PROJECT SUMMARY Antibiotics represent the most successful class of drugs developed by modern science. They have spurred numerous medical advances by facilitating invasive surgeries with minimal risk of infection. Today, a major healthcare crisis looms as we are fast approaching a post-antibiotic era. Historically, it has proven to be much more difficult to find agents that are active against Gram-negative pathogens compared to Gram-positive pathogens. The primary reason is that Gram-negative bacteria have a unique asymmetric outer membrane (OM) in addition to an inner membrane. The targets for most antibiotics reside beyond the OM, and thus these molecules need to penetrate the OM to be active. Yet, the OM is uniquely effective in blocking the translocation of small molecules, thus creating a major challenge for the field. The Golden Era of antibiotics leveraged naturally abundant small molecules that were readily identified using traditional methods; however, this methodology has proven to be much more difficult to be further mined for new antibiotics during the past several decades. The next phase of antibiotic drug discovery has the potential to be supported by our increasing collection of proteomics, genomics, and metabolomics data that will reveal promising drug targets. Academia and industry could potentially exploit these data sets to design small molecule agents that are potent and of high specificity. To accomplish this, the field fundamentally requires guiding principles describing the molecular determinants of permeation into bacterial cells akin to the Lipinski’s rules of 5 (Ro5). We propose to develop a novel fluorescence assay that measures the accumulation of small molecules in Gram- negative pathogens based on a combination of HaloTag expression and anchoring of a biorthogonal epitope within HaloTag. Our team will systematically (testing established antibiotics with known permeation profiles) and broadly (screening a unique library of small molecules modified with an azide tag) apply this approach to measurably grow our fundamental understanding of the molecular determinants of permeation. Additionally, we will utilize the same platform to provide unprecedented spatial resolution of the distribution of small molecules (including known antibiotics) in subcellular compartments.