Multi-drug resistance is one of the most pressing issues in treating bacterial infections. Antibiotics areextruded from cells and cannot reach high enough intracellular concentrations to exert a therapeutic effect.This problem is most formidable with Gram-negative (Gram (-)) bacteria, due to their double-membranestructure. While efforts have focused on targeting one efflux pump at a time, resistance mutations can quicklydevelop. We propose to target the m1G37-tRNA methylation catalyzed by TrmD to inhibit protein synthesis ofmultiple pumps simultaneously, thus reducing drug efflux and accelerating bactericidal action. TrmD is abacteria-specific S-adenosyl-methionine (AdoMet)-dependent methyl transferase that controls the accuracy ofprotein-synthesis reading frame. Loss of TrmD increases +1 frameshifts and terminates protein synthesisprematurely. We have discovered that genes for multiple membrane proteins and efflux pumps in E. coli andother Gram (-) bacteria contain TrmD-dependent codons near the start of the reading frame. We hypothesizethat targeting TrmD will reduce protein synthesis of all of these genes. By reducing multiple membrane andefflux proteins at once, we propose that targeting TrmD offers a novel solution to an unmet medical need.While AstraZeneca (AZ) has attempted to target TrmD, progress has stalled, because isolated inhibitors lackedboth the selectivity against the human counterpart (Trm5) and the activity against bacterial growth. Wehypothesize that successful targeting must explore novel chemical space and diversity to capture the uniqueconformation of AdoMet when bound to TrmD. To test this hypothesis, our multi-PI team will use E. coli TrmD(EcTrmD) as a model and apply a series of high-throughput screening (HTS) assays, each unique to ourteam, to isolate potent and selective inhibitors. In Aim 1, we will use an enzyme-based fluorescence assay toisolate active inhibitors of EcTrmD. This fluorescence assay is HTS-ready, has all of the required reagents inhand, and exhibits advantages over the radioactivity-based (3H-AdoMet) assay. We will screen the collection of~370,000 compounds in the NCATS SMR (small molecular repository) at Sanford Burnham Prebys (SBP) andwill apply human Trm5 in a counter screen to remove non-selective compounds. In Aim 2, we will usecheminformatics to prioritize hits. We will assess hits in a multitude of secondary assays to determine theirinhibition potency and modality. In Aim 3, we will screen hits with our whole-cell assays to isolate compoundsthat inhibit cell growth and display phenotypes specific to TrmD deficiency, including reduced drug efflux. Wewill assess the structure-activity relationship of each hit by analysis of ~20 analogs from commercial vendorsand determine the binding modality using a computer-aided approach based on our ternary TrmD crystalstructure in complex with a bound tRNA and sinefungin (a non-reactive analog of AdoMet). We will determinehits for specificity of targeting EcTrmD inside E. coli cells. These hits will serve as powerful chemical probes ina new paradigm of antibiotic discovery that inhibits Gram (-) bacterial drug efflux by targeting TrmD.