A very good correlation, with a correlation coefficient of 0.90, is obtained between the calculated free energy differences and the experimental data (Figure ). In the subgroup with compound L30 as the reference, most of the predicted binding free energy differences are negative, indicating that the modifications to the compound are likely to improve the binding potencies (ΔΔG<0). The calculations correctly predict compound L2 (Figure ) as one of the best binders. As L2 already has a very favourable binding free energy (-10.49 kcal/mol), it is not surprising that most of the modifications from it deteriorate the binding affinities (ΔΔG>0). For all of the 103 compound pairs, 94% of the TIES predictions agree directionally with the experimental observations, that is, the calculated binding free energy differences have the same sign as those from experimental measurements. The relative binding free energy differences from TIES agree well with those from ESMACS (Figure ), with a correlation coefficient of 0.62. The predicted ΔΔG values for the two subgroups, paired with L30 and L2, are clearly distinct from both of the protocols. The predicted ΔΔG results from ESMACS