Concluding Remarks

In this chapter we have described the binding modes of some CK2 inhibitors that target the ATP-binding site. These inhibitors belong to different chemical families and have inhibitory constants in the low micromolar range. Two of them, namely TBB and IQA, display a significant selectivity among panels of tens of different kinases, and can be considered an encouraging starting point for the development of compounds of potential pharmacological interest.

For all the inhibitors here described, the main energetic contribution to the binding is attributed to hydrophobic interaction with the apolar surface region of the CK2 binding cleft. In this respect, the shape and the reduced dimension of the CK2 active site in comparison with other kinases are essential in explaining the selectivity of these inhibitors as well as the anomalous low potency of staurosporine. In particular, isoleucine/valine-66 and isoleucine-174, residues unique to CK2, play an important role as demonstrated by mutagenesis studies. The aromatic nature of the inhibitors can be considered a key characteristic of these compounds, providing the main energetic contribution to the binding.

In general, the ATP-binding sites of kinases present both polar and hydrophobic portions. While the characteristics of the polar moiety are quite conserved among the family, as, for instance, some key residues in the hinge regions, in the phosphate anchor region, or other residues like lysine-68, glu-tamate-81, and aspartate-175 (CK2 numbering), the hydrophobic ones have a higher degree of variability in the amino acid composition. Consequently, the selectivity is ensured by targeting the hydrophobic portion of the bind ing site while the presence of polar interactions (hydrogen bonds, salt bridges) enhances the potency of the inhibitor. The inhibitors here described can be considered more selective than potent, and this is in agreement with the above considerations in that they bind to the enzyme mainly through hydro-phobic interactions. As noticed for the four anthraquinones, the presence of polar interactions is essential to increase the binding potency; in this respect, the interactions with the hinge region seem more effective (see the case of DAA in comparison with emodin). To produce inhibitors in the nanomolar range, as would be desirable, our aim is now concentrated in the increase of the number and strength of the polar interactions between the inhibitors and the enzyme, without depleting the hydrophobic interactions. Other important points are the increase of the solubility of these compounds in water and the analysis of their potential pharmacological properties.

The estimate of the selectivity of a kinase inhibitor is always a difficult task; in fact, while the number of kinases has been estimated around 500 from the human genome, the enzymes really available for inhibition assays are only a fraction of that (around some tens). The development of enzyme mutants resistant to the inhibitory activity of a specific compound but with a catalytic activity similar to the wild-type protein [i.e., mutants Ile66Ala and Ile174Ala for CK2 (Sarno et al. 2002)] can be very useful in the analysis of its selectivity. In fact, the recovery of the original kinase activity by means of such mutants in the presence of the specific inhibitor in in vivo assays can be considered a validation of the involvement of just that kinase in the process under study.


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