Exploiting Riboswitch Sensors to Reveal Antibiotics Uptake and Retention in Gram Negative Bacteria

利用核糖开关传感器揭示革兰氏阴性细菌中抗生素的摄取和保留

• 批准号: 10084798
• 负责人: RONALD R BREAKER
• 金额: $ 109.08万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-02-19 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10084798
• 关键词: Affect; Anti-Bacterial Agents; Antibiotic Therapy; Antibiotics; Bacteria; Bacterial Physiology; Binding; Biochemical; Biochemical Pathway; Biochemistry; Biological Process; Cells; Cellular Assay; Chemical Structure; Chemicals; Chemistry; Data; Development; Dihydrofolate Reductase Inhibitor; Enzymes; Escherichia coli; Exposure to; Folic Acid; Foundations; Gene Expression; Genes; Gram-Negative Bacteria; Growth; Immunocompromised Host; Infection; Ions; Knowledge; Lead; Libraries; Ligands; Measurable; Mediating; Messenger RNA; Metabolic Pathway; Metabolism; Modeling; Monitor; Multi-Drug Resistance; New Agents; Organism; Pathway interactions; Penetration; Permeability; Pharmaceutical Chemistry; Pharmacopoeias; Physiological Processes; Physiology; Prevalence; Pseudomonas aeruginosa; RNA; Regimen; Reporter; Reporter Genes; Reporting; Resistance; Screening Result; Signaling Molecule; Structure; Structure-Activity Relationship; System; Systems Analysis; Universities; Untranslated RNA; Validation; absorption; analog; antimicrobial; base; cell envelope; cheminformatics; high throughput screening; model development; multidrug-resistant Pseudomonas aeruginosa; next generation; novel; novel strategies; opportunistic pathogen; pathogen; pharmacophore; programs; response; screening; sensor; small molecule; small molecule inhibitor; small molecule libraries; targeted agent; therapeutic protein; therapeutic target; uptake

ABSTRACT Infections caused by MDR Pseudomonas aeruginosa and other Gram-negative pathogens challenge clinicians to find safe and effective antibiotic regimens that can eradicate these opportunistic pathogens from the frail, often immunocompromised hosts that they target. Two features of the P. aeruginosa cell envelope - its limited permeability to small molecules and the large number of both constitutive and inducible efflux systems it contains - render this pathogen intrinsically resistant to many available antimicrobials and contribute to acquired resistance toward the small set of existing anti-Pseudomonal antibiotics. This seriously limits the ability to identify “hits” with antibiotic activity using whole-cell assays - as compounds that penetrate and can inhibit key metabolic pathways are often effluxed out before they measurably inhibit bacterial growth or viability. Approaches that identify novel small molecule inhibitors of key bacterial enzymes often fail when these small molecules cannot achieve effective intrabacterial concentrations - and our understanding of the chemistries that would allow for penetration and retention is woefully incomplete. In this application we use a diverse array of riboswitches, sensitive and specific RNA-based small molecule sensors, as rapid and quantitative indicators that bacterial physiology has been perturbed. By multiplexing several riboswitches that report on accumulation of the alarmones ZTP and ppGpp, as well as the toxic product of increased SAM utilization, SAH, we can effectively screen for “signatures” of a bacterial response to sub- MIC levels of small molecules. Our approach places these riboswitch reporters in isogenic MDR and efflux- deficient P. aeruginosa strains, simultaneously yielding information about both physical and structural chemical features that allow penetration and efflux-avoidance and identifying “hit” molecules that can be developed as leads for new antibacterial agents. Our medicinal chemistry approach will build on both types of knowledge, allowing novel anti-Pseudomonal compounds to be identified and optimized.

摘要 耐多药铜绿假单胞菌和其他革兰氏阴性病原体引起的感染挑战着临床医生 为了找到安全有效的抗生素方案，可以从体弱者身上根除这些机会致病菌， 通常是免疫功能低下的宿主铜绿假单胞菌细胞被膜的两个特征-其有限的 渗透性的小分子和大量的组成和诱导外排系统， 含有-使这种病原体对许多可用的抗菌剂具有内在抗性，并有助于 对现有的一小部分抗假单胞菌抗生素产生了耐药性。这严重限制了 使用全细胞测定法鉴定具有抗生素活性的“命中物”的能力-作为渗透并可 被抑制关键代谢途径通常在它们可测量地抑制细菌生长或 生存能力鉴定关键细菌酶的新型小分子抑制剂的方法通常会失败， 这些小分子不能达到有效的细菌内浓度-我们对细菌内浓度的理解是， 允许渗透和保留的化学物质非常不完整。 在这个应用中，我们使用了各种各样的核糖开关，敏感和特异性的RNA基小分子 传感器，作为细菌生理学受到干扰的快速和定量指标。通过复用 几个核糖开关报告了Alarmones ZTP和ppGpp的积累，以及有毒产物 增加SAM利用，SAH，我们可以有效地筛选细菌对亚硝酸盐的反应的“签名”， 小分子的MIC水平。我们的方法将这些核糖开关报告基因置于同基因MDR和外排- 缺陷铜绿假单胞菌菌株，同时产生关于物理和结构化学的信息， 允许渗透和避免流出的特征，以及识别可以发展为 新的抗菌剂的领导者。我们的药物化学方法将建立在这两种类型的知识， 允许鉴定和优化新的抗假单胞菌化合物。