Relative binding enthalpies (∆∆H) were estimated by the direct method , which involves taking differences in mean (Boltzmann-averaged) potential energies for simulated systems of interest. With the direct method, the absolute protein-ligand binding enthalpy can be computed by running separate simulations of the ligand in solvent, the protein in solvent, and the protein-ligand complex in solvent, and subtracting the mean energies, while ensuring that the composition of the bound-state system is identical to that of the unbound state systems; for example, the number of water molecules must match exactly between the bound and free states. Here, however, we computed the relative binding free energies of a series of similar ligands. This avoids the requirement of converging the mean energy of the free protein, which might be particularly difficult due to possible conformational shifts on removal of ligands from the binding site. The relative binding enthalpies considered in this study were computed according to the following equations: