PROJECT SUMMARY The sodium-dependent NADH: ubiquinone oxidoreductase (Na+-NQR) is the main ion transporter in hundreds of pathogenic bacteria, including Vibrio cholerae, the causal agent of cholera, a devastating gastrointestinal disease with a worldwide distribution that has developed multidrug-resistant phenotypes. Na+- NQR fulfills two essential roles in V. cholerae cell physiology, as a respiratory enzyme, providing energy to the cell, and as the main sodium pump, energizing the membrane and driving nutrient uptake, pH regulation, elimination of drugs, cell motility, secretion of toxins and other homeostatic processes. Na+-NQR is an optimal drug target due to its critical role in bacterial metabolism and because it is absent in mammalian cells. Moreover, Na+-NQR has unique structural motifs, not found in any human protein, which allow the discovery of drugs that can act specifically on this enzyme. In addition, Na+-NQR inhibitors could increase the susceptibility of V. cholerae to other drugs by de-energizing the membrane, and may be used in a combination dosing approach to rescue obsolete antibiotics. Our group has now identified two novel compound leads, ubiquinone analogs (UQAs) and phenothiazines, as inhibitors of this enzyme that are suitable for drug development. The three UQAs analogs characterized have antimalarial properties and show specific and potent inhibitory effects on Na+-NQR, with strong antibiotic activity against V. cholerae. These compounds not only abolish V. cholerae Na+-NQR enzymatic activity, but also trigger the overproduction of reactive oxygen species, which is lethal to microbes. The structures of these inhibitors and docking methods were used to identify the pharmacophore and the binding modes of the molecules in the UQ binding site, which allow us to pursue lead development to obtain inhibitors of high potency and specificity. In addition, we have identified three phenothiazine-like compounds with anti- psychotic properties that show potent inhibitory activity against Na+-NQR and that could be optimized into antibiotics. The main aim of this project is the development of a novel class of antibiotics to specifically target the Na+-NQR complex. The inhibitors that we have identified will be fully characterized, to understand their mechanism of action, binding sites, potency and antibiotic properties. Moreover, toxicity towards human cells and mitochondria, as well as their pharmacologic properties, will be assessed to evaluate the potential of these compounds to treat human infections. The data obtained from toxicity studies, enzymatic and microbiological characterizations will be used to guide the design and synthesis of analogs with high potency and low toxicity. Lead optimization will be carried out by our medicinal chemistry team guided by pharmacophore analysis, docking and binding free energy calculations. The structures of the newly-identified inhibitors will be used to build compound libraries carrying the active core with different substitution patterns, which will be iteratively screened and characterized. The data generated in this proposal is critical to the discovery of urgently-needed antibiotics with a new mechanism of action effective against V. cholerae and many other Na+-NQR bearing pathogens.

PROJECT NARRATIVE This project aims to develop a new class of antibiotics targeted against Vibrio cholerae’s respiratory complex Na+-NQR. This enzyme is essential for several bacterial pathogens and it is absent in the human cell, which makes it an ideal drug target.