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的基本代谢过程，也是开发新抗生素的目标。这项建议的目标是开发一种从铜绿假单胞菌中合成蛋白质的系统，这将使我们能够通过生物化学的方法筛选出能够特异性地抑制铜绿假单胞菌蛋白质合成的化合物。下一步，这个系统可以用来识别通过全细胞筛选发现的候选药物的目标。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质合成系统的每个组成部分以及整个系统进行严格的检查。使用铜绿假单胞菌作为模型系统，我们将建立在我们之前从大肠杆菌构建氨基酰化/翻译(A/T)系统所获得的经验的基础上。将开发一个由苯基-tRNA合成酶(PHERS)、核糖体和核糖体延长因子组成的最小PolyU指导的A/T系统。该系统将用于高通量筛选小的、有重点的化合物文库，以识别抑制铜绿假单胞菌蛋白质合成的化合物。A/T系统将以微滴定板形式进行优化，以进行高通量筛选，并将筛选化合物文库，以确定其抑制多-苯丙氨酸多肽合成的能力。将为A/T系统的每个组件开发功能分析，以检测抑制的目标。从生化筛选出来的HIT化合物将被检测它们在培养中抑制铜绿假单胞菌生长的能力。将确定HIT化合物对铜绿假单胞菌和其他临床相关病原体的最低抑制浓度(MIC)。HIT化合物的类似物将被设计和开发，以提高在培养中对细菌的生化特异性、效力和效力。类似物的构效关系(SAR)将在生化分析和细菌抑制培养两个方面进行分析。选定的化合物将在培养中针对真核细胞进行测试，以确定其毒性的可能性。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。

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.