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，WHO和CDC将耐碳青霉烯类肠杆菌科（CRE）指定为“1级关键” 超级细菌“和”紧急威胁“，并警告说，新的治疗超级细菌，杀死近5万人 美国人和欧洲人一年的时间，如果只靠市场力量，是不可能及时发展起来的。几 留下了治疗CRE的治疗选择，并且出现了对“泛耐药”CRE的恐惧。目前，大多数CRE 感染发生在医院环境中，但CRE在社区中的潜在传播也存在。为流动 治疗是在医院环境中静脉内给药的，未来提供口服给药的药物将 减少住院时间和整体医疗费用。具有新作用机制的新药物逐渐消失; 因此，不受临床相关耐药机制影响的机制性新抗生素和合适的抗生素， 口服降压治疗的方法。我们的建议旨在建立一个机制， 用于治疗抗生素敏感性和多重耐药性引起的感染的新型IV和PO给药药剂 肠杆菌科，包括产超广谱β-内酰胺酶菌和CRE。使用创新的 通过在大肠杆菌（Escherichia coli，Ec）中进行基于过表达的共培养筛选，我们鉴定了一个小分子抑制剂系列 LspA是一种重要的、广泛保守的革兰氏阴性脂蛋白信号肽酶， 在外膜脂蛋白生物合成中起重要作用。本提案中提出的初步工作确定 这一系列的巨大潜力和目标。我们将通过以下方式继续发展这一计划： 在一场“点击领先”运动中的目标： 目标1 -开发工具集，以起诉LspAi命中到铅运动。(1)合成25 mg LspAi筛选 命中以及关键的中间体，以询问弹头的性质，并促进目标2中的类似物合成。 (2)在Ec LspA体外生物化学测定中测试LspAis以建立体外效力。(3)展开FOR分析 Ec ΔtolC。(4)在肺炎克雷伯菌（Kp）ΔtolC中进行FOR和作用机制（MOA）分析， 示出MOA延伸到Kp。 目标2 -命中-电极导线活动，以确定具有WT活性和体内疗效的1个电极导线系列。(1)击中- 领导医学化学模拟合成。(2)针对WT和配对外排和渗透性的MIC测定 修正Ec和Kp。(3)体外IC 50测定。(4)评估血浆蛋白结合率（ppb），哺乳动物HepG 2 细胞毒性和溶血活性。(5)FOR测定、扩展MOA分析、杀灭曲线和多粘菌素 协同测定。(6)测量PK（IV、SC和PO）。(7)用于体内研究的化合物放大。(8)进行剂量- 范围小鼠PK研究。(9)证明WT Ec在鼠败血症模型中的体内疗效。 目标3 -与SSGCID合作获得Ec LspA 1-LspA X射线共晶体结构，以实现 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。相关目标里程碑见研究策略。