Features and potentials of ATP-site directed CK2 inhibitors

Biochimica et Biophysica Acta 1754 (2005) 263 – 270 http://www.elsevier.com/locate/bba

Review

Features and potentials of ATP-site directed CK2 inhibitors Stefania Sarno a,c, Mauro Salvi a, Roberto Battistutta b,c, Giuseppe Zanotti b,c, Lorenzo A. Pinna a,c,* a

Department of Biological Chemistry, University of Padova, Viale G. Colombo 3, 35129 Padova, Italy b Department of Chemistry, The University of Padova, Padova, Italy c Venetian Institute for Molecular Medicine, Padova, Italy Received 22 June 2005; received in revised form 18 July 2005; accepted 20 July 2005 Available online 12 September 2005

Abstract A panel of quite specific, fairly potent and cell-permeable inhibitors of protein kinase CK2 belonging to the classes of condensed polyphenolic compounds, tetrabromobenzimidazole/triazole derivatives and indoloquinazolines have been developed, with K i values in the submicromolar range. Nine structures have been solved to date of complexes between the catalytic a subunit of CK2 and a number of these compounds, many of which display a remarkable specificity toward CK2 as compared to a panel of >30 kinases tested. The structural basis for such selectivity appears to reside in the shape and size of a hydrophobic pocket adjacent to the ATP binding site where these ATP competitive ligands are entrapped mainly by van der Waals interactions and by an energy contribution derived from the hydrophobic effect. In CK2, this cavity is smaller than in the majority of other protein kinases due to a number of unique bulky apolar residues. Consequently, the replacement of two of these residues (V66 and I174) in human CK2 alpha with alanines gives rise to mutants, which are markedly less susceptible than wild type to these classes of inhibitors. Cell-permeable CK2 inhibitors have been successfully employed, either alone or in combination with CK2 mutants refractory to inhibition, to dissect signalling pathways affected by CK2 and/or to validate the identification of in vivo targets of this pleiotropic kinase. Moreover, the remarkable pro-apoptotic efficacy of these compounds toward cell lines derived from a wide spectrum of tumors, disclose the possibility that in perspective CK2 inhibitors might become leads for the development of anti-cancer drugs. D 2005 Elsevier B.V. All rights reserved. Keywords: Protein kinase CK2; Casein kinase-2; Protein kinase inhibitor; Apoptosis; Neoplasia

1. Introduction The development of specific cell-permeable inhibitors of individual protein kinases is generally motivated by two aims: providing a mean to dissect the cellular functions of the kinase, and generating molecules which in perspectives could display therapeutic potential whenever the aberrant activity of the kinase of interest is suspected to have pathological consequences [1 –3]. The former approach is especially valuable in the case of constitutively active protein kinases which, unlike

Abbreviations: CK2, casein kinase 2; Dyrk1A, dual specificity tyrosine phosphorylation-regulated kinase; DMAT, 4,5,6,7-tetrabromo-2-(dimethylamino)benzimidazole; GSK3, glycogen synthase kinase 3; HSP90, heat shock protein; MNX, 1,8-dihydroxy-4-nitro-xanthen-9-one; PI3 kinase, phosphoinositide-3 kinase * Corresponding author. Department of Biological Chemistry, University of Padova, Viale G. Colombo 3, 35129 Padova, Italy. Tel.: +39 049 8276108; fax: +39 049 8073310. 1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2005.07.043

the majority of the other members of the family are not activated in response to specific stimuli nor exist within the cell as inactive and active forms, generally characterized by different phosphorylation degree. Therefore alternative tools like physiological agonists/antagonists, phosphospecific antibodies and constitutively active mutants are not applicable to disclose the signalling and metabolic pathways affected by these protein kinases, and cell -permeable, highly selective inhibitors represent one of the few strategies exploitable to gain information about their cellular functions. One could argue, on the other hand, that a kinase whose constitutive activity is not the outcome of a pathogenic mutation, but is required for normal cell life, is unlikely to represent a valuable pharmacological target. This may be not always the case however, as exemplified by protein kinase CK2, an acronym derived from the misnomer ‘‘casein kinase 2’’ [4]. Owing to a number of structural features [5], the activity of CK2 catalytic subunits (a and/or a_) is constantly on, either in the absence or presence of the regulatory h subunits with

