  We here report on non-equilibrium targeted Molecular Dynamics simulations as
tool for the estimation of protein-ligand unbinding kinetics. Correlating
simulations with experimental data from SPR kinetics measurements and X-ray
crystallography on two small molecule compound libraries bound to the
N-terminal domain of the chaperone Hsp90, we show that the mean non-equilibrium
work computed in an ensemble of trajectories of enforced ligand unbinding is a
promising predictor for ligand unbinding rates. We furthermore investigate the
molecular basis determining unbinding rates within the compound libraries. We
propose ligand conformational changes and protein-ligand nonbonded interactions
to impact on unbinding rates. Ligands may remain longer at the protein if they
exhibit strong electrostatic and/or van der Waals interactions with the target.
In the case of ligands with rigid chemical scaffold that exhibit longer
residence times however, transient electrostatic interactions with the protein
appear to facilitate unbinding. Our results imply that understanding the
unbinding pathway and the protein-ligand interactions along this path is
crucial for the prediction of small molecule ligands with defined unbinding
