Avoiding re-hydration of the binding site. In our ABFE thermodynamic cycle, the trapped water is alchemically decoupled in edge E, i.e., its vdw and Coulomb interactions are turned off. This creates a cavity in the binding site. If the binding site has some level of solvent accessibility, it may re-hydrate at different stages of the thermodynamic cycle, which would invalidate the thermodynamic cycle (e.g., by replacing the trapped water with a different water in some intermediate states but not others). To avoid the re-hydration of the cavity, we added a repulsive potential for the solvent at the position of the trapped water in our ABFE thermodynamic cycle (edge C). This repulsive potential is only removed at the end of the thermodynamic cycle, in edge G, after the trapped water is decoupled. Re-hydration of the cavity can occur in different parts of the thermodynamic cycle. Without a solvent restraint, it could occur during the NES switching simulations (which would likely lead to poor convergence since the paths including re-hydration may not reach the expected end states), or more likely, during the (longer) equilibrium simulation of the end state of the NES. If re-hydration would occur in the end state, the switching NES simulations would be ill-defined, since some or all of the start-ing structures of the reverse NES (for coupling the trapped water) would now have a resident water present before trying to couple the noninteracting trapped water. A dry cavity is also paramount to create space for inserting a bigger ligand in the thermodynamic cycle for ligand transformation (Figures and).