Differential Docking of Methotrexate and Metabolite on Renal Transporters and Dihydrofolate Reductase

Abstract

Introduction: Methotrexate is widely used for cancer and autoimmune disease therapy, but its use is often limited by nephrotoxicity, particularly during high-dose treatment. This toxicity is linked to its metabolite, 7-hydroxymethotrexate, although the precise molecular mechanisms remain unclear.

Objectives: In this study, we aimed to employ molecular docking methods to compare the binding of methotrexate and 7-hydroxymethotrexate to key renal proteins—OAT1, OAT3, and DHFR.

Material and methods: In this study, we used structures of methotrexate and 7-hydroxymethotrexate to key renal proteins, OAT1, OAT3, and DHFR from AlphaFold, RCSB PDB, and PubChem. AutoDock Vina and PyMOL were used for docking and interaction analysis.

Results: Results showed that 7-hydroxymethotrexate binds more strongly to all targets, especially OAT3 (docking score: -9.7 vs. -8.6 kcal/mol), and forms unique hydrogen bonds with residues Tyr356, Ser442, and Arg469 in OAT3. While both ligands had good affinity for DHFR, differences were more pronounced for the renal transporters.

Conclusions: These findings suggest that enhanced binding of 7-hydroxymethotrexate to OAT3 may contribute to its nephrotoxic risk, supporting the importance of transporter-mediated drug interactions in methotrexate therapy.