Discovery of a potential CERS2 inhibitor: hit compound identification via structure—based virtual screening and molecular dynamics simulations

Ceramides, central bioactive mediators of sphingolipid metabolism, critically regulate signal transduction and essential cellular processes. Among six ceramide synthases (CERS1-6), CERS2 preferentially synthesizes very-long-chain ceramides (>C22-24) and holds exceptional promise as a pharmacological target for modulating ceramide levels and composition, with therapeutic potential in metabolic disorders, neurodegenerative diseases, and cardiovascular pathologies. Inhibitors specifically targeting CERS2 remain an urgent unmet need for targeted therapeutics modulating ceramide subclasses. In this study, we firstly performed a multi-step virtual screen of over 10 million drug-like compounds from the ZINC20 database to identify hits against CERS2. Compounds were hierarchically ranked by Glide HTVS/SP/XP docking scores (thresholds: − 6.5/ − 7.0/ − 7.5 kcal/mol), MM-GBSA binding free energies, and ligand-based clustering. Next, the top two candidates were selected for molecular dynamics (MD) simulations, which revealed that Hit-325144 formed highly stable interactions with key catalytic residues His212 and His213 in the CERS2 active site, indicating strong binding affinity. Finally, experimental validation employing LC–MS/MS-based ceramide quantification and fluorescence-coupled enzymatic assays using recombinant human CERS2 protein confirmed Hit-325144’s dose-dependent inhibition of CERS2 activity. This study establishes a high-throughput, cost-effective virtual screening framework for CERS2 inhibitor discovery and provides a structurally validated lead compound Hit-325144 as a foundation for developing small-molecule therapeutics targeting CERS2-mediated ceramide dysregulation in associated diseases.