Theoretical foundation Binding affinity is often quantified using the equilibrium dissociation constant (Kd), defined as: $$K_mathrmd = left[ mathrmP right]left[ mathrmL right]/left[ mathrmP:mathrmL right]$$ (1) where [P], [L] and [P:L] are the concentrations of protein, ligand and the protein–ligand complex, respectively. Computationally, the absolute binding free energy (ΔG°), which is the standard molar free energy of binding, is more convenient to calculate. The dissociation constant and the absolute binding free energy are related via $$Delta G^circ = RTln fracK_mathrmd1,mathrmM$$ (2) where R is the gas constant, T is the temperature and 1 M is 1 molar concentration. Various strategies have been used to estimate ΔG°, some of which were briefly discussed above.…