Protein synthesis is an essential metabolic process in all bacteria and a target for the development of new antibiotics. In our initially funded SCORE grant we developed an amino acylation/translation (A/T) system from P. aeruginosa based on polyU mRNA directed protein synthesis. This system was optimized and developed into a platform to perform high throughput screening of chemical compounds against the activity of the system. During this period a number of inhibitory compounds (six) were identified and characterized and developed into lead compounds. A small (5-6) congener group was built around six of the lead compounds. The goal of the current proposal is to develop a lead series around each of these lead compounds as well as to continue discovery of additional compounds that inhibit growth of pathogenic bacteria. To develop a lead series we will carry out structure activity relationship (SAR) studies to increase the inhibitory potency, solubility, ADME and abilit to inhibit biofilm formation. At the same time we will attempt to decrease the potential for drug-drug induced enzyme inhibition and toxicity to human cell lines. In the continued discovery process, we will actively recruit collaborators doing work in isolation of natural compounds to test against our system for identification of new inhibitory compounds as well as obtain small focused synthetic compound libraries. The IC50 as well as the MIC against a panel of pathogenic bacteria will initially be determined for each new inhibitor discovered. These data will determine if new hit compounds enter SAR studies as described above. Next, we propose to expand the A/T minimal protein synthesis system into a more natural like protein synthesis system. During the initial grant period, we were able to accomplish much more than was proposed. We have cloned, expressed, and isolated 11 additional proteins involved in protein synthesis: seven additional aaRS proteins, three translation initiation factors (IF-1, IF-2, and IF 3) and the methionyl-tRNA formyltransferase. Incorporation of these proteins into the A/T assay, along with a designer natural like mRNA, allowing translation of a short peptide will allow us to screen for inhibitors of the ribosome and 15 accessory proteins in one assay. We have functional assays developed for each component of the system and will be able to quickly determine the molecular target of an inhibitor. This system will be developed and optimized based on the experience gained in the initial funding period. Screening of compound libraries will continue to be carried out in a high throughput format using scintillation proximity assays (SPA). Four of the lead compounds inhibit protein synthesis by inhibiting the activity of PheRS. To determine mechanism of action of these inhibitory compounds we will evaluate promising lead compounds for their ability to generate spontaneous mutants or develop resistant after serial sub-culturing of hypersensitive strains of P. aeruginosa. We will screen for mutations in the gene encoding PheRS. The structure for P. aeruginosa PheRS has been solved and we will collaborate with Dr. Kotsikorou at UTPA and use the crystal structure and molecular modeling methods to explain the effects of the mutations on resistance to the antibacterial compounds. We will also model the binding of the compounds in the active site of PheRS to better understand the mechanism of inhibitory activity.
Completion of these goals 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. Particularly in cystic fibrosis, where P. aeruginosa respiratory infections ae 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.
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