ESMACS. We used the ESMACS (enhanced sampling of molecular dynamics with approximation of continuum solvent) protocol for the simulations and analyses. The protocol uses replica simulations to obtain reproducible binding affinity predictions with robust uncertainty estimates. It is based on the molecular mechanics Poisson-Boltzmann surface area (MMPBSA), which is an approximate method for calculating absolute binding affinities from molecular dynamics trajectories. It is an endpoint free energy calculation, in which the difference in binding free energy, ∆G, is calculated using