A directory of the thermodynamic averages calculated from these tables and Eq. is given in Dining table IV. Both DG 0 bind and DGbind precisely predicted indirubin 3 0 oxime to be a more potent inhibitor than indirubin, and that 2-ME2 2-Methoxyestradiol KT5720 and staurosporine were consequently better inhibitors than indirubin 3 0 oxime. But, the ranking of inhibitor binding affinities for KT5720 and staurosporine were reversed in comparison to kinetics, with KT5720 predicted to become stronger by 1. 7 kcal mol21 using DGbind. This was reduced to 0. 7 kcal mol21 for DGbind, on inclusion of the increasing loss of ligand VRT entropy upon binding. Sales for entropy effects, needlessly to say, brought the free energy values significantly closer in degree with their experimental values. The solved rating of KT5720 and staurosporine inhibitor potencies calculated using MM GBSA can be accounted for. Given the additional flexibility of the KT5720 alkyl chain, we are able to estimate an upper bound for the loss of KT5720 conformational entropy by attributing an entropy loss of 0. 30 0. 54 kcal mol21 64 to each rotatable sp3 relationship of atoms 16-24. This increases the KT5720 binding free energy by 1. 8 3. 2 kcal mol21, bringing the DGbind importance relative to staurosporine to 1. 2 2. 6 kcal mol21 in support of staurosporine, and consistent with the rankings from experiment. Further, although staurosporine does not have any important receptor ligand linking waters, KT5720 was firmly bridged by exchanging water molecules with the receptor. The loss associated with a bound water molecule in protein ligands complexes is suggested by Dunitz to have an upper bound of 2 kcal mol21 free energy charge at 300 K. 65 Therefore, the relative free energy in favor of staurosporine could be increased even more because of benefits from the loss of bound waters in the complex. The MM/GBSA method employed here and the relevant MM PBSA method are computationally efficient, end point free energy which have been widely used to examine protein ligand binding affinities. pifithrin 52,66 73 Even though they lack the sound theoretical basis of recently developed, computationally challenging overall appreciation free power methods,69,74 78 their experience of statistical thermodynamics has been established. 79 They should be regarded as approximate, as they mix a molecular mechanics energy function using a procession electrostatics treatment of solvation effects, they contain solute conformational entropy effects in a approximate manner80 and ignore the solvent molecular structure. For the indirubins, the break down of binding free energy contributions revealed that the supply of the more binding affinity of indirubin 3 0 oxime in comparison with indirubin is electrostatic, which is often caused by favorable indirubin 3 0 oxime NOH group interactions.