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which they form a heterotetrameric holoenzyme [5 – 7]. It has been suggested that this may reflect the extraordinary pleiotropy of CK2, whose endless list of phosphorylatable targets already includes more than 300 proteins implicated in the regulation of many cellular functions, notably gene expression and signal transduction, and whose restless activity may generate a substantial proportion of the eukaryotic phosphoproteome [8]. Despite its global implication in so many aspects of cell life evidence is accumulating that CK2 may represent a valuable pharmacological target to combat cancer and infectious diseases. Its activity, invariably elevated in a wide spectrum of tumors [9], is exploited by many viruses for the phosphorylation of proteins, which are essential to their life cycle [8,9]. In several experimental models, moreover, the transfection of CK2 catalytic subunits contributes to the enhancement of the tumor phenotype, consistent with the rising concept that CK2 plays a general role as anti-apoptotic agent [10] and by doing that it may promote cell survival ‘‘at all costs’’ even in circumstances where programmed cell death would be desirable for the organism as a whole [11]. For a number of reasons, therefore, the development of potent, selective and cell-permeable inhibitors of CK2 is receiving increasing attention: these reagents on one side will provide a powerful tool for dissecting the biological functions of this pleiotropic and in many respects still enigmatic protein kinase, on the other, they may become the leads for anti-cancer and anti-infectious drugs. In this paper, an overview will be presented of recent progress in the field of ATP site-directed CK2 inhibitors, with special reference to the structural features underlying their specificity and their exploitation to gain information about the biological role and pathogenic potential of this pleiotropic kinase. 2. Classification of ATP site-directed CK2 inhibitors Most protein kinase inhibitors of practical interest, including many of those that have entered clinical practice, are competitive with respect to the phosphodonor substrate, ATP, which means that their binding and that of ATP are mutually exclusive. This may come as a surprise, considering on one side the high conservation of the ATP binding site across all protein kinases, on the other the specificity of the most successful inhibitors toward an individual or few protein kinases. An explanation for this apparent paradox was provided by the solution of the crystal structure of protein kinases in complex with ATP sitedirected inhibitors, revealing that although these ligands partially occupy the ATP binding pocket, they make specific interactions with surrounding elements, notably the hinge segment and two hydrophobic regions which display significant variability from one protein kinase to the others. This network of potential interactors responsible for the specific binding of ATP competitive inhibitors is sometimes referred to as the ‘‘pharmacophore’’ of the protein kinase [12] and these ligands are often termed ATP-mimetics even if their chemical structure has little to share with that of ATP. Their affinity for the kinase however, commonly expressed by IC50 values (the concentra-

