The Trisubstituted Isoxazole MMV688766 Exerts Broad-Spectrum Activity against Drug-Resistant Fungal Pathogens through Inhibition of Lipid Homeostasis

Candida species are among the most prevalent causes of systemic fungal infection, posing a growing threat to public health. While Candida albicans is the most common etiological agent of systemic candidiasis, the frequency of infections caused by non-albicans Candida species is rising. Among these is Candida auris, which has emerged as a particular concern. Since its initial discovery in 2009, it has been identified worldwide and exhibits resistance to all three principal antifungal classes. Here, we endeavored to identify compounds with novel bioactivity against C. auris from the Medicines for Malaria Venture’s Pathogen Box library. Of the five hits identified, the trisubstituted isoxazole MMV688766 emerged as the only compound displaying potent fungicidal activity against C. auris, as well as other evolutionarily divergent fungal pathogens. Chemogenomic profiling, as well as subsequent metabolomic and phenotypic analyses, revealed that MMV688766 disrupts cellular lipid homeostasis, driving a decrease in levels of early sphingolipid intermediates and fatty acids and a concomitant increase in lysophospholipids. Experimental evolution to further probe MMV688766’s mode of action in the model fungus Saccharomyces cerevisiae revealed that loss of function of the transcriptional regulator HAL9 confers resistance to MMV688766, in part through the upregulation of the lipid-binding chaperone HSP12, a response that appears to assist in tolerating MMV688766-induced stress. The novel mode of action we have uncovered for MMV688766 against drug-resistant fungal pathogens highlights the broad utility of targeting lipid homeostasis to disrupt fungal growth and how screening structurally-diverse chemical libraries can provide new insights into resistance-conferring stress responses of fungi.