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Kinetics of protein–ligand unbinding: Predicting pathways, rates, and rate-limiting steps

Significance A crucial factor for drug efficacy is not just the binding affinity, but also the mean residence time in the binding pocket, usually quantified by its inverse, koff. This is an important parameter that regulates the time during which the drug is active. Whereas the calculation of the binding affinity is by now routine, the calculation of koff has proven more challenging because the timescales involved far exceed the limits of standard molecular dynamics simulation. We propose a metadynamics-based strategy that allows reaching timescales of seconds, and estimate koff along with unbinding pathways and associated dynamical bottlenecks. The protocol is exemplified for trypsin–benzamidine unbinding. This work is a step towards a more effective computer-based drug design. The ability to predict the mechanisms and the associated rate constants of protein–ligand unbinding is of great practical importance in drug design. In this work we demonstrate how a recently introduced metadynamics-based approach allows exploration of the unbinding pathways, estimation of the rates, and determination of the rate-limiting steps in the paradigmatic case of the trypsin–benzamidine system. Protein, ligand, and solvent are described with full atomic resolution. Using metadynamics, multiple unbinding trajectories that start with the ligand in the crystallographic binding pose and end with the ligand in the fully solvated state are generated. The unbinding rate koff is computed from the mean residence time of the ligand. Using our previously computed binding affinity we also obtain the binding rate kon. Both rates are in agreement with reported experimental values. We uncover the complex pathways of unbinding trajectories and describe the critical rate-limiting steps with unprecedented detail. Our findings illuminate the role played by the coupling between subtle protein backbone fluctuations and the solvation by water molecules that enter the binding pocket and assist in the breaking of the shielded hydrogen bonds. We expect our approach to be useful in calculating rates for general protein–ligand systems and a valid support for drug design.