tion required for 50% inhibition of activity) is indeed dependent on ATP concentration in the assay, which normally varies between 10 and 100 AM. This should be born in mind everytime IC50 values are compared, if they were calculated under different experimental conditions. A more reliable indicator of inhibitory potency is provided by the inhibition constant, K i, a theoretical IC50 value extrapolated to zero ATP concentration [13]. Often, however, investigators in the fields of biochemical pharmacology and medicinal chemistry tend to provide only IC50 values, as these better reflect ‘‘real’’ experimental conditions where the kinase is working with a given ATP concentration. Also, in the case of CK2, the most successful inhibitors usable for in cell studies are competitive with respect to ATP (and GTP, which, in the case of CK2, can replace ATP as phosphate donor). If we restrict our analysis to ATP sitedirected inhibitors displaying IC50 values <1 AM (generally calculated in the presence of 20 –40 AM ATP) they roughly fall into the following chemical categories: (i) Flavonoids; (ii) Derivatives of hydroxyantraquinones/xantenones; (iii) Derivatives of hydroxycoumarines; (iv) Derivatives of tetrabromotriazole/imidazole; (v) Derivatives of indoloquinazolines. Each category is represented in Fig. 1 by one compound, selected among those displaying low K i and IC50 values. The properties of these different classes of CK2 inhibitors can be summarized as follows. 2.1. Flavonoids These are in general broad specificity inhibitors of many classes of protein kinases. Even if they are fairly potent, therefore, they are likely to be poorly specific. This also applies to apigenin which is held and marketed as a first choice inhibitor of CK2 since it was shown that an affinity resin made by attachment of apigenin to sepharose 6B specifically bound CK2 present in cell lysates [14]. When tested on a panel of 33 protein kinases, however, apigenin inhibited 7 kinases as efficiently or even more efficiently than CK2 [15]. A similar behaviour was reported of another flavonoid, quercetin [16] whose efficacy on CK2 is slightly higher than that of apigenin [17]. While the broad specificity of these flavonoids should refrain from using them as first choice inhibitors of CK2, they can prove useful, in combination with other compounds, to corroborate the view that CK2 is implicated in specific physiological or pathological processes [18]. 2.2. Hydroxyantraquinone/xantenone derivatives The prototype of these compounds can be considered emodin, the active principle of Rheum palmatum, whose biological effects on cultured cells has been related to its ability to inhibit Herb-2 neu and other receptor protein tyrosine kinases [19,20] before it was realized that CK2 is inhibited even more efficiently by emodin, with IC50 values around 1 AM [21]. The potency and selectivity of emodin toward CK2 could be further improved [22], e.g., by derivatization of the hydroxyantraquinone scaffold with nitro group(s) (as exempli-

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Fig. 1. Compounds representative of the different classes of CK2 inhibitors.

fied in Fig. 1 by MNX), whose beneficial effect is due to their ability to increase the acidic dissociation of the phenolic groups in para position [23]. 2.3. Hydroxycoumarines Several hydroxycoumarinic compounds display IC50 (and K i) values significantly lower than 1 AM [22]. The most efficient inhibitor is DBC (3,8-dibromo-7-hydroxy-4-methylchromen-2-one) with a K i value below 100 nM (see Fig. 1). An advantage of compounds like DBC is that their coumarinic scaffold does not act as a DNA intercalator, a property that reduces side effects with respect to the emodin related inhibitors having antraquinone scaffolds. 2.4. Halogenated benzimidazole(triazole) derivatives The ancestor of this numerous family of CK2 inhibitors can be considered the nucleoside DRB (di-cloro-ribofuranosylbenzimidazole) firstly described in 1986 by Zandomeni et al. [24] and still marketed as a CK2 inhibitor despite its relatively high IC50 value (13 AM) and rather modest specificity. Later it was shown that the inhibitory efficiency of DRB could be substantially improved by replacing the two Chlorine atoms with 4 Bromines, while the sugar moiety could be removed without any detrimental effect [25,26]. This led to a compound, TBB (tetrabromo-benzotriazole) with an IC50 value around 1 AM and, more important, a remarkable selectivity for just CK2 among a panel of >30 protein kinases originally tested [27]. These properties, in conjunction with cell permeability, have made TBB the first choice inhibitor used for studying the biological role of CK2. Recently however it has been shown that the inhibitory potency of TBB can be further improved by generating adducts in which N2 is replaced by a carbon atom bound to a variety of polar functions [28]. The most efficient of these derivatives is the 2-dimethylamino one, DMAT [29] whose

