Project Summary There is a worldwide need for improved treatment of systemic, life-threatening fungal infections. Current therapies are limited by the small number of approved drugs, toxicities, drug-drug interactions, mode of administration, and growing problems of drug resistance and emerging pathogens. Treatment also suffers from a lack of rapid clinical diagnoses, leading to dependence on broad-spectrum antifungal drugs. Moreover, existing antifungal drug classes target membrane and cell wall integrity, and there is a need to develop drugs against targets with new modes of action to minimize cross resistance. Available evidence suggests dihydrofolate reductase (DHFR) might be one such target. Commercial DHFR inhibitors are available for other therapeutic indications (immunology, oncology, antibacterial, antiparasitic). Bactrim® is the only available DHFR inhibitor for fungal infections but has a very narrow spectrum of activity (Pneumocystis jirovecii and Paracoccidioides spp.), despite DHFR being an essential enzyme that is highly conserved in fungal pathogens. As demonstrated in the current work, Candida spp., Aspergillus spp., Cryptococcus spp., rare molds, Mucorales and dermatophytes are susceptible to DHFR inhibition, and the DHFR mechanism of action is complementary to the targets of existing antifungals. Fungal DHFR inhibitors have been identified that inhibit the enzyme selectively and fungal cell growth in vitro, and fungal DHFRs differ in structure sufficiently from human DHFR indicating that development of selective DHFR drugs is feasible. The specific aims of this Fast Track proposal are to (1) reduce serum antagonism of our initial fungal DHFR inhibitors and demonstrate in vivo efficacy, (2) optimize a lead fungal DHFR compound or series to select a potential development candidate and (3) validate a fungal DHFR lead (or leads) and select a single candidate for IND-enabling studies. The approach will enable a robust lead optimization program to accomplish the aims and will include analog synthesis and computational chemistry, biochemical, structural, and modeling studies, whole-cell assays, in vivo models (i.e., efficacy, pharmacokinetic and pharmacodynamic) and in vitro ADME/Safety profiling to synthesize and test inhibitors against fungal and human DHFR enzymes, fungal pathogens, and mammalian cells. The outcomes will advance this program into IND-enabling studies (Phase IIB). The long-term objective is to develop fungal-selective DHFR inhibitors into a new class of broad-spectrum antifungal drugs.