Impact of glycosylation position on flavonoid isomers' inhibition of breast cancer-associated enzyme CYP1B1.

Isomerism in natural flavonoids critically influences enzyme inhibitory activity, particularly toward cytochrome P450 enzymes. Mangiferin and isomangiferin, two C-glycosyl flavonoid isomers differing only in glycosidic substitution position on the xanthone skeleton, serve as ideal model compounds for probing structure-activity relationships against CYP1B1, a breast cancer-associated enzyme involved in carcinogen activation. However, whether such glycosidic positional isomerism affects CYP1B1 inhibition and binding behavior remains unknown. Here, the inhibitory effects and molecular mechanisms of mangiferin and isomangiferin toward CYP1B1 were systematically investigated using enzyme kinetics, quantum chemical calculations, molecular docking, and machine learning-based ADMET prediction. Kinetic analysis revealed that mangiferin inhibited CYP1B1 over 100-fold more potently than isomangiferin, exhibiting mixed-type inhibition, whereas isomangiferin showed non-competitive behavior. Quantum chemical calculations demonstrated that altered glycosidic positions shifted HOMO-LUMO energy distributions and electron density, affecting intermolecular interactions. Molecular docking identified key residues responsible for stronger mangiferin binding. ADMET prediction indicated favorable pharmacokinetics and low toxicity for both compounds. These results demonstrate that subtle changes in glycosidic substitution position can profoundly influence CYP1B1 inhibitory potency and binding mode of mangiferin and isomangiferin, providing mechanistic insight into structure-activity relationships of such isomers.