Protocol for screening inhibitors of the fungal sphingolipid-metabolizing enzyme ceramide synthase

Fungal sphingolipids have emerged as potent antifungal targets based on cumulative evidence demonstrating that fungal sphingolipid metabolism is essential to the virulence of pathogenic fungi. Here, we present a protocol to screen for inhibitors of the sphingolipid-metabolizing enzyme ceramide synthase. We describe steps for harvesting microsomes from Saccharomyces cerevisiae, performing in vitro reactions, and performing lipid extractions. We then detail the use of a high-performance liquid chromatography (HPLC)-based fluorometric assay to screen large chemical libraries for ceramide synthase inhibitors. For complete details on the use and execution of this protocol, please refer to Dasilva et al.¹.