Antibiotic-resistant Gram-negative infections pose a major threat to human health. A defining feature of Gram-negative organisms is the presence of a second membrane, the outer membrane (OM), which regulates access of molecules to the periplasm. The OM is the reason that antibiotics that are effective against Gram-positive organisms, such as vancomycin, are not effective against Gram-negatives even though Gram-negatives contain the same targets. The OM is composed of an asymmetric bilayer containing phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. LPS on the cell surface creates a polyelectrolyte mesh that acts as a formidable barrier to passage of both hydrophilic and hydrophobic molecules. Preventing proper LPS biosynthesis and assembly is often lethal since LPS is essential in most Gram-negative organisms. Those organisms that are viable in the presence of LPS assembly inhibitors have OM defects that render them sensitive to antibiotics that cannot normally penetrate the OM barrier. In this grant, we propose to develop a comprehensive approach involving both target- and cell-based screens to identify small molecule inhibitors of OM biogenesis in Pseudomonas aeruginosa and Acinetobacter baumannii, two opportunistic pathogens for which multi-drug resistance is rampant. Aim 1 will use a target-based screen to identify inhibitors of LptB, the essential ATPase that powers the transfer of LPS from the inner membrane to proteins that translocate it to the OM. Aim 2 will use cell-based reporter assays to identify inhibitors of OM biogenesis in P. aeruginosa. Aim 3 will exploit the conditional essentiality of late stage enzymes involved in OM biogenesis in A. baumannii to develop a cell-based, pathway-speciflc screen to discover small-molecule inhibitors of LPS biogenesis. A novel fluorescence-based assay that reports on properly assembled LPS on the cell surface will be used to show that inhibitors found in the pathway-specific screen lead to defects in LPS assembly. We will validate that the hit compounds found in all aims are on target using novel biochemical and microbiological approaches developed in our labs. The most promising hit compounds will be subjected to optimization and in vivo efficacy studies in collaboration with the Discovery and Translational Services (DTS) Core. Using this combination of target- and cell-based screens we hope to identify new antibiotics to treat Gram-negative infections as well as compounds that potentiate clinically used antibiotics by rendering the OM leaky.