A Multifaceted Mechanistic Approach to Assess the Inhibitory Potential of Broccoli-Derived Glucosinolates Against Tumor-Associated Carbonic Anhydrase IX

Abstract

Hypoxia-induced carbonic anhydrase IX (CA IX) is a clinically validated anticancer target; yet, few natural, isoform-selective inhibitors have been described. We isolated three glucosinolates—glucoraphanin, 1,4-dimethoxyglucobrassicin, and 4-methoxyglucobrassicin—from Brassica oleracea and interrogated their CA IX inhibitory potential through an integrated experimental-computational workflow. Enzyme assays revealed mixed-type inhibition with nanomolar potency (IC₅₀ = 65–221 nM), while counterscreens against housekeeping CA I/II demonstrated > 40-fold selectivity. Molecular docking, 500-ns molecular-dynamics simulations, free-energy-landscape mapping, and MM/PBSA calculations consistently ranked the ligands as 4-methoxyglucobrassicin > glucoraphanin > 1,4-dimethoxyglucobrassicin, attributing superior binding to deep hydrophobic insertion, persistent hydrogen bonding, and favorable van der Waals and electrostatic contributions (ΔG_MM/PBSA = −25 kJ mol⁻¹). In silico ADMET profiling indicated low hERG and CYP liabilities but flagged limited oral absorption and moderate hepatotoxicity, suggesting the need for targeted delivery or scaffold optimization. Collectively, these findings position 4-methoxyglucobrassicin as a promising natural-product lead for selective CA IX inhibition and provide a mechanistic framework for structure-guided analog design.