A Protein Synthesis System from Pseudomonas aeruginosa for Screening for Antibact

用于筛选抗菌剂的铜绿假单胞菌蛋白质合成系统

• 批准号: 8837647
• 负责人: James M Bullard
• 金额: $ 3.53万
• 依托单位: UNIVERSITY OF TEXAS RIO GRANDE VALLEY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-19 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8837647
• 关键词: Adverse effects; American; Amino Acyl-tRNA Synthetases; Aminoacylation; Anti-Bacterial Agents; Antibiotics; Bacteria; Bacterial Infections; Bacterial Proteins; Benign; Biochemical; Biological Assay; Biological Models; Cell Survival; Cells; Chemicals; Chronic; Colorado; Cystic Fibrosis; Development; Distant; Drug resistance; EEF1A1 gene; Elongation Factor; Enteral; Escherichia coli; Escherichia coli Proteins; Eukaryotic Cell; Evolution; Genome; Goals; Growth; Human; In Vitro; Infection; Lead; Libraries; Medical; Messenger RNA; Metabolism; Minimum Inhibitory Concentration measurement; Modification; Montana; Outcome; Patients; Peptide Elongation Factor G; Peptides; Pharmaceutical Preparations; Probability; Procedures; Protein Biosynthesis; Protein Synthesis Inhibition; Pseudomembranous Colitis; Pseudomonas aeruginosa; Public Health; Research; Resistance; Respiratory Tract Infections; Ribosomes; Series; Specificity; Structure-Activity Relationship; System; Systems Development; Testing; Texas; Time; Toxic effect; Translations; Universities; Vendor; Work; analog; bacterial resistance; clinically relevant; cystic fibrosis patients; design; drug candidate; drug discovery; experience; high throughput screening; improved; inhibitor/antagonist; nano; novel; pathogen; pressure; programs; screening; tissue culture

DESCRIPTION (provided by applicant): Protein synthesis is an essential metabolic process in all bacteria and a target for the development of new antibiotics. The goal of this proposal is to develop a protein synthesis system from Pseudomonas aeruginosa that will permit us to screen biochemically for compounds that will specifically inhibit protein synthesis in P. aeruginosa. Next, this system can be used to identify the targets for drug candidates discovered using whole cell screens. Finally, development of this system will allow us, for the first time, to critically inspect each component of P. aeruginosa protein synthesis system separately as well as the system in its entirety. Using P. aeruginosa as a model system we will build upon the experience gained from our previous work of constructing an aminoacylation/translation (A/T) system from E. coli. A minimal polyU directed A/T system composed of phenyl-tRNA synthetases (PheRS), ribosomes and ribosomal elongation factors will be developed. This system will be used in high- throughput screens of small, focused chemical compound libraries to identify compounds that inhibit protein synthesis in P. aeruginosa. The A/T system will be optimized in a microtiter plate format for high-throughput screening and compound libraries will be screened for their ability to inhibit synthesis of poly-Phe peptides. Functional assays will be developed for each component of the A/T system to detect the target of inhibition. Hit compounds coming out of the biochemical screens will be assayed for their ability to inhibit growth of P. aeruginosa in cultures. Minimum inhibitory concentrations (MIC) will be determined for hit compounds against P. aeruginosa and other clinically relevant pathogens. Analogs of the hit compounds will be designed and developed to increase biochemical specificity, potency and efficacy against bacterial in cultures. The structure activity relationship (SAR) of analogs will be analyzed in both biochemical assays and in inhibition of bacteria in cultures. Selected compounds will be assayed against eukaryotic cells in culture to determine the probability of toxicity. Completion of this project will provide unique new platform for identifying antibacterial as well as an in-depth understanding of protein synthesis in P. aeruginosa.

描述（由申请人提供）：蛋白质合成是所有细菌的重要代谢过程，也是开发新抗生素的目标。该提案的目标是开发一种来自铜绿假单胞菌的蛋白质合成系统，该系统将使我们能够通过生化筛选特异性抑制铜绿假单胞菌中蛋白质合成的化合物。接下来，该系统可用于识别通过全细胞筛选发现的候选药物的靶标。最后，该系统的开发将使我们首次能够分别严格检查铜绿假单胞菌蛋白质合成系统的每个组件以及整个系统。使用铜绿假单胞菌作为模型系统，我们将借鉴我们之前从大肠杆菌构建氨酰化/翻译（A/T）系统的工作中获得的经验。将开发由苯基-tRNA合成酶（PheRS）、核糖体和核糖体延伸因子组成的最小的polyU定向A/T系统。该系统将用于小型、集中的化合物库的高通量筛选，以鉴定抑制铜绿假单胞菌中蛋白质合成的化合物。 A/T 系统将以微量滴定板形式进行优化，以进行高通量筛选，并且将筛选化合物库抑制多聚苯丙氨酸肽合成的能力。将为 A/T 系统的每个组件开发功能测定，以检测抑制目标。将分析来自生化筛选的命中化合物抑制培养物中铜绿假单胞菌生长的能力。将确定针对铜绿假单胞菌和其他临床相关病原体的命中化合物的最低抑制浓度 (MIC)。将设计和开发命中化合物的类似物，以提高针对培养物中细菌的生化特异性、效力和功效。类似物的结构活性关系（SAR）将在生化测定和培养物细菌抑制中进行分析。将针对培养物中的真核细胞对选定的化合物进行测定，以确定毒性的可能性。该项目的完成将为鉴定抗菌物质以及深入了解铜绿假单胞菌蛋白质合成提供独特的新平台。