院环境中，但是CRE在社区中的潜在传播也存在。作为最新 治疗是在医院环境中静脉注射的，未来提供口服的特工将 降低住院和整体医疗保健费用。具有新颖的动作机制的全新代理人； 因此，机械新颖的抗生素不受临床相关的抗性机制的影响和合适 迫切需要口服口服的渐进式疗法。我们的建议旨在以机械的方式发展 小说，IV和PO施用的药物治疗抗生素易感和多种耐药性引起的感染 肠杆菌科，包括扩展的光谱β-内酰胺酶生产者和CRE。使用创新 基于过表达的大肠杆菌（EC）中的基于过表达的共培养筛查，我们确定了一个小分子抑制剂序列 靶向LSPA，必不可少的且宽的革兰氏阴性脂蛋白信号肽 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建立了 这个系列和目标的巨大潜力。我们试图通过以下 瞄准命中率广告： 目标1-开发工具集以起诉LSPAI命中率广告系列。 （1）合成25毫克LSPAI筛选 命中和关键中间体会询问弹头的性质并促进AIM 2中的模拟合成。 （2）在EC LSPA中测试LSPAIS在体外生化测定中以建立体外效力。 （3）扩展以进行分析 ECΔTOLC。 （4）在肺炎（KP）ΔTOLC中进行作用和作用机理（MOA）分析 显示MOA延伸到KP。 AIM 2 - 命中率广告活动，以识别具有WT活动和体内效率的1个领先系列。 （1）命中 - 铅中化学模拟合成。 （2）针对WT和匹配的PAIR排出和渗透性的MIC测定 修改后的EC和KP。 （3）体外IC50测定。 （4）评估血浆蛋白结合（PPB），哺乳动物HEPG2 细胞毒性和溶血活性。 （5）用于确定，扩展MOA分析，杀死曲线和多牙素 协同测定法。 （6）测量PK（IV，SC和PO）。 （7）体内研究的复合缩放。 （8）进行剂量 - 小鼠PK研究。 （9）证明了WT EC的鼠败血症模型的体内效率。 AIM 3-与SSGCID合作获取EC LSPAI-LSPA X射线共晶结构 基于结构的药物设计方法。 SSGCID领导的EC LSPA异源表达，蛋白质纯化， 和结晶研究以实现SBDD策略。有关相关目标里程碑的研究策略。