Network pharmacology and experimental validation for deciphering the action mechanism of D. Don constituents in suppressing breast carcinoma.

D. Don is a well-known medicinal plant of Kashmir Himalaya. Traditionally, it has been used to treat several diseases, including cancer. However, the molecular mechanism behind anticancer activity remains unclear. Therefore, in the present study, we have performed high performance-liquid chromatography-mass spectrometry (HR-LC/MS), network pharmacology, molecular docking and molecular dynamic (MD) simulation methods were used to explore the underlying molecular mechanism of for the treatment of breast cancer (BC). The targets of for treating BC were predicted using databases like SwissTargetPrediction, Gene Cards and OMIM. Protein-protein interaction analysis and network construction were performed using the Search Tool for the Retrieval of Interacting Genes/Proteins programme, and analysis of Gene Ontology term enrichment and Kyoto Encyclopedia of Genes and Genomes pathway enrichment was done using the Cytoscape programme. In addition, molecular docking was used to investigate intermolecular interactions between the compounds and the proteins using the Autodock tool. MD simulations studies were also used to explore the stability of the representative AKT1 gene peiminine and Imperialine-3-β-glucoside. In addition, experimental treatment of was also verified. HR-LC/MS detected the presence of several secondary metabolites. Afterward, molecular docking was used to verify the effective activity of the active ingredients against the prospective targets. Additionally, Peiminine and Imperialine-3-β-glucoside showed the highest binding energy score against AKT-1 (-12.99 kcal/mol and -12.08 kcal/mol). AKT1 with Peiminine and Imperialine-3-β-glucoside was further explored for MD simulations. During the MD simulation study at 100 nanoseconds, a stable complex formation of AKT1 + Peiminine and Imperialine-3-β-glucoside was observed. The binding free energy calculations using MM/GBSA showed significant binding of the ligand with protein (Δ: -79.83 ± 3.0 kcal/mol) between AKT1 + Peiminine was observed. The principal component analysis exhibited a stable converged structure by achieving global motion. Lastly, extracts also exhibited momentous anticancer activity through studies. Therefore, present study revealed the molecular mechanism of constituents for the effective treatment of BC by deactivating various multiple gene targets, multiple pathways particularly the PI3K-Akt signaling pathway. These findings emphasized the momentous anti-BC activity of constituents.Communicated by Ramaswamy H. Sarma.