Identification of Flavonoid-based Hypoxia-inducible Factor-2 Alpha Inhibitors for the Treatment of Breast Cancer– In silico and In vitro Evidence

Background: Breast cancer (BC) is a common malignancy that poses a serious threat to women's health. The hypoxic tumor microenvironment in BC promotes drug resistance, making hypoxia-targeted therapies crucial. Targeting hypoxia-inducible factors (HIFs), particularly HIF-2α, has emerged as a promising approach to inhibit tumor growth and improve response to chemotherapy and radiotherapy. However, further research is required to fully understand the role of HIF-2α to develop more effective treatments for BC. Aim: The aim of this study is to identify phytochemicals that target HIF-2α and evaluate their effects on the MCF-7 breast cancer cell line under hypoxic conditions. Methods: Molecular docking identified phytochemicals targeting HIF-2α, with high-affinity compounds undergoing stability evaluation via GROMACS molecular dynamics simulations. ADMET and toxicity assessments were performed using SwissADME and ProTox-3.0. In-vitro assays on hypoxic MCF-7 cells examined cell viability and gene expression. The expression of HIF-2α-regulated genes (VEGFA, CCND1, GLUT1) was analyzed by using qRT-PCR. Results: Molecular docking revealed that naringin (-8.2 Kcal/mol) and morin (-7.1 Kcal/mol) showed better binding affinity than the standard drug, belzutifan (-7.7 Kcal/mol). Dynamic simulations, including RMSD, RMSF, Hbond interactions, Rg, SASA, and PE, confirmed their strong binding potential. Morin, in particular, demonstrated more H-bond interactions and met Lipinski's Rule of Five, making it a promising candidate for in vitro studies. It reduced cell viability with an IC50 of 118 μM and significantly downregulated HIF-2α-associated genes. Conclusion: Morin demonstrated promising anti-cancer activity under hypoxic conditions by inhibiting HIF-2α in the hypoxia signaling pathway.