IQA [[5-oxo-5,6-dihydro-indolo(1,2-a)quinazolin-7-yl]acetic acid] is a novel ATP/GTP site-directed inhibitor of CK2 ('casein kinase 2'), a pleiotropic and constitutively active protein kinase whose activity is abnormally high in transformed cells. The K (i) value of IQA (0.17 microM) is lower than those of other CK2 inhibitors reported so far. Tested at 10 microM concentration in the presence of 100 microM ATP, IQA almost suppresses CK2 activity in vitro, whereas it is ineffective or weakly effective on a panel of 44 protein kinases and on phosphoinositide 3-kinase. In comparison, other CK2 inhibitors, notably apigenin and quercetin, are more promiscuous. The in vivo efficacy of IQA has been assessed by using the fact that treatment of Jurkat cells with IQA inhibits endogenous CK2 in a dose-dependent manner. IQA has been co-crystallized with maize CK2alpha, which is >70% identical with its human homologue, and the structure of the complex has been determined at 1.68 A (1 A=0.1 nm) resolution. The inhibitor lies in the same plane occupied by the purine moiety of ATP with its more hydrophobic side facing the hinge region. Major contributions to the interaction are provided by hydrophobic forces and non-polar interactions involving the aromatic portion of the inhibitor and the hydrophobic residues surrounding the ATP-binding pocket, with special reference to the side chains of V53 (Val53), I66, M163 and I174. Consequently, mutants of human CK2alpha in which either V66 (the homologue of maize CK2alpha I66) or I174 is replaced by alanine are considerably less sensitive to IQA inhibition when compared with wild-type. These results provide new tools for deciphering the enigmatic role of CK2 in living cells and may pave the way for the development of drugs depending on CK2 activity.

Two novel crystal structures of Zea mays protein kinase CK2␣ catalytic subunit, one in complex with the specific inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) and another in the apo-form, were solved at 2.2 Å resolution. These structures were compared with those of the enzyme in presence of ATP and GTP (the natural cosubstrates) and the inhibitor emodin. Interaction of TBB with the active site of CK2␣ is mainly due to van der Waals contacts, with the ligand fitting almost perfectly the cavity. One nitrogen of the five-membered ring interacts with two charged residues, Glu 81 and Lys 68, in the depth of the cavity, through two water molecules. These are buried in the active site and are also generally found in the structures of CK2␣ enzyme analyzed so far, with the exception of the complex with emodin. In the N-terminal lobe, the position of helix ␣C is particularly well preserved in all the structures examined; the Gly-rich loop is displaced from the intermediate position it has in the apo-form and in the presence of the natural cosubstrates (ATP/GTP) to either an upper (with TBB) or a lower position (with emodin). The selectivity of TBB for CK2 appears to be mainly dictated by the reduced size of the active site which in most other protein kinases is too large for making stable interactions with this inhibitor.

The structure of a complex between the catalytic subunit of Zea mays CK2 and the nucleotide binding sitedirected inhibitor emodin (3-methyl-1,6,8-trihydroxyanthraquinone) was solved at 2.6-Å resolution. Emodin enters the nucleotide binding site of the enzyme, filling a hydrophobic pocket between the N-terminal and the C-terminal lobes, in the proximity of the site occupied by the base rings of the natural co-substrates. The interactions between the inhibitor and CK2␣ are mainly hydrophobic. Although the C-terminal domain of the enzyme is essentially identical to the ATP-bound form, the ␤-sheet in the N-terminal domain is altered by the presence of emodin. The structural data presented here highlight the flexibility of the kinase domain structure and provide information for the design of selective ATP competitive inhibitors of protein kinase CK2.

Protein kinases play key roles in signal transduction and therefore are among the most attractive targets for drug design. The pharmacological aptitude of protein kinase inhibitors is highlighted by the observation that various diseases with special reference to cancer are because of the abnormal expression/activity of individual kinases. The resolution of the three-dimensional structure of the target kinase in complex with inhibitors is often the starting point for the rational design of this kind of drugs, some of which are already in advanced clinical trial or even in clinical practice. Here we present and discuss three new crystal structures of ATP site-directed inhibitors in complex with "casein kinase-2" (CK2), a constitutively active protein kinase implicated in a variety of cellular functions and misfunctions. With the help of theoretical calculations, we disclose some key features underlying the inhibitory efficiency of anthraquinone derivatives, outlining three different binding modes into the active site. In particular, we show that a nitro group in a hydroxyanthraquinone scaffold decreases the inhibitory constants K i because of electron-withdrawing and resonance effects that enhance the polarization of hydroxylic substituents in paraposition.