Leveraging the MMV Pathogen Box to Engineer an Antifungal Compound with Improved Efficacy and Selectivity against .

Fungal infections pose a significant and increasing threat to human health, but the current arsenal of antifungal drugs is inadequate. We screened the Medicines for Malaria Venture (MMV) Pathogen Box for new antifungal agents against three of the most critical species (, , and ). Of the 14 identified hit compounds, most were active against and . We selected the pyrazolo-pyrimidine MMV022478 for chemical modifications to build structure-activity relationships and study their antifungal properties. Two analogues, and , with distinct fluorine substitutions, greatly improved the efficacy against and inhibited fungal replication inside immune cells. Additionally, analogue had improved selectivity toward fungal killing compared to mammalian cytotoxicity. Evolution experiments generating MMV022478-resistant isolates revealed a change in morphology from oblong to round cells. Most notably, the resistant isolates blocked the uptake of the fluorescent dye rhodamine 6G and showed reduced susceptibility toward fluconazole, indicative of structural changes in the yeast cell surface. In summary, our study identified a promising antifungal compound with activity against high-priority fungal pathogens. Additionally, we demonstrated how structure-activity relationship studies of known and publicly available compounds can expand the repertoire of molecules with antifungal efficacy and reduced cytotoxicity to drive the development of novel therapeutics.