K i value (40 nM) is the lowest so far reported of a CK2 inhibitor (see Fig. 1). The inclusion of additional enzymes in the panel of protein kinases, used to profile the specificity of inhibitors, has revealed that TBB and TBB derivatives (DMAT included) inhibit another protein kinase, DYRK1a, with efficiency comparable to CK2 [15,28]. 2.5. Indoloquinazoline derivatives A virtual screening of the Novartis compounds collection led to the identification of compound CGP029482 (5-oxo-5,6dihydroindolo-(1,2-a)quinazolin-7-yl)acetic acid) as a powerful CK2 inhibitor [30]. This indoloquinazoline derivative, later renamed IQA (see Fig. 1), displays an IC50 value (0.39 AM), which is somewhat lower than those of emodin and TBB. It is not as potent as the TBB derivative DMAT, but it is somewhat more specific especially as far as its modest effect on DYRK1a (IC50 = 6 AM) is concerned [15]. A potential drawback of IQA, however, is its instability in water where its lactam ring slowly undergoes hydrolysis. Attempts to derivatize IQA in order to stabilize its scaffold and/or to improve its inhibitory efficiency were not successful to date. 3. Structural aspects The structures of the catalytic subunit of Zea mays CK2 (whose active site is nearly identical to that of its human homologue) in complex with 9 ATP site-directed inhibitors have been solved to date, as summarized in Table 1. These include emodin [31] and three emodin-related compounds [23], TBB [32] and three TBB derivatives and IQA [15]. Two general concepts can be drawn from the information available: firstly, as outlined in Fig. 2A where the structures of emodin, TBB and IQA are superimposed, the same hydrophobic pocket, which in CK2 is smaller than in the majority of other protein kinases, accommodates all these ligands regard-

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Table 1 Available structures of complexes between Zea mays CK2a and inhibitors CK2 complexes Apo CK2 Emodin MNA MNX DAA IQA TBB DMAT = K25 K44 K37

Crystallization method

Max. ˚) resolution (A

PDB code

Soaking Co-crystallization Co-crystallization Co-crystallization Co-crystallization Soaking Co-crystallization Co-crystallization Co-crystallization

2.10 2.63 2.00 1.79 1.70 1.68 2.19 1.76 1.80 2.26

1JAM 1FOQ 1M2P 1M2Q 1M2R 1OM1 1J91 1ZOE 1ZOH 1ZOG

less to substantial differences in their scaffolds. The reason why CK2 can efficiently accommodate different scaffolds into the same pocket is that the binding interaction is mainly apolar in nature (van der Waals forces and hydrophobic effect) involving the aromatic and/or apolar portions of the inhibitor always present in substantial amount, with polar interactions marginally contributing to the energetic of binding. The second general concept arising from the inspection of CK2-inhibitor complexes available is that strictly related compounds can enter the same site with remarkably different orientations, thus accounting in some cases for substantial variations in K i values. This is exemplified in Fig. 2B for three emodin related compounds and in Fig. 2C for three TBB derivatives. Note that the molecules are always co-planar, but in the former case they are displaced laterally so that they overlap different sections of the hydrophobic cavity, in the latter they enter the cavity deeper than the parent compound TBB, reaching the hinge region, where they establish halogen bonds with the backbone, and they are rotated with respect to each other. A detailed analysis reveals that in both situations weak polar interactions dictate the different orientation, which in turn is instrumental to the establishment of hydrophobic contacts that ultimately are responsible for the binding potency. This is different from what is generally observed with the inhibitors of other kinases, whose potency is primarily grounded on polar interactions, notably hydrogen bonds.

