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DESCRIPTION: 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. Related clinically important Gram-negative [Gr(-)] nonfermenters include species of the genera Burkholderia, Acinetobacter, and Stenotrophomonas, which are prevalent in severe nosocomial infections, such as pneumonia, bacteremia and urinary tract infections. Multidrug resistance is increasing among Gr(-) nonfermenters, and as a last resort, polymyxins such as colistin have been revived for use against these infections. The discovery of new chemical entities that are not subject to existing target- based resistance mechanisms is an important strategy to address this unmet need, and screening for inhibitors of new or under-exploited targets is a useful approach. The overall goal of this research is discover a novel class of drugs that target the bacterial isoprenoidbiosynthetic pathway for therapy of Gr(-) nonfermenter infections. Isoprenoids are essential for electron transport and cell wall biosynthesis in bacteria. Many bacterial species, including the Gr(-) nonfermenters, utilize an alternate isoprenoid synthesis pathway, the 2-C- methyl-D-erythritol 4-phosphate (MEP) pathway, which is quite distinct from that found in humans, the mevalonate (MVA) pathway, and provides a high likelihood of selectivity for inhibitors. While screens for MEP inhibitors have been reported, this remains an underexploited pathway, in part because availability of substrates for research is quite limited and because most previous strategies have relied on biochemical enzymatic screens, yielding inhibitors with poor cellular activity. Thenovel approach of this study is to screen for P. aeruginosa MEP pathway inhibitors with a sensitive cellular bioluminescent reporter assay and to profile resulting hits with biochemical assays to validate compounds as specific MEP inhibitors and to identify the precise targets. This is feasible because of the experience of the team with building and utilizing these types of screens and because of the unique access of the team to synthesized substrates of each of the seven enzymatic reactions in the pathway. In Phase I, we will develop and apply a P. aeruginosa MEP pathway cellular reporter screen to a diverse library of gt300,000 discrete small molecules. Hits will be confirmed in the screening assay and evaluated for specificity for the MEP pathway by differential growth inhibition of strains carrying MEP vs. MEP+MVA pathway enzymes. The specific targeted MEP reaction will be identified for pathway-validated non-cytotoxic hits in biochemical assays utilizing bacterial cytosol and radiolabeled substrates foreach enzymatic step. Inhibitors will be evaluated for potency, mode of inhibition and spectrum in biochemical and MIC assays and prioritized by their antibacterial spectrum. In Phase II, we will examine analogs of the highest priority hits and chemicallyoptimize the most promising of these structures to develop lead compounds for efficacy and toxicity testing in animal models. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: This research is aimed at discovering new antibiotics against pathogenic Pseudomonas aeruginosa and related Gram-negative species. New drugs that inhibit the bacterial-specific isoprenoid biosynthetic pathway are unlikely to be subject to existig resistance mechanisms or to exhibit target-related toxicity. These new inhibitors may be developed into vital new antibiotics to combat drug-resistant bacteria and improve therapy.