SUMMARY/ABSTRACT Candida albicans is the most common pathogen causing invasive fungal infections. These infections are dreaded complications of serious illnesses, and are estimated to lead to death in about 20% of patients. There are very few treatment options for fungal infections. This is because many cellular components whose function could be disrupted by drugs are similar between fungi and humans, leading to unacceptable toxicities to human cells. We have identified a potential drug target in C. albicans which has no human homologs, and whose inactivation leads to potentiation of drugs in 2 of the 3 major antifungal classes. This target is conserved among 3 phyla of pathogenic fungi, suggesting that small-molecule inhibitors could potentiate antifungal activity against other fungal pathogens. Because it is a cell-surface transporter, inhibitors of this target are not susceptible to a major multidrug-resistance mechanism of C. albicans, the induction of drug efflux pumps. Other features that render it impervious to development of resistance are that it is not an essential protein, diminishing the selective pressure during drug exposure, and that part of the mechanisms of action of its inhibitors may be its role in virulence activities of the fungus. Virulence factors like hyphal growth and oxidative stress resistance are not required during commensal growth, which comprises the majority of the C. albicans life cycle. We engineered a C. albicans strain in which inhibition of our target of interest induces GFP expression. This reporter strain is amenable to high-throughput screening using the HyperCyt platform developed at The University of New Mexico. We propose to screen library collections of chemically well-defined compounds with desirable physicochemical properties, and with existing data for other indications. Active compounds identified in the screen will be prioritized by their effect in potentiating the “gold standard” antifungal amphotericin, by their potency in blocking virulence factors (e.g. hyphal growth), as well as their toxicity window. In addition, prioritization will involve compound physicochemical properties, chemical tractability, and binding efficiencies using state-of-the-art post-screen triage approaches. Further validation tests will employ an engineered “test tube cell” Saccharomyces cerevisiae strain, in which the target is expressed at the plasma membrane as the only transporter of its substrate. Structure-activity relationships for chemotypes of prioritized hits will be established early. Hit-to-lead medicinal chemistry will be performed on prioritized hits with a focus on low host cell toxicity and physicochemical properties that support the advancement of compounds into in vivo pharmacokinetics and efficacy experiments. This project will provide high-quality lead compounds for further development as an antifungal potentiator, and potentially as a virulence-modifying single agent.