Integrated systems pharmacology-based exploration of sanguinarine: Comprehensive metabolite identification, enzyme...

Sanguinarine, a natural benzophenanthridine alkaloid with promising anticancer potential, has an incompletely defined metabolic and mechanistic landscape. Here, we present an integrated study that systematically maps its metabolism and explores the systemic pharmacological implications of its metabolites. Using a ²H-isotope-labeling LC-MS/MS strategy, we identified 40 metabolites in microsomal incubations (12 Phase I and 28 Phase II), 31 of which are newly reported. Recombinant enzyme phenotyping revealed CYP1A2, 2C19, 3A4 and 2D6 as the principal Phase I enzymes, while UGT1A1 and UGT1B4 dominated Phase II glucuronidation. Molecular docking simulations confirmed stable binding modes between sanguinarine and these key CYP450 enzymes, supporting their direct interactions. Beyond metabolic profiling, network pharmacology analysis predicted that the major metabolites collectively target a protein network centered on hubs such as PIK3CA and SRC. This network was significantly enriched in key oncogenic and regulatory pathways, most prominently "Pathways in cancer" and "Chemical carcinogenesis - receptor activation", along with "positive regulation of MAPK cascade" and "Aryl hydrocarbon receptor pathway". Collectively, this work provides a comprehensive metabolic atlas of sanguinarine, pinpoints its key metabolizing enzymes, and proposes a multi-target, multi-pathway systems pharmacology framework for its polypharmacology, offering a robust foundation for future development.