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: 8267427
• 负责人: James M Bullard
• 金额: $ 10.77万
• 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 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8267427
• 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; Screening procedure; 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; 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. PUBLIC HEALTH RELEVANCE: Completion of this goal will provide an additional means to identifying compounds that are potential drug candidates against a major human pathogen at a time of widespread growth of bacterial resistance. This system will also be used to identify targets of antibacterial compounds identified in whole cell discovery systems as well as provide a system for in-depth study of protein synthesis in an important pathogen. Broad spectrum antibiotics disrupt the normal enteric flora, leading to undesirable side effects, including potentially serious conditions such as antibiotic induced pseudomembranous colitis (Andrejak et al., 1991). Particularly in cystic fibrosis, where P.aeruginosa respiratory infections are often chronic, the availability of a P. aeruginosa specific antibacterial treatment would be a positive step forward both in terms of patient outcome (by reducing side effects) and in terms of public health (by reducing the selective pressure for the evolution of drug resistant pathogens). In addition, by developing drugs against a novel target, we hope to forestall the emergence of resistance for an extended period. There is clearly an unmet medical need for new and better treatments of P. aeruginosa infections.

描述（由申请人提供）：蛋白质合成是所有细菌的基本代谢过程，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，该系统将允许我们以生物化学方式筛选将特异性抑制铜绿假单胞菌中蛋白质合成的化合物。接下来，该系统可用于识别使用全细胞筛选发现的候选药物的靶点。最后，该系统的开发将使我们能够第一次严格检查铜绿假单胞菌蛋白质合成系统的每个组成部分，以及整个系统。以铜绿假单胞菌为模型系统，我们将建立在我们以前从E.杆菌将开发由苯基-tRNA合成酶（PheRS）、核糖体和核糖体延伸因子组成的最小polyU定向A/T系统。该系统将用于小的、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，用于高通量筛选，并筛选化合物文库抑制聚-Phe肽合成的能力。将为A/T系统的每个组件开发功能试验，以检测抑制靶点。将测定来自生物化学筛选的命中化合物在培养物中抑制铜绿假单胞菌生长的能力。将确定命中化合物对铜绿假单胞菌和其他临床相关病原体的最小抑菌浓度（MIC）。将设计和开发命中化合物的类似物，以增加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）将在生化测定和细菌抑制培养物中进行分析。将针对培养物中的真核细胞测定所选化合物，以确定毒性概率。该项目的完成将为鉴定抗菌药物以及深入了解铜绿假单胞菌的蛋白质合成提供独特的新平台。 公共卫生关系：这一目标的完成将提供一种额外的手段，以确定在细菌耐药性广泛增长的时候，针对主要人类病原体的潜在候选药物的化合物。该系统还将用于鉴定在全细胞发现系统中鉴定的抗菌化合物的靶标，并为深入研究重要病原体中的蛋白质合成提供系统。广谱抗生素破坏正常的肠道植物群，导致不期望的副作用，包括潜在的严重病症，例如抗生素诱导的假膜性结肠炎（Andrejak et al.，1991年）。特别是在囊性纤维化中，其中铜绿假单胞菌呼吸道感染通常是慢性的，铜绿假单胞菌特异性抗菌治疗的可用性在患者结果（通过减少副作用）和公共卫生（通过减少耐药病原体进化的选择压力）方面都将是向前迈出的积极一步。此外，通过开发针对新靶点的药物，我们希望在较长时间内防止耐药性的出现。对于铜绿假单胞菌感染的新的和更好的治疗，显然存在未满足的医疗需求。