Understanding the governing factors of fast or slow inhibitor binding/unbinding assists in developing drugs with preferred kinetic properties. For inhibitors with the same binding affinity targeting different binding sites of the same protein, the kinetic behavior can profoundly differ. In this study, we investigated unbinding kinetics and mechanisms of fast (type-I) and slow (type-II/III) binders of p38α mitogen-activated protein kinase, where the crystal structures showed that type-I and type-II/III inhibitors bind to pockets with different conformations of the Asp-Phe-Gly (DFG) motif. The work used methods that combine conventional molecular dynamics (MD), accelerated molecular dynamics (AMD) simulations, and the newly developed pathway search guided by internal motions (PSIM) method to find dissociation pathways. The study focuses on revealing key interactions and molecular rearrangements that hinder ligand dissociation by using umbrella sampling and post-MD processing to examine changes in free energy during ligand unbinding. As anticipated, the initial dissociation steps all require breaking interactions that appeared in crystal structures of the bound complexes. Interestingly, for type-I inhibitors such as SB2, p38α keeps barrier-free conformational fluctuation in the ligand-bound complex and during ligand dissociation. In contrast, with a type-II/III inhibitor such as BIRB796, with the rearrangements of p38α in its bound state, ligand unbinding features energetically unfavorable protein-ligand concerted movement. Our results also show that the type-II/III inhibitors preferred dissociation pathways through the allosteric channel, which is consistent with an existing publication. The study suggests that the level of required protein rearrangement is one major determining factor of drug binding kinetics in p38α systems, providing useful information for development of inhibitors.
This work describes an example of using Accelerated MD in kinetic calculations.