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.

Drug-induced liver injury (DILI) remains a serious problem in clinics for both diagnoses and treatment decisions. It is a result of accumulated drugs in human bodies metabolized into toxic constituents generating reactive metabolites, and then arise initial consequences of oxidative stress, organelle stress responses, and lethal consequences (liver necrosis or apoptosis). However, the idiosyncratic nature of DILI complexes its mechanistic studies and still little is known of its potential etiopathogenesis for certain. Single-cell omics technology and approaches serve as powerful tools for investigating cellular heterogeneity and relationships from measurements of up to millions of individual cells at an unprecedented resolution, which are achieved by advances in genome, epigenome, transcriptome, proteome, and metabolism technologies. As liver contains heterogeneous cell types of distinct spatial, molecular, and functional properties, they interact with each other to precede cell type-specific omics reprogramming and play an irreplaceable role in liver cells with heterogeneous properties upon encountering toxic insults. Single-cell omics, especially single-cell transcriptomics and single-cell proteomics, have been utilized for exploring the mechanisms of DILI and prediction for risk factors. In this review, we discuss the recent development and future perspectives of single-cell omics-based technologies for DILI-related research.