Therapeutics targeting fatty acid synthesis in P. aeruginosa

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DESCRIPTION (provided by applicant): Pseudomonas aeruginosa is a common and extremely virulent cause of serious infections in immune- compromised/suppressed patients (e.g., HIV and cancer), cystic fibrosis patients, and those on mechanical ventilation or with burn wounds. Frequent antibiotic resistance and the highly virulent nature of P. aeruginosa make it deadlier than most other bacterial species. New chemical classes of antibiotics acting on novel targets are crucial for continued effective therapy against P. aeruginosa because such drugs will not be subject to existing resistance mechanisms. Recent identification of inhibitors of fatty acid biosynthesis (FAB) targets FabI (enoyl-ACP reductase), FabF (condensing enzyme), and AccD (acetyl-CoA carboxylase) in several species has validated this macromolecular synthesis pathway as an under-exploited potentially useful route for discovery of new antibacterials. However, these inhibitors fail to block growth or viability of P. aeruginosa for two primary reasons -- (a) cell permeability and efflux obstacles and (b) the existence of a second enoyl-ACP reductase FabK, which is non-homologous to, but duplicates the function of FabI. The strategy of this project is to develop new antibacterials, which are active against P. aeruginosa FAB, by using cellular reporter screens to detect inhibitors of any of several steps in the FAB pathway. These will be based on fusion of the Photorhabdus luminescens luxCDABE luciferase operon to promoters which are up-regulated in response to depletion of three different essential FAB targets -- AccD, FabF, and the second reductase in the cycle, FabG, which is structurally related to FabI but is essential in P. aeruginosa. Such screens will detect inhibitors capable of entering P. aeruginosa cells and capable of escaping efflux sufficiently to generate a report. A diverse collection of synthetic and natural product compounds will be screened with the FAB pathway-specific cellular reporter strain. Hits will be confirmed by repeat assay vs. the reporter strain, demonstration of concentration dependent activity, and inhibition of an in vitro biochemical assay consisting of the entire re-constituted fatty acid biosynthesis cycle. The precise target of each inhibitor will be identified by determination of FAB precursors that accumulate in inhibited biochemical assays of the FAB cycle, and confirmation will be accomplished in direct assays of the putative target in the presence of the compound. Validated hits will be prioritized by demonstrating MICs, minimal inhibition of mammalian cell viability, minimal disruption of cell membranes, and by determining the spectrum of anti-bacterial activity. Prioritized validated hits will be optimized in Phase II to generate lead compounds for drug development. PUBLIC HEALTH RELEVANCE: This research is aimed at discovering new antibiotics effective against a common dangerous clinical bacterial pathogen, Pseudomonas aeruginosa. Cellular luminescent assays will be used to identify inhibitors of the essential fatty acid biosynthesis pathway. Inhibitors will be verified to act on this pathway by testing them in a biochemical assay of the entire fatty acid synthesis pathway.