Computational discovery and validation of 4'-O-methylochnaflavone as a novel HSP90AB1 inhibitor for hepatocellular carcinoma treatment.

Hepatocellular carcinoma (HCC) is a global health issue with limited therapeutic efficacy. Heat shock proteins (HSPs) could enhance adaptation to the oxygen-deprived tumor microenvironment (TME), leading to chemotherapy resistance. Regulating HSP to improve the immune microenvironment may enhance tumor treatment efficacy. However, pharmacological agents targeting HSPs to reverse the immunosuppressive TME remain scarce. In this study, we integrated bioinformatic analysis, virtual screening, molecular docking, molecular dynamics (MD) simulations, and experimental validation to identify therapeutic targets and potential natural product-derived inhibitors. Differentially expressed hypoxia-related immune genes (HRIGs) associated with HCC prognosis were first identified using bioinformatic analysis. Venn diagram revealed HSP90AB1 as the candidate target protein. Virtual screening of the TargetMol and MedChemExpress (MCE) compound libraries was performed, and preliminary molecular docking identified 4'-O-methylochnaflavone as the lead compound. The interaction between 4'-O-methylochnaflavone and HSP90AB1 was further evaluated by MD simulations and experimental validation. Surface plasmon resonance (SPR) assay demonstrated that 4'-O-methylochnaflavone could stably bind HSP90AB1. In vitro CCK-8 assays demonstrated that 4'-O-methylochnaflavone inhibited Hep3B cell proliferation in a time- and dose-dependent manner. Finally, Western blot analysis confirmed hypoxia-induced upregulation of HSP90AB1, which was significantly suppressed by 4'-O-methylochnaflavone. In conclusion, our study identifies 4'-O-methylochnaflavone is a promising HSP90AB1-targeting therapeutic candidate for HCC treatment.