Artesunate directly targets glucosylceramidase to suppress hepatocellular carcinoma proliferation and trigger apoptosis

Our previous preclinical study determined artesunate as a candidate drug for hepatocellular carcinoma (HCC) and identified glucosylceramidase (GBA) as one of its direct targets. This research aimed to identify the binding sites of GBA with artesunate and the potential anti-HCC mechanisms, which remain unclear. Artesunate effectively suppressed cell viability and proliferation, and enhanced apoptosis of HCC cell lines with more sensitivity in HepG2 than MHCC-97H cells. Network calculation and a series of in vivo and in vitro experimental data demonstrated that the apoptosis-related GBA-ceramide-CTSD-BID-BAX signaling was one of the key putative target pathways by which artesunate may inhibit the malignant progression of HCC. Furthermore, through integrated computational and experimental approaches, we identified Y313, E340, and N396 as critical binding residues within the GBA active site. Mutagenesis studies revealed that these residues were indispensable for the interaction, with E340R and N396R mutations exhibiting the most pronounced impairment in binding affinity and enzymatic activity, respectively. Crucially, disrupting this binding interface abolished artesunate’s ability to modulate the downstream apoptotic pathway. Our findings provide the first structural and mechanistic elucidation of artesunate’s target engagement with GBA, unveiling a specific signaling cascade for its anti-HCC activity and establishing a foundational framework for developing novel GBA-targeted therapies.