Project Summary Multi-drug resistant infections are a major threat to global health and resistance has been observed for every clinically-used antibiotic, even those considered to be the last lines of treatment. Further compounding the antimicrobial resistance crisis is the lack of new antibiotics entering the pipeline, particularly for Gram- negative pathogens which have an impermeable outer membrane, limiting small molecule accumulation, and promiscuous efflux pumps, which can recognize and expel most small molecules from the cell. A basic-science understanding of favorable chemical properties required to enhance compound accumulation and decrease efflux propensity is needed to develop antibacterial candidates with whole-cell activity against Gram-negative pathogens. Initial efforts in the Hergenrother lab have identified the physicochemical traits needed for compound permeation in E. coli and successfully applied these guidelines to convert several Gram-positive only antibiotics to broad-spectrum agents. While this strategy improves Gram-negative antibacterial activity, compound efflux is still detrimental to efficacy and prevents development of these leads into potent antibiotics. It is imperative to understand the physicochemical properties governing compound recognition and efflux to provide a novel design platform to engineer efflux susceptibility out of drug candidates. The objective of this proposal is to identify the parameters that define compound efflux in Gram-negative bacteria and apply these findings to remove efflux liability out of promising antibacterial candidates. Work proposed herein will build upon preliminary studies of the efflux propensity of ~200 compounds utilizing a novel LC-MS/MS-based accumulation (Efflux Propensity EvaLuation (EXPEL)) assay which can detect small changes in efflux susceptibilities irrespective of antibacterial activity and a chemoinformatic model which can accurately classify 50% of compounds as efflux substrates and non-substrates. In Specific Aim 1, additional physicochemical properties determined important for compound efflux by the random forest model will be probed through synthesis of a targeted library of side-by-side comparisons. These compounds will be added to the dataset and iterative cycles of compound synthesis, EXPEL assay, and chemoinformatic model validation will be performed. Utilizing the EXPEL assay and the initial properties identified as correlating to efflux ratios, derivatives of an exciting FabI inhibitor will be explored to identify promising antibacterials with decreased efflux liabilities in Specific Aim 2. The therapeutic potential of these compounds will be explored through toxicity studies, determination of pharmacokinetic profile, and evaluation of efficacy in mouse infection models. Specific Aims 1 and 2 will run concurrently and completion of these studies will significantly impact antibacterial research and remedy attrition points in the antibacterial clinical pip...