Recently, selective inhibitors for CDK4 have gained substantial interest. For example the orally active small molecule PD0332991, which induces G1 arrest in primary myeloma cells, prevents tumor growth by specific inhibition of CDK4/6 and is now in Phase 2 clinical trials. 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 ?, 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. 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 dynamics simulations which predict the physical properties of the protein-ligand complexes based on atomistic structural models. The energetic consequences of small structural Staurosporine 62996-74-1 changes in inhibitor complexes have been successfully studied using thermodynamic integration. An added benefit of TI calculations, as compared to empirical ligand Sorafenib Raf inhibitor docking algorithms is that the former include accurate estimates of binding entropy as well as enthalpy, based on rigorous statistical thermodynamics. In this work, we specifically address the contribution of the positive charge of fascaplysin to selectivity by applying thermodynamic integration calculations. In silico, fascaplysin can be modified easily by the iso-electronic substitution of the positively charged nitrogen to a charge neutral carbon atom, resulting in a compound, which for clarity and 
simplicity we refer to as carbofascaplysin. By calculating the energetic effect of this substitution for the protein-inhibitor complexes of both CDK2 and CDK4, we can quantify the impact of the positive charge of fascaplysin on its specificities towards CDK2 and CDK4.