The impact of inhibitor size and flexibility on the binding pathways to c-Src kinase

Considering dynamical aspects of protein-drug binding processes is inevitable in current drug compound design. Conformational plasticity of protein kinases poses a challenge for the design of their inhibitors, and therefore, atomistic molecular dynamics (MD) simulations have often been utilized. While protein conformational changes have been increasingly discussed, a fundamental yet non-trivial question remains for the effect of drug compound flexibility, which is hardly detectable from experiments. In this study, we apply two-dimensional replica-exchange MD simulations as enhanced sampling to investigate how c-Src kinase can bind PP1, a small inhibitor, and dasatinib, a larger inhibitor with greater flexibility. 600 microseconds simulations in total sample binding and unbinding events of these inhibitors much more frequently than conventional MD simulation, resulting in statistically converged binding pathways. While the two inhibitors adopt a similar mechanism of multiple binding pathways, the non-canonical binding poses become less feasible for dasatinib. A notable difference is apparent in their energetics where dasatinib stabilizes at intermediate states more than PP1 to raise the barrier toward the canonical pose. Conformational analysis shows that dasatinib adopts linear and bent forms for which relative populations are altered upon binding. We further find hidden conformations of dasatinib at intermediate regions, and unexpectedly one of them could efficiently bypasses the intermediate-to-bound state transition. The results demonstrate that inhibitor size and flexibility impact the binding mechanism, which could potentially modulate inhibitor residence time.