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.
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.
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