Umbrella sampling was performed on each ligand-protein complex to obtain an estimate of their relative binding energies. The position of the ligand within the binding cavities were sampled using 15-30 windows. In each window, a harmonic potential of the form 𝑉 = 1 2 ⁄ 𝑘(𝑥 -𝑥 0 ) 2 with a force constant of 5.0 kcal/mol/Å 2 was used to apply the distance restraints between a ligand atom and a protein atom located at the bottom of the binding pocket.The centers of the umbrella potential windows were spaced by 0.8 Å. Sampling was performed for 5.2 ns in each bin, of which the first 1.2 ns were used to enable the ligand to relax into the position defined by the new equilibrium distance of the harmonic potential and were therefore eliminated from the analyses. Statistics were then collected every 0.5 ps, for 4 ns. The potentials of mean force were obtained from the statistical distribution of the distances between the ligand and protein atoms in the restrained coordinate through the weighted histogram analysis method (WHAM) using a bin size of 0.2 Å.