Biophysical and Computational Evidence for Ibuprofen Binding to and Inhibition of MARK4

MARK4 is a serine/threonine kinase implicated in neurodegenerative disorders and cancer, making it an attractive target for therapeutic intervention and drug repurposing. In this study, we investigated the inhibition and interaction of ibuprofen with MARK4 using an integrated experimental and computational approach employing steady-state fluorescence and UV-vis spectroscopy, circular dichroism, molecular docking, and molecular dynamics simulations. The ATPase assays showed that ibuprofen produces dose-dependent inhibition of MARK4 ATPase activity. The ATPase activity of MARK4 was inhibited by >93% with an IC50 of 4.02 ± 0.33 µM. The fluorescence titrations demonstrated quenching of MARK4 intrinsic fluorescence upon addition of ibuprofen. The thermodynamic analysis indicated spontaneous binding with favorable entropy and involvement of hydrophobic forces in the binding. The observed reduction in ellipticity suggests alterations in the secondary structure of MARK4 upon ibuprofen binding. The binding energy from docking was obtained as −7.0 kcal/mol. Ibuprofen was docked within the ATP or inhibitor binding site, with two hydrogen bonds. MD simulations confirmed the dynamic stability of the MARK4–ibuprofen complex over 100 ns. The complex retained overall compactness, with nominal increases in RMSD and RMSF relative to the apo protein. The findings demonstrate the potential of ibuprofen to inhibit MARK4 ATPase activity, which may further guide drug discovery.