The natural compound fascaplysin, originally isolated from the sponge Fascaplysinopsis Bergquist, is a kinase inhibitor with enticing selectivity for CDK4 relative to the close homolog CDK2, and also shows approximately eightfold selectivity over CDK6. Approximating the dissociation constant KD with IC50 and using the relation DG0=2RTlnKD, the difference in the free energy of binding between the CDK4/fascaplysin and CDK2/fascaplysin complexes can be calculated to 4.2 kcal/mol. Considering the close structural similarity of the active sites of CDK2, CDK4 and CDK6, and the relatively small size and rigid structure of fascaplysin, the observed selectivity is remarkable. Chemically, fascaplysin is a planar, aromatic compound with no freely rotatable single bonds. It comprises five condensed rings, the central ring includes a positively charged imminium nitrogen. An indol-NH and a carbonyl can act as H-bond donor and H-bond acceptor, respectively. The H-bond donor and H-bond acceptor in fascaplysin are oriented in parallel spaced at,2.6 , a feature shared with other kinase inhibitors. The fascaplysin framework has been used to synthesise a series of selective CDK4 inhibitors, though in most cases selectivity was partially lost in the redesign process. So what are the features that could explain the remarkable selectivity of fascaplysin? There is a considerable amount of structural information on CDKs available to help addressing this question. More than 100 CDK2 structures in complex with small molecules are deposited in the protein databank. However, compared to CDK2, structural information on CDK6 and CDK4 with inhibitors bound is scarce, in fact 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 threonines correspond to Lys89CDK2. The negatively charged residues Asp97CDK4 and Asp102CDK6 have His84CDK2 in the equivalent position, and finally glutamate Glu144CDK4 is corresponding to MCE Chemical 1252003-15-8 Gln131CDK2 and Gln149CDK6 �C 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 and more recently by 917879-39-1 Mascarenhas . 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.
