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）是β-内酰胺类药物的靶点，但对β-内酰胺类抗生素具有耐药性， 例如碳青霉烯类和头孢菌素类，已经导致了全球性的健康问题。特别是，扩展- 产超广谱β-内酰胺酶（ESBL）或对碳青霉烯类耐药的肠杆菌科，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 由这些病原体引起的感染具有高死亡率。β-内酰胺类抗生素耐药性 机制在E.大肠杆菌中的β-内酰胺酶是多种的，并且包括例如可以降解 β-内酰胺、孔蛋白缺失和外排泵过表达。抵抗力正在发展， 针对新的β-内酰胺/β-内酰胺酶抑制剂组合。这种令人担忧的阻力促使人们需要 开发不同的PBP抑制机制来打破这种抗性循环。 PBP 1b是E.大肠杆菌，具有两种肽聚糖（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， 机械强度和细菌的形状。PBP 1b通过LpoB与PBP 1b结合而活化，导致 构象变化刺激PBP 1b的两种活性。我们的目标是开发一种新的方法， 通过LpoB靶向激活PBP 1b来抑制PBP 1b。LpoB缺失或LpoB突变 破坏PBP 1b结合导致对某些β-内酰胺抗生素的超敏反应。 目的1：我们计划通过筛选和开发化合物来开发PBP 1b活化抑制剂 其通过基于片段的结构方法与激活剂LpoB上的PBP 1b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文库化合物。安打 从这些正交分析中进一步使用剂量-反应测量，生物物理工具， 以及TG活性测定，测试LpoB介导的PBP 1b激活的减少。 目的2：将片段命中作为与LpoB复合物的晶体学分析的目标。将合并的 结构信息、亲和力、活性和热位移数据将用于设计新的LpoB定向的 以迭代的方式抑制剂。顶级铅化合物将进入微生物检测。 我们全面的高风险/高回报PPI目标定位方法的成功完成将导致 一种新的策略，重新敏感的PBP靶向抗生素，这是迫切需要的，鉴于 抗生素耐药性问题。LpoB：PBP 1b系统在肠杆菌科中是保守的，因此我们的 结果可能会扩展到其他病原体。此外，这一提议的成功结果可能导致 到抗生素开发的范式转变，重新集中精力靶向细菌病原体中的PPI。