4. Generation and expression of CK2 mutants refractory to inhibition As mentioned above the selectivity of CK2 inhibitors related to either emodin or TBB or IQA is mainly ensured by the size and shape of a hydrophobic pocket partially overlapping the ATP/GTP binding site: this pocket in CK2 is smaller than in the great majority of other protein kinases, thus making possible van der Waals contacts between the surface of the ligands and the internal side chains. A few of these bulky side chains are unique to CK2, being replaced by smaller and/or polar ones in the other members of the kinase family. Especially notable are, in this respect, Val/Ile66, which is invariably replaced by Ala in all protein kinases other than CK2, and Ile174, whose structural homologue in the other protein kinases is not highly conserved but is generally endowed with a side chain which is smaller and/ or less hydrophobic than that of Ile (Ser, Ala, Thr etc). Consequently, human CK2a mutants in which either Val66 or Ile174 or both have been replaced by alanine invariably display a reduced sensitivity to the inhibitors of the emodin, TBB and IQA classes tested so far [17,28,15] with increments in IC50 values ranging between 10 and >100-fold, reflecting the different modes of binding of the individual molecules. By sharp contrast staurosporine, a potent wide spectrum protein kinase inhibitor whose efficacy on CK2 is abnormally low, displays toward the double mutant V66A,I174A a 10-fold reduced IC50 value [33] supporting the concept that the smaller size of the CK2 hydrophobic pocket not only accounts for its selective inhibition by emodin, TBB, IQA and related molecules, but also for its modest sensitivity to staurosporine. Mutants refractory to specific inhibitors represent a valuable tool for validating the implication of a kinase in cellular functions, which are altered by treatment with the inhibitors [1]. To this aim, we have now generated CHO cell lines in which human CK2a, either wild type or V66A,I174A mutant, have been ectopically expressed with a Myc-His-tag which makes possible to discriminate them from endogenous CK2a. As shown in Fig. 3A, the levels of ectopically expressed CK2a w.t. and V66A,I174A are similar and about 5-fold higher than that of endogenous CK2. As expected from the notion that the V66A,I174A mutant is normo-active [28] the activity data (Fig.

Fig. 2. Superposition of ATP-binding site directed CK2 inhibitors. (A) Emodin (red), TBB (yellow) and IQA (blue); (B) MNA (red), MNX (yellow) and DAA (blue); as reference, position of the parent compound Emodin is traced in white; (C): K37 (red), DMAT = K25 (yellow) and K44 (blue); TBB is traced in dark grey as reference. Polar interactions between residues E114 and V116 of the hinge region and DAA (2B, two hydrogen bonds) and the three TBB-derivatives (2C, two halogen bonds) are shown in white (dotted lines).

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Fig. 3. Inhibition of CK2 activity in cell transfected with pcDNAi 3.1/myc-His vector with CK2a wild type and the double mutant V66AI174A. (A) 5 Ag of total lysate proteins were loaded on 11% SDS/PAGE, blotted on Immobilon-P membrane (Millipore), and processed in Western Blot with a CK2a antibody. (B) 1 Ag of cell lysate was used to test CK2 activity. CK2 activity was measured in a final volume of 25 Al containing 50 mM Tris – HCl pH 7.5, 50 mM NaCl, 12 mM MgCl2, 1 mM synthetic peptide substrate RRRADDSDDDDD and 0.02 mM [g33P-ATP] (500 – 1000 cpm/pmol) in presence or absence of specific inhibitors, TBB and K44, for 10 min at 37 -C.

3B) correlate quite well with western blots. Fig. 3B also shows that while CK2 activity of cells transfected with CK2a w.t. is readily inhibited by TBB and K44, the activity of cells transfected with V66A,I174A is unaffected by TBB and K44 (N1,N2-ethylene-2-methylamino-4,5,6,7-tetrabromo-benzi-imidazole) up to 5 and 100 AM, respectively. In contrast, inhibition by staurosporine is reached more readily with cells transfected with the double mutant (not shown). 5. Usage of inhibitors to dissect CK2 cellular functions Given its permeability and remarkable selectivity TBB is the inhibitor that has been most frequently used in recent cell studies on CK2. In particular, it has been successfully employed either alone or in combination with other cell-permeable inhibitors to validate the identification of a number of cellular targets of CK2. These include the scaffold protein XRCC1, implicated in the machinery committed with repair of DNA single strand breaks [34], the cell cycle regulatory protein geminin [35], the cromatin protein DEK [36], yeast immunophilin Fpr3 [37], the molecular co-chaperone cdc37 [38], the C9 component of the complement system [39], the h isoform of ecto-nucleoside triphosphate diphosphohydrolase-2 [40], the human La antigen [41], a target of autoimmune antibodies in patients suffering from connective tissue disorders and the protein kinase Akt/PKB [42]. Phosphorylation of Akt by CK2 came as a surprise and disclosed an unanticipated role of CK2 in the Akt signalling

