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(PBPs)是已证实的β-内酰胺类药物靶标，但对β-内酰胺类抗生素耐药， 例如碳青霉烯类和头孢菌素类，已导致全球健康问题。特别是，扩展了- 产超广谱β-内酰胺酶或耐碳青霉烯类的肠杆菌科细菌，包括 大肠埃希氏菌是严重的威胁，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死亡率很高。β-内酰胺类抗生素耐药性 在大肠杆菌中的机制是多方面的，包括，例如，可以降解的β-内酰胺酶的表达 β-内酰胺类、孔蛋白缺失和外排泵的过度表达。抗药性甚至正在发展 对抗新的β-内酰胺/β-内酰胺酶抑制剂组合。这种令人震惊的抵抗促使人们有必要 开发不同的PBP抑制机制来打破这种抗性循环。 PBP1b是大肠杆菌中的关键多肽之一，具有两种与肽聚糖(PG)相关的催化活性：a 转糖基酶活性和转肽酶活性。这两项活动都构建了PG网状网，以提供关键的 细菌的机械强度和形状。PBP1b由LpoB与PBP1b结合激活，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的方法来 通过靶向LpoB激活PBP1b来抑制PBP1b。LpoB缺失或LpoB突变 这会破坏PBP1b的结合，导致对某些β-内酰胺类抗生素的过敏。 目的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 它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。我们会 用热位移法和裂解荧光素酶互补法筛选片段文库化合物。点击量 使用剂量-效应测量、生物物理工具、 TG活性测定检测LpoB介导的PBP1b活化减少。 目的2：片断将被用于LpoB络合物的结晶学分析。合并后的 结构信息、亲和力、活性和热位移数据将被用来设计新的LpoB导向的 以迭代的方式使用抑制剂。顶级先导化合物将进入微生物测试。 我们全面的高风险/高回报PPI目标方法的成功完成将导致 一种使PBP重新敏感的新战略-靶向抗生素，这是鉴于 目前的抗生素耐药性问题。LpoB：PBP1b系统在肠杆菌科中是保守的，所以我们的 结果可能延伸到其他病原体。此外，这项提议的成功结果可能导致 抗生素开发的范式转变，重新将工作重点放在针对细菌病原体的PPI上。