Network pharmacology-guided identification and validation of ganoderic acids A and D from Ganoderma lucidum against esophageal squamous cell carcinoma.

BACKGROUND/OBJECTIVE: Esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy with unsatisfactory prognosis and ongoing therapeutic challenges. Ganoderma lucidum has been widely investigated for anticancer activity, but the specific monomeric ganoderic acids that may contribute to anti-ESCC effects and their potential target context remain insufficiently characterized. METHODS: Compounds and targets were prioritized through network pharmacology, ADME evaluation, disease-target integration and TCGA-based clinical bioinformatics. Molecular docking and 100-ns molecular dynamics (MD) simulations were used to evaluate putative ligand-target binding stability. The effects of ganoderic acid A (GA-A) and ganoderic acid D (GA-D) were assessed in ESCC and normal esophageal epithelial cells using image-based cell counting, EdU incorporation assays, flow cytometric cell-cycle and apoptosis analyses, and immunoblotting of mTORC1-associated readouts. RESULTS: Nine compounds and 191 drug-disease intersection targets were identified. GA-A and GA-D were prioritized as core ganoderic acid monomers based on network connectivity, pharmacological relevance and experimental feasibility. Enrichment analysis implicated cancer-related signaling, including PI3K-Akt/mTOR-associated pathways. Docking and MD simulations supported stable putative interactions between GA-A/GA-D and mTOR. In vitro, both compounds reduced cell number and EdU incorporation in ECSS cells, and induced G0/G1 accumulation and apoptosis, with GA-D showing stronger anti-proliferative activity than GA-A under the tested conditions. HEEC cells were less sensitive under the tested conditions. GA-A and GA-D also reduced phosphorylated S6 and 4EBP1 levels. CONCLUSION: This study identifies GA-A and GA-D, particularly GA-D, as candidate anti-proliferative monomers from G. lucidum against ESCC. The findings provide a focused, experimentally supported basis for further mechanistic evaluation of ganoderic acids, including target engagement assays and drug-treated transcriptomic profiling.