The first CDK4 structures have only been published recently. Most residues in the active sites of CDK2, CDK4 and CDK6 are remarkably conserved. A key difference is the presence of a histidine residue in CDK4/6 while CDK2 comprises a phenylalanine in the equivalent position. The His95CDK4/His100CDK6 side-chain is in a position where it potentially can donate or accept a H-bond from an inhibitor. Other differences are in Val96CDK4 and Val101CDK6 corresponding to Leu83CDK2. This residue is capable of forming H-bonds to inhibitors with both backbone NH and carbonyl group, but as its side chain is pointing away from the binding site and is not in direct contact with inhibitors the Val/Leu variation appears to be less relevant for selectivity. Other differences in the binding site are residues Thr120CDK4 and Thr107CDK6, these 924416-43-3 threonines correspond to Lys89CDK2. The negatively charged residues Asp97CDK4 and Asp102CDK6 have His84CDK2 in the equivalent position, and finally glutamate Glu144CDK4 is corresponding to Gln131CDK2 and Gln149CDK6 the latter being the only position where CDK4 and CDK6 have different residues. Interestingly, in all three of these positions CDK4 gains a negative charge relative to CDK2. The potential role of charge as a determinant of CDK4 inhibitor specificity has been pointed out originally by McInnes et al. and more recently by Mascarenhas et al.. In this work, we have studied this example of charge-determined protein-ligand interactions using a variety of methods from the molecular modelling and drug design fields. The binding of inhibitors to protein receptors with high affinity and specificity is central to structure-based drug design applications. The quest for the calculation of binding affinities remains one of the main goals of modern computational biophysical methods. The most accurate methods for calculating binding free energies are based on molecular Ellipticine dynamics simulations which predict the physical properties of the protein-ligand complexes based on atomistic structural models. The energetic consequences of small structural changes in inhibitor complexes have been successfully studied using thermodynamic integration. An added benefit of TI calculations, as compared to empirical ligand docking algorithms is that the former inc
