Targeting Escherichia coli PBP1b using fragment-based approaches

使用基于片段的方法靶向大肠杆菌 PBP1b

• 批准号: 10374158
• 负责人: FOCCO VAN DEN AKKER
• 金额: $ 24.15万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 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 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10374158
• 关键词: Affinity; Antibiotic Resistance; Antibiotics; Antimicrobial Resistance; Autolysis; Bacteria; Bacterial Antibiotic Resistance; Binding; Biological Assay; Carbapenems; Cell Death; Cell Wall; Centers for Disease Control and Prevention (U.S.); Cephalosporins; Complement; Complex; Crystallization; Data; Development; Dose; Drug Targeting; Enterobacteriaceae; Escherichia coli; Escherichia coli Proteins; Extended-spectrum β-lactamase; Genes; Goals; Hypersensitivity; Label; Lead; Libraries; Light; Link; Luciferases; Measurement; Mechanics; Mediating; Membrane; Microbiology; Molecular Conformation; Molecular Weight; Monobactams; Mutation; Nosocomial Infections; Outcome; Penicillin-Binding Proteins; Peptides; Peptidoglycan; Peptidyltransferase; Protein Inhibition; Proteins; Resistance; Resistance development; Resort; Scanning; Sepsis; Shapes; Site; Structure; System; Testing; Up-Regulation; Urinary tract infection; VDAC1 gene; base; beta-Lactam Resistance; beta-Lactamase; beta-Lactams; biophysical tools; carbapenem-resistant Enterobacteriaceae; crosslink; design; efflux pump; fitness; global health; high reward; high risk; inhibitor; mortality; novel; novel strategies; novel therapeutic intervention; overexpression; pathogen; pathogenic bacteria; periplasm; resistance mechanism; response; screening; success

Penicillin-binding proteins (PBPs) are proven β-lactam drug targets yet resistance to β-lactam antibiotics, such as carbapenems and cephalosporins, has resulted in a global health problem. In particular, extended- spectrum β-lactamase (ESBL) producing or carbapenem-resistant Enterobacteriaceae, which includes Escherichia coli, are serious threats and are often linked to hospital-acquired infections. Bloodstream infections caused by these pathogens have a high mortality rate. β-lactam antimicrobial resistance mechanism in E. coli are multiple and include, for example, the expression of β-lactamases that can degrade β-lactams, deletion of porins, and the overexpression of efflux pumps. Resistance is developing even against new β-lactam/β-lactamase inhibitor combinations. This alarming resistance spurs the need to develop different mechanisms of PBP inhibition to break this resistance cycle. PBP1b, one of the key PBPs in E. coli, has two peptidoglycan (PG)-related catalytic activities: a transglycosylase activity and a transpeptidase activity. Both activities build the PG mesh that provides critical mechanical strength and shape for bacteria. PBP1b is activated by LpoB binding to PBP1b, leading to a conformational change that stimulates both activities of PBP1b. Our goal is to develop a novel approach to inhibiting PBP1b, by targeting the activation of PBP1b by LpoB. Deletion of LpoB or mutations in LpoB that disrupt PBP1b binding leads to hypersensitivity to certain β-lactam antibiotics. Aim 1: We propose to develop inhibitors of PBP1b activation by screening and developing compounds that bind to the PBP1b-recognition site on activator LpoB via a fragment-based structural approach. We will use thermal shift and split luciferase complementation assays to screen fragment library compounds. Hits from these orthogonal assays are further probed using dose-response measurements, biophysical tools, and a TG activity assay testing for a decrease of LpoB-mediated activation of PBP1b. Aim 2: Fragment hits will be targeted for crystallographic analysis in complex with LpoB. The combined structural information, affinity, activity, and thermal shift data will be used to design novel LpoB-directed inhibitors in an iterative fashion. Top lead compounds will advance to microbiological testing. The successful completion of our comprehensive high-risk/high-reward PPI targeting approach will lead to a new strategy of re-sensitizing PBP-targeting antibiotics, which is urgently needed in light of the current antibiotic resistance problem. The LpoB:PBP1b system is conserved in Enterobacteriaceae so our results could extend to other pathogens. Furthermore, the successful outcome of this proposal could lead to a paradigm shift in antibiotic development, re-focusing efforts on targeting PPIs in bacterial pathogens.

青霉素结合蛋白 (PBP) 已被证明是 β-内酰胺药物靶标，但对 β-内酰胺抗生素具有耐药性， 碳青霉烯类、头孢菌素类等抗生素已导致全球健康问题。特别是，扩展- 产β-内酰胺酶（ESBL）或耐碳青霉烯类肠杆菌科细菌，其中包括 大肠杆菌是严重的威胁，通常与医院获得性感染有关。血流 这些病原体引起的感染死亡率很高。 β-内酰胺抗菌药物耐药性 大肠杆菌中的机制是多种的，包括例如可以降解的β-内酰胺酶的表达 β-内酰胺、孔蛋白的缺失和外排泵的过度表达。抵抗甚至正在发展 针对新的β-内酰胺/β-内酰胺酶抑制剂组合。这种令人震惊的阻力促使我们需要 开发不同的 PBP 抑制机制来打破这种耐药循环。 PBP1b 是大肠杆菌中的关键 PBP 之一，具有两种与肽聚糖 (PG) 相关的催化活性： 转糖基酶活性和转肽酶活性。这两项活动都构建了 PG 网格，该网格提供了关键的 细菌的机械强度和形状。 LpoB 与 PBP1b 结合可激活 PBP1b，从而导致 刺激 PBP1b 两种活性的构象变化。我们的目标是开发一种新颖的方法 通过 LpoB 靶向激活 PBP1b 来抑制 PBP1b。 LpoB 缺失或 LpoB 突变 破坏 PBP1b 结合会导致对某些 β-内酰胺抗生素过敏。 目标1：我们建议通过筛选和开发化合物来开发PBP1b激活抑制剂 通过基于片段的结构方法与激活剂 LpoB 上的 PBP1b 识别位点结合。我们将 使用热位移和裂解荧光素酶互补测定来筛选片段库化合物。点击数 使用剂量反应测量、生物物理工具进一步探讨这些正交测定的结果， TG 活性测定检测 LpoB 介导的 PBP1b 激活的减少。 目标 2：目标片段将用于与 LpoB 复合物的晶体学分析。合并后的 结构信息、亲和力、活性和热位移数据将用于设计新型 LpoB 导向的 以迭代方式抑制抑制剂。顶级先导化合物将进入微生物测试阶段。 我们全面的高风险/高回报 PPI 目标方法的成功完成将引领 鉴于目前迫切需要一种使 PBP 靶向抗生素重新敏感的新策略 当前的抗生素耐药性问题。 LpoB:PBP1b 系统在肠杆菌科细菌中是保守的，因此我们的 结果可以扩展到其他病原体。此外，该提案的成功结果可能会导致 抗生素开发的范式转变，重新集中精力针对细菌病原体中的 PPI。