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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的由MDR假单胞菌和其他革兰氏阴性病原体挑战临床医生引起的感染为了找到可以从虚弱中造成这些机会性病原体的安全抗生素方案，它们的靶向寄生虫的两种特征通常是铜绿。对小分子的渗透性以及大量的征兵和诱导外排系统包含 - 这种病原体本质上对许多可用的抗微生物剂的抗性，并促成。获得了对现有伪造抗生素的一小部分的抵抗力。使用整个细胞测定 - 作为穿透并可以使用抗生素活性识别抗生素活性的“命中”的能力 - 抑制关键代谢途径在可测量的抑制细菌生长或可行性。这些小分子无法获得有效的细菌浓度 - 我们对允许穿透和保留的化学条件严重不完整。在此应用中，我们使用各种核糖开关，敏感和特定的基于RNA的小分子传感器，作为迅速和定量的指标，表明双臂物理学已通过多重。该报道的几个核糖开关ZTP和PPGPP SAH的SAM利用率增加，我们可以有效地筛选出对亚的“签名”的“签名” 小分子的麦克风水平。缺乏铜绿假单胞菌菌株，同时产生有关物理和结构化学的信息可以开发允许穿透和避免外排出并识别“命中”分子的特征新的抗菌剂的铅。允许鉴定和优化新型的抗峰值化合物。