Understanding the binding interaction between methotrexate and human alpha-2-macroglobulin: Multi-spectroscopic and computational investigation

Methotrexate (MTX) is advised in the treatment of solid tumours, hematologic malignancies and autoimmune disorders. On reaching the circulation, 60% of MTX is bound to the proteins present in serum. Alpha-2-macroglobulin (alpha M-2) is a plasma proteinase inhibitor with numerous functions such as binding, transportation and targeting of molecules. Our studies are the first attempt to investigate the binding interaction of pharmacologically important drug MTX, and highly abundant proteinase inhibitor- alpha M-2. The protein functional activity assay shows 53% decrease in antiproteolytic potential of alpha M-2 upon drug interaction. The binding of MTX with alpha M-2 was studied by various biophysical methods. UV-visible absorption spectroscopy reveals hyperchromicity of alpha M-2 spectra upon drug binding. The intrinsic fluorescence spectra show quenching in fluorescence intensity of alpha M-2 and the mechanism of quenching was found to be static in nature. Far UV-CD spectra unveil slight alteration in secondary structure of alpha M-2 upon drug binding. Isothermal titration calorimetry (ITC) reveals the value of thermodynamic parameters and which affirms the binding process to be spontaneous and exothermic. Molecular docking illustrates that Asn173, Leu1298, Gly172, Lys1240, Gln1325, Ser1327, Glu913, Asn1139, Lys1236, Leu951 and Arg1297 were the key residues involved during interaction process. Molecular dynamics (MD) simulation studies suggest that MTX form a stable complex with alpha M-2. Our study assumes importance from the fact that MTX is known to bind plasma proteins quite efficiently.