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: 10217694
• 负责人: FOCCO VAN DEN AKKER
• 金额: $ 20.13万
• 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 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10217694
• 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/antagonist; 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）是被证明的β-内酰胺药物靶标，但对β-内酰胺抗生素的抗性， 例如碳青霉烯和头孢菌素，导致了全球健康问题。特别是扩展 频谱β-内酰胺酶（ESBL）产生或抗碳青霉烯型肠杆菌科，其中包括 大肠杆菌是严重威胁，通常与医院获得的感染有关。血液 这些病原体引起的感染具有高死亡率。 β-内酰胺抗菌抗性 大肠杆菌中的机制是多种的，包括可以降解的β-内酰胺酶的表达 β-内酰胺，porin的缺失和外排泵的过表达。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激发了 开发PBP抑制的不同机制以打破这种抗性周期。 PBP1B是大肠杆菌中的关键PBP之一，具有两个辣椒（PG）相关的催化活性：A 转糖基化酶活性和转肽酶活性。这两种活动都建立了提供关键的PG网格 细菌的机械强度和形状。 PBP1B被LPOB与PBP1B结合激活，导致A 构象变化刺激PBP1B的两种活动。我们的目标是开发一种新颖的方法 通过靶向LPOB靶向PBP1B的激活来抑制PBP1B。 LPOB或LPOB突变的删除 破坏PBP1b的结合会导致对某些β-内酰胺抗生素的过敏。 目标1：我们建议通过筛选和开发化合物来开发PBP1B激活的抑制剂 通过基于片段的结构方法，激活剂LPOB上的PBP1B-识别位点结合。我们将 使用热偏移和分裂荧光素酶完成测定法进行筛选片段库化合物。命中 从这些正交分析中，使用剂量反应测量，生物物理工具，进一步探测 以及LPOB介导的PBP1B激活减少的TG活性评估测试。 AIM 2：片段命中将被针对与LPOB复合物进行晶体学分析。组合 结构信息，亲和力，活动和热偏移数据将用于设计新型LPOB指导 以迭代方式抑制剂。顶级铅化合物将推进微生物学测试。 成功完成我们全面的高风险/高回报PPI定位方法将领导 为重新敏感PBP靶向抗生素的新策略，鉴于 当前的抗生素抗性问题。 LPOB：PBP1B系统是在肠杆菌科中配置的 结果可以扩展到其他病原体。此外，该提议的成功结果可能会导致 为了改变抗生素发育的范式，重新集中了针对细菌病原体中PPI的努力。