PROJECT SUMMARY/ABSTRACT Candida albicans is the most frequently isolated fungus causing invasive disease in the United States. These infections are dreaded complications of serious illnesses, especially in hospitalized or immunocompromised patients. C. albicans infections are challenging to eradicate, and are still estimated to lead to death in 20% of the affected patients. Currently only 3 classes of antifungal drugs are available to treat invasive fungal infections. Antifungal drug development is difficult because of the similarity between fungal and human cells, which leads to unacceptable toxicity of many compounds that damage or kill fungi. Developing antifungal agents whose targets are absent in human cells would circumvent this difficulty. Increasing potency of antifungal drugs could also lead to better outcomes. A paradigm for increased antimicrobial potency is the important combination antibiotic Cotrimoxazole, whose two components target sequential steps in the biosynthesis of tetrahydrofolate. The goal of this proposal is to find compounds that can be developed into specific inhibitors of two cellular processes unique to fungi, whose combination could give rise to a more potent antifungal agent. Two fungal cellular processes that are fundamentally different or absent in humans are phosphate homeostasis and cell wall construction. We previously found that the major C. albicans high-affinity phosphate transporter Pho84 is required for normal nutritional (Target of Rapamycin-) signaling, cell wall stress- and oxidative stress resistance, hyphal growth and virulence. Since humans manage their phosphate balance completely differently from fungi, blocking Pho84 is not predicted to impact human cellular functions. Pho84 is highly conserved across the fungal kingdom, including in emerging pathogens like Candida auris. Cells that lack Pho84 contain diminished concentrations of nucleotides, whose production requires ample intracellular phosphate supplies, and of their downstream metabolites, nucleotide sugars. Nucleotide sugars are precursors for biosynthesis of the major cell wall polymers. We propose to take advantage of this defect to sequentially perturb major steps in cell wall biosynthesis by combining inhibition of Pho84 with inhibition of glucan biosynthesis. To do this, we established a novel high-throughput assay system to detect specific inhibitors of these fungal targets. We will prioritize hit compounds according to their biological effects in virulence-associated or essential cellular processes, and according to their chemical features. This work will lay the ground to apply the paradigm of stepwise inhibition of a critical biosynthetic process to antifungal drug development. The proposal is intended to select screen hits that meet defined biological and medicinal chemistry criteria for further development.