Project Summary The long-term goals of this project are to uncover molecular mechanisms of antibiotic resistance and to design inhibitors as therapeutics against disease-causing bacteria. Widespread antibiotic resistance among hospital and community associated bacteria, coupled with the lack of new antibiotics, poses an urgent threat to global public health. For these reasons, antibiotic resistance is the silent pandemic, which has been mounting since the introduction of antibiotics in the clinic. The situation is urgent for several pathogens and will become more acute for other pathogenic bacteria in the coming years. There is no magic bullet solution to solve the antibiotic resistance problem; rather, a concerted effort is required that includes new antibiotics, tackling resistance mechanisms head-on, and better stewardship of antibiotics and antiseptics. Bacteria possess several mechanisms to confer resistance to the host organism, including modification of the antibiotic, mutation to the target site, reduced uptake of antibiotics, and the presence of membrane protein efflux pumps. This proposal is aimed at understanding molecular mechanisms of efflux, which is a first-line resistance mechanism used by bacteria to lower the intracellular drug concentration, thereby facilitating pathogen survival. Inhibitors of efflux pumps have therapeutic potential as antibiotic adjuvants by directly targeting a central mechanism of resistance in pathogenic bacteria. However, the lack of clinically approved efflux pump inhibitors underscores a key challenge in the field: how to design specific inhibitors of a substrate binding pocket that promiscuously binds structurally diverse drugs? We recently uncovered a promising solution to this problem through the discovery of antibody inhibitors to B. subtilis Bmr and S. aureus NorA where a loop inserts into the substrate binding pocket. We hypothesize the substrate binding pockets of transporters can be systematically targeted for inhibition by peptide mimics of the antibody paratope. By using efflux pumps within the Bacillus genus, Bmr and Blt, this proposal will probe how efflux pumps recognize multiple substrates (Aim 1), will reveal cryptic pockets within the substrate binding pocket of efflux pumps by using antibodies as tools (Aim 2), and develop a systematic approach for creating conformationally defined mimics of the antibody b-hairpin paratope (Aim 3). Since Bmr and Blt share ~80% sequence homology yet display different substrate specificities, this system serves as a good comparative group for determining whether transporter-selective inhibitors can be designed. The approaches developed in this grant will establish the long-term viability of novel efflux pump inhibitors to the treatment of antibiotic resistant organisms.