Integrated systems pharmacology reveals the multitarget therapeutic mechanisms of pancratistatin against cholangiocarcinoma

Cholangiocarcinoma (CCA) is a highly aggressive biliary tract malignancy with poor prognosis and limited therapeutic options. Pancratistatin, a bioactive Amaryllidaceae alkaloid, exhibits selective cytotoxicity toward cancer cells while largely sparing normal tissues. However, its molecular mechanisms against CCA remain unclear. This study aimed to elucidate the therapeutic targets and molecular mechanisms of pancratistatin against CCA using an integrated systems pharmacology approach. Potential targets of pancratistatin and CCA-associated genes were retrieved from public databases, and a protein-protein interaction network was constructed to identify key hub genes using five cytoHubba algorithms. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed, followed by gene expression, immune cell infiltration, and molecular docking analyses. Nine hub genes (HSP90AA1, CASP3, HIF1A, NFKB1, TLR4, PIK3R1, PRKACA, CDK1, and NOS2) were identified as central regulators of the therapeutic network. KEGG enrichment analysis highlighted significant involvement in the HIF-1 signaling pathway, PD-L1 expression and PD-1 checkpoint pathway in cancer, and central carbon metabolism in cancer. Most hub genes were significantly dysregulated in CCA tissues, and immune infiltration analysis supported their immunological relevance within the tumor microenvironment. Molecular docking demonstrated favorable binding affinities of pancratistatin toward multiple hub proteins, particularly HSP90AA1 (-8.11 kcal/mol), followed by PRKACA (-6.86 kcal/mol) and TLR4 (-6.30 kcal/mol). Collectively, these findings identify HSP90AA1 as a promising therapeutic target and provide mechanistic evidence supporting pancratistatin as a potential multitarget agent for cholangiocarcinoma.