Molecular\nDynamics Unveils Multiple-Site Binding of\nInhibitors with Reduced Activity on the Surface of Dihydrofolate Reductase

The sensitivity to protein inhibitors\nis altered by modifications\nor protein mutations, as represented by drug resistance. The mode\nof stable drug binding to the protein pocket has been experimentally\nclarified. However, the nature of the binding of inhibitors with reduced\nsensitivity remains unclear at the atomic level. In this study, we\nanalyzed the thermodynamics and kinetics of inhibitor binding to the\nsurface of wild-type and mutant dihydrofolate reductase (DHFR) using\nmolecular dynamics simulations combined with Markov state modeling.\nA strong inhibitor of methotrexate (MTX) showed a preference for the\nactive site of wild-type DHFR with minimal binding to unrelated (secondary)\nsites. Deletion of a side-chain fragment in MTX largely destabilized\nthe active site-bound state, with clear evidence of binding to secondary\nsites. Similarly, the F31V mutation in DHFR diminished the specificity\nof MTX binding to the active site. These results reveal the presence\nof multiple-bound states whose stabilities are comparable to or higher\nthan those of the unbound state, suggesting that a reduction in the\nbinding affinity for the active site significantly elevates the fractions\nof these states. This study presents a theoretical model that more\naccurately interprets the altered drug sensitivity than the traditional\ntwo-state model.