Understanding the structural and energetic requisites of ligand binding toward its molecular target is of paramount relevance in drug design. In recent years, atomistic free energy calculations have proven to be a valid tool to complement experiments in characterizing the thermodynamic and kinetic properties of protein/ligand interaction. Here, we investigate, through a recently developed metadynamics-based protocol, the unbinding mechanism of an inhibitor of the pharmacologically relevant target p38 MAP kinase. We provide a thorough description of the ligand unbinding pathway identifying the most stable binding mode and other thermodynamically relevant poses. From our simulations, we estimated the unbinding rate as k = 0.020 ± 0.011 s. This is in good agreement with the experimental value (k = 0.14 s). Next, we developed a Markov state model that allowed identifying the rate-limiting step of the ligand unbinding process. Our calculations further show that the solvation of the ligand and that of the active site play crucial roles in the unbinding process. This study paves the way to investigations on the unbinding dynamics of more complex p38 inhibitors and other pharmacologically relevant inhibitors in general, demonstrating that metadynamics can be a powerful tool in designing new drugs with engineered binding/unbinding kinetics.

This article reviews different formulations of the thermodynamic cycles used for the prediction of pK a values, their advantages, and disadvantages with special emphasis on the limitations resulting from the necessity of gas-phase calculations, which allow introducing some difficult cases that motivated alternative strategies. Before introducing the protocols that do not consider gas-phase calculations, the two current opinions available in the literature on the debate about the correct formalism for the calculation of free energies in solution are briefly introduced. Then, the isodesmic proton exchange reaction in solution is reviewed by analyzing its performance on difficult cases for thermodynamic cycles such as carbon acids and amino acids. The pK a values predicted by the isodesmic reaction for common acid species are also reviewed to compare their accuracy results in relation with those of thermodynamic cycles. Linear regressions between experimental pK a values and the calculated free energies obtained with the isodesmic reaction provide expressions for the dependence of the error in the calculated pK a s on the pK a difference between the studied acid and the reference species. Finally, it is shown that linear regressions correct the calculated free energies of the isodesmic reaction, when high constant precision is required in a large pK a range. The deviations from the expected behavior are equivalent to those reported previously for different pK a calculation protocols and are determined by the inaccuracies of continuum solvent models on the interactions with ionic species.

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