pathway initiated by PI3 kinase, by showing that phosphorylation of Akt Ser129 by CK2 correlates both in vitro and in vivo with further up-regulation of Akt already active for having been phosphorylated at the canonical residues Thr308 and Ser473 [42]. The conclusions of this study were reinforced by the combined usage of structurally unrelated CK2 inhibitors, notably TBB and IQA which were shown to have only minor effects of the phosphorylation of Akt T308 and S473, while preventing S129 phosphorylation and substantially reducing the phosphorylation of a number of Akt targets, namely GSK3, FKHR and AFX [42]. Given the central role of Akt to ensure cell survival, these data provide a rationale to explain, at least in part, the anti-apoptotic potential of CK2. Also worthy of attention is the identification among the numerous substrates of CK2 of the co-chaperone protein cdc37 which, in combination with HSP90 plays a fundamental role in subtracting a large array of protein kinases, including among others Src, Raf, cdk4, Akt, Aurora, from ubiquitination and proteasome-mediated destruction. Miyata and Nishida were able to demonstrate by various criteria, in primis usage of the specific inhibitor TBB that phosphorylation of cdc37 Ser13, which is essential for interaction with the protected protein kinases, is catalyzed by CK2 [38]. Indirectly, therefore, CK2 deeply affects the fate of a battery of protein kinases which play crucial roles in cell signalling and homeostasis. This adds a new level of complexity to the pleiotropy of CK2, which through cdc37 ultimately impinges on a variety of proteins,

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which do not belong to the repertoire of its substrates, being instead the targets of many other protein kinases whose stability is dependent on CK2 activity. 6. Pharmacological potential of CK2 inhibitors The concept that CK2 inhibitors in perspective may develop into therapeutic tools stems from the observation that CK2 on one side is a global promoter of cell survival, on the other is exploited by many viruses for the phosphorylation of proteins which are essential to their life cycle. Pertinent to this is the mounting evidence that CK2 activity counteracts apoptosis [43], a property which links CK2 to those kinds of tumors, exemplified by prostate carcinoma, whose key feature is precisely deregulation of programmed cell death. Based on these premises, a common feature of cellpermeable CK2 inhibitors would be to enhance apoptosis of cancer cells where a high level of CK2 is suspected to contribute to the tumor phenotype by dysregulating the apoptotic signals. Consistent with this prediction, TBB [44] and IQA [15] were shown to induce apoptosis of Jurkat cells. More recently, it has been shown that the same applies also to several emodin related compounds and TBB derivatives whose pro-apoptotic efficacy (measured by DC50 values) correlates fairly well with their inhibitory efficiency on CK2 (expressed by IC50 values) [22]. This observation strengthens the idea that the cytotoxic effect of these compounds is indeed mediated by CK2 inhibition, although the proof of concept will require experimentation with cells transfected with CK2 mutants refractory to inhibition (see above). In addition to Jurkat cells, which have been routinely used to test the apoptotic efficacy of CK2 inhibitors, a variety of other cancer cell lines have been shown to undergo accelerated apoptosis upon treatment with a number of these compounds. These include prostate cancer cells [45] and a wide spectrum of haematopoietic tumors, e.g., BrcAbl190 expressing lymphoma [46], Burkitt lymphomas and multiple myelomas [47]. An unanticipated efficacy of CK2 inhibitors to counteract angiogenesis and retinal neovascularization has been recently disclosed by studies on a mouse model of oxygen-induced retinopathy (OIR). In this model, the extent of retinal neovascularization was drastically reduced after treatment with emodin or with TBB [18]. Interestingly, the main vascular tree had minimal changes while the neovascular tufts were greatly reduced in number or absent after treatment with CK2 inhibitors. Incidentally, the protocol used for these experiments also provided evidence that mice can be treated for long periods of time with TBB and emodin without any severe side effect. In the same study [18], it was also shown that a number of broad specificity kinase inhibitors, including the flavonoids quercetin and apigenin, are capable of preventing tube formation and collapse, secondary sprouting and migration of cultured retinal endothelial cells from fresh bovine eyes. These compounds shared the property of being potent CK2 inhibitors. It is possible therefore that the reported anti-tumor activity of quercetin, apigenin and emodin can be at least partially accounted for by their ability to adversely

