We did not consider a positively charged imidazole side chain as this would unfavourably interact with the positively charged fascaplysin. However, alternative positioning of hydrogens in His95CDK4 was considered in the ligand docking process. ChemScores were by a small margin higher for the His95CDK4Nd-H/fascaplysin complex indicating a slight preference for the side-chain conformation in which the Nd-hydrogen of the imidazol ring forms an additional H-bond to the carbonyl of FAS and CRB, respectively. This conformation is different from the His95 conformation found in the experimentally determined CDK4 structures, but such a conformational change could occur upon ligand binding, when the alternative His95CDK4 side chain conformation is stabilised by the interaction with the inhibitor. The idea of His95 as a key player for CDK4 specificity is Glesatinib (hydrochloride) supported by the notion that CDK6 also has a histidine residue in the equivalent position. Any energetic contribution of the additional His95-Nd H-bond to the free energy of GW-610742 supplier binding should also feature in CDK6, and indeed the IC50 of CDK6/fascaplysin is, while being,8 times higher than CDK4/fascaplysin, still,100 times lower than CDK2/fascaplysin. However, there is a problem with this notion, as if correct, the interaction in question should occur for most inhibitors, essentially for any ligand that forms a H-bond with the backbone NH of Val96CDK4. If His95CDK4 was indeed the key to the observed fascaplysin CDK4 specificity we would expect this to be rather generic feature, rendering most CDK inhibitors more specific for CDK4 as CDK2. This is however not the case and hence it is unlikely that the difference between His95CDK4 and Phe82CDK2 can account fully for the differential binding of fascaplysin. The inaccuracy of docking scoring functions for estimating free energies of binding is a major short coming of typical ligand docking approaches. To obtain more accurately calculated values for free energies of binding thermodynamic integration was used. A key feature of fascaplysin is its positive charge. Docking scoring functions are limited in accounting for long-range electrostatic interactions; Thermodynamic Integration however d
