Targeting ceramide synthases for the development of new antifungals.

Invasive fungal infections (IFIs) caused by pathogenic fungi are a major public health concern, particularly across various immunocompromised populations. Effective clinical management is currently hindered by limited treatment options. Fungal sphingolipids have emerged as potential antifungal targets based on cumulative evidence demonstrating that fungal sphingolipid metabolism is key to the virulence of pathogenic fungi. This study focuses on the sphingolipid metabolizing enzyme ceramide synthase. We developed an enzymatic assay to examine ceramide synthase activity and devised a high-throughput screening platform. Two synthetic compounds were identified that preferentially inhibit the fungal vs. the mammalian ceramide synthase activity. Further studies indicate that these compounds block fungal growth, with in silico and mutagenesis investigations revealing insights into the interactions between the inhibitors and the ceramide synthase active site. Together, our study establishes fungal ceramide synthase as a promising antifungal target and paves the way for new structure-activity relationship studies leveraging fungal sphingolipid metabolism.