A Protein Synthesis System from Pseudomonas aeruginosa for Screening for Antibact

描述（由申请人提供）：蛋白质合成是所有细菌必不可少的代谢过程，也是开发新抗生素的目标。本提案的目标是开发一种铜绿假单胞菌的蛋白质合成系统，使我们能够从生化角度筛选特异性抑制铜绿假单胞菌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标。最后，该系统的开发将使我们第一次能够对铜绿假单胞菌蛋白合成系统的每个组成部分进行单独以及整体的严格检查。利用铜绿假单胞菌作为模型系统，我们将在之前从大肠杆菌中构建氨基酰化/翻译（a /T）系统的工作中获得的经验基础上进行构建。将开发一个由苯基trna合成酶（PheRS）、核糖体和核糖体延伸因子组成的小型多u定向A/T系统。该系统将用于小的、集中的化学化合物文库的高通量筛选，以鉴定铜绿假单胞菌中抑制蛋白质合成的化合物。A/T系统将在微滴板上进行优化，以进行高通量筛选，并对化合物文库进行筛选，以确定其抑制多肽合成的能力。将为A/T系统的每个组成部分开发功能分析，以检测抑制目标。从生化筛选中得到的Hit化合物将被用于测定其在培养物中抑制铜绿假单胞菌生长的能力。最低抑制浓度（MIC）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。将设计和开发hit化合物的类似物，以提高生物化学特异性，效力和对培养细菌的功效。类似物的结构活性关系（SAR）将在生化分析和细菌抑制培养中进行分析。选定的化合物将对培养的真核细胞进行测定，以确定毒性的可能性。该项目的完成将为铜绿假单胞菌的抗菌鉴定以及对蛋白质合成的深入了解提供独特的新平台。

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.