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）已被证实是β-内酰胺类药物靶点，但对β-内酰胺类抗生素具有耐药性。