affecting tumor neovascularization, through inhibition of CK2. Another field where CK2 inhibitors might have pharmacological applications is that of human protozoan parasites. These organisms are often equipped with ecto-protein kinases, in primis CK2, which are suspected to be instrumental to their invasive strategies. These may represent especially vulnerable targets for externally added inhibitors. In this respect the development of cell impermeable CK2 inhibitors might represent a successful strategy. Another promising hint is provided from the sequence of CK2 catalytic subunit from Leishmania species, which are responsible for a spectrum of human diseases [48]. Interestingly, one of the residues of the hydrophobic pocket which is conserved in all animal and plant CK2s and plays a role in the binding ATP site-directed CK2 inhibitors, Met163, in Leishmania CK2s is replaced by Cys. This discloses the possibility of designing new molecules capable to hit the parasite but not the host CK2. 7. Conclusions and perspectives A battery of ATP site directed ligands capable to inhibit CK2 with remarkable specificity and IC50 values in the submicromolar range are available to date. Several of them are fairly stable and cell permeable and have been successfully used to validate the implication of CK2 in the phosphorylation of endogenous proteins and to demonstrate its functional link with signaling pathways. The crystal structures of complexes between CK2 catalytic subunit and 9 ATP site-directed inhibitors have been solved revealing that the selectivity of inhibition is dictated by the size and shape of a hydrophobic cavity which in CK2 is smaller than in the great majority of other protein kinases and where the ligands are entrapped, regardless of substantial differences in their scaffolds, by virtue of van der Waals interactions and hydrophobic effects. Consequently, by replacing with alanines two bulky residues which contribute to the small size of the cavity a CK2 mutant refractory to inhibition has been generated and expressed in CHO cells which will prove useful to provide the proof of the concept that CK2 is implicated in cell functions which are perturbed by treatment with the inhibitors. Among these functions are the Akt/PKB signaling pathway and apoptosis, which are down regulated and enhanced, respectively, by CK2 inhibitors. The proapoptotic efficacy of CK2 inhibitors has been invariably observed with a variety of tumor cell lines where CK2 activity is abnormally elevated and corroborates the view that increased CK2 can enhance the tumor phenotype by promoting cell survival ‘‘at all cost’’. This in perspective discloses the possibility that CK2 inhibitors might develop into leads of anti-cancer drugs, all the more so considering that the detailed knowledge of the mode of binding of these compounds provides valuable hints about how to improve their efficacy and selectivity and to reduce undesired sideeffects. Other therapeutical fields where CK2 inhibitors may prove useful are those of anti viral and anti parasite drugs.

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Many viruses in fact are dependent on the host cell CK2 for the phosphorylation of proteins, which are essential to their life cycle. On the other side, protozoan parasites not only encode their own CK2 essential to survival, but also are often equipped with ecto-CK2 suspected to be instrumental to their invasive strategy. Interestingly, the CK2 pharmacophore of some protozoan parasites is significantly different from that of human CK2. In the case of Leishmania species, responsible for world wide diseases, one of the residues implicated in apolar interactions with ATP site-directed inhibitors, M163, is replaced by Cys, a circumstance which discloses the possibility of designing CK2 inhibitors able to discriminate between the parasite and the host cell CK2.

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