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<p>Cruciferous vegetables such as turnip (<i>Brassica rapa</i>) are rich in bioactive glucosinolates with potential health-promoting and anticancer properties. Here, 4-methoxyglucobrassicin, glucobrassicin and neoglucobrassicin were purified from hot-methanol extracts of <i>B. rapa</i> roots by acidic-alumina and Sephadex LH-20 chromatography. Recombinant human carbonic anhydrase IX (CA IX) esterase activity was measured with a 4-nitrophenyl-acetate microplate assay, and inhibition constants were extracted from Lineweaver–Burk plots. Protein–ligand interactions were investigated by AutoDock Vina docking (PDB 5FL4), followed by 200 ns CHARMM36m molecular-dynamics simulations in GROMACS; binding free energies were obtained with the MM/PBSA method, and conformational stability was evaluated through free-energy-landscape analysis. In vitro enzyme-inhibition assays revealed that neoglucobrassicin and glucobrassicin exhibited potent CA IX inhibition, with IC<sub>50</sub> values of 48.5 ± 2.1 nM and 65.06 ± 2.8 nM, respectively. Kinetic studies indicated a mixed-type inhibition mechanism, suggesting multiple interaction modes with the enzyme. Molecular docking confirmed strong polar interactions within the CA IX active site, and MD/MM-PBSA ranked neoglucobrassicin as the most favorable binder (ΔG<sub>total</sub> = − 34.74 ± 2.47&#xa0;kcal mol⁻¹). Free-energy-landscape analysis further showed that neoglucobrassicin maintains a highly stable conformational state during the simulation. Overall, this integrated isolation, enzyme-assay, and computational workflow identifies turnip-derived indole glucosinolates as promising natural CA IX inhibitors and supports their potential role in functional foods and nutraceuticals aimed at cancer prevention and tumor-micro-environment modulation.</p>

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Glucosinolate-Mediated Inhibition of Carbonic Anhydrase IX: A Study on Turnip (Brassica rapa) and its Potential in Cancer Prevention

  • 期刊论文

  • 发表日期：

    2025年6月27日

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  • Emadeldin M. Kamel,
  • Ahmed A. Allam,
  • Hassan A. Rudayni,
  • Saleh Alkhedhairi,
  • Faris F. Aba Alkhayl,
  • May Bin-Jumah,
  • Doaa A. Abdelrheem,
  • Al Mokhtar Lamsabhi

摘要

Cruciferous vegetables such as turnip (Brassica rapa) are rich in bioactive glucosinolates with potential health-promoting and anticancer properties. Here, 4-methoxyglucobrassicin, glucobrassicin and neoglucobrassicin were purified from hot-methanol extracts of B. rapa roots by acidic-alumina and Sephadex LH-20 chromatography. Recombinant human carbonic anhydrase IX (CA IX) esterase activity was measured with a 4-nitrophenyl-acetate microplate assay, and inhibition constants were extracted from Lineweaver–Burk plots. Protein–ligand interactions were investigated by AutoDock Vina docking (PDB 5FL4), followed by 200 ns CHARMM36m molecular-dynamics simulations in GROMACS; binding free energies were obtained with the MM/PBSA method, and conformational stability was evaluated through free-energy-landscape analysis. In vitro enzyme-inhibition assays revealed that neoglucobrassicin and glucobrassicin exhibited potent CA IX inhibition, with IC50 values of 48.5 ± 2.1 nM and 65.06 ± 2.8 nM, respectively. Kinetic studies indicated a mixed-type inhibition mechanism, suggesting multiple interaction modes with the enzyme. Molecular docking confirmed strong polar interactions within the CA IX active site, and MD/MM-PBSA ranked neoglucobrassicin as the most favorable binder (ΔGtotal = − 34.74 ± 2.47 kcal mol⁻¹). Free-energy-landscape analysis further showed that neoglucobrassicin maintains a highly stable conformational state during the simulation. Overall, this integrated isolation, enzyme-assay, and computational workflow identifies turnip-derived indole glucosinolates as promising natural CA IX inhibitors and supports their potential role in functional foods and nutraceuticals aimed at cancer prevention and tumor-micro-environment modulation.