An Unbiased Evaluation of CK2 Inhibitors by Chemoproteomics

Abstract

Recently protein kinases have emerged as some of the most promising drug targets; and therefore, pharmaceutical strategies have been developed to inhibit kinases in the treatment of a variety of diseases. CK2 is a serine/threonine-protein kinase that has been implicated in a number of cellular processes, including maintenance of cell viability, protection of cells from apoptosis, and tumorigenesis. Elevated CK2 activity has been established in a number of cancers where it was shown to promote tumorigenesis via the regulation of the activity of various oncogenes and tumor suppressor proteins. Consequently the development of CK2 inhibitors has been ongoing in preclinical studies, resulting in the generation of a number of CK2-directed compounds. In the present study, an unbiased evaluation of CK2 inhibitors 4,5,6,7-tetrabromo-1H-benzotriazole (TBB), 4,5,6,7-tetrabromo-1H-benzimidazole (TBBz), and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) was carried out to elucidate the mechanism of action as well as inhibitor specificity of these compounds. Utilizing a chemoproteomics approach in conjunction with inhibitor-resistant mutant studies, CK2α and CK2α′ were identified as bona fide targets of TBB, TBBz, and DMAT in cells. However, inhibitor-specific cellular effects were observed indicating that the structurally related compounds had unique biological properties, suggesting differences in inhibitor specificity. Rescue experiments utilizing inhibitor-resistant CK2 mutants were unable to rescue the apoptosis associated with TBBz and DMAT treatment, suggesting the inhibitors had off-target effects. Exploitation of an unbiased chemoproteomics approach revealed a number of putative off-target inhibitor interactions, including the discovery of a novel TBBz and DMAT (but not TBB) target, the detoxification enzyme quinone reductase 2 (QR2). The results described in the present study provide insight into the molecular mechanism of action of the inhibitors as well as drug specificity that will assist in the development of more specific next generation CK2 inhibitors. Recently protein kinases have emerged as some of the most promising drug targets; and therefore, pharmaceutical strategies have been developed to inhibit kinases in the treatment of a variety of diseases. CK2 is a serine/threonine-protein kinase that has been implicated in a number of cellular processes, including maintenance of cell viability, protection of cells from apoptosis, and tumorigenesis. Elevated CK2 activity has been established in a number of cancers where it was shown to promote tumorigenesis via the regulation of the activity of various oncogenes and tumor suppressor proteins. Consequently the development of CK2 inhibitors has been ongoing in preclinical studies, resulting in the generation of a number of CK2-directed compounds. In the present study, an unbiased evaluation of CK2 inhibitors 4,5,6,7-tetrabromo-1H-benzotriazole (TBB), 4,5,6,7-tetrabromo-1H-benzimidazole (TBBz), and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) was carried out to elucidate the mechanism of action as well as inhibitor specificity of these compounds. Utilizing a chemoproteomics approach in conjunction with inhibitor-resistant mutant studies, CK2α and CK2α′ were identified as bona fide targets of TBB, TBBz, and DMAT in cells. However, inhibitor-specific cellular effects were observed indicating that the structurally related compounds had unique biological properties, suggesting differences in inhibitor specificity. Rescue experiments utilizing inhibitor-resistant CK2 mutants were unable to rescue the apoptosis associated with TBBz and DMAT treatment, suggesting the inhibitors had off-target effects. Exploitation of an unbiased chemoproteomics approach revealed a number of putative off-target inhibitor interactions, including the discovery of a novel TBBz and DMAT (but not TBB) target, the detoxification enzyme quinone reductase 2 (QR2). The results described in the present study provide insight into the molecular mechanism of action of the inhibitors as well as drug specificity that will assist in the development of more specific next generation CK2 inhibitors. Targeting protein kinases for therapeutic applications has become a rapidly evolving field with a number of successful inhibitors in clinical studies or approved for treatment of various cancers. Loss of regulation of protein kinase activity has been identified as a key event in the development of cancer and has therefore become an avenue of intense research. Specifically the success of targeted therapies such as Gleevec (imatinib) for the treatment of chronic myelogenous leukemia has provided a model for the development of novel inhibitors targeting a wide variety of protein kinases (1Jabbour E. Cortes J.E. Giles F.J. O'Brien S. Kantarjian H.M. Current and emerging treatment options in chronic myeloid leukemia.Cancer. 2007; 109: 2171-2181Crossref PubMed Scopus (107) Google Scholar). Recently protein kinases have emerged as some of the most promising drug targets, representing as much as 20% of the “druggable” genome (2Hopkins A.L. Groom C.R. The druggable genome.Nat. Rev. Drug Discov. 2002; 1: 727-730Crossref PubMed Scopus (2655) Google Scholar, 3Pagano M.A. Cesaro L. Meggio F. Pinna L.A. Protein kinase CK2: a newcomer in the ‘druggable kinome'.Biochem. Soc. Trans. 2006; 34: 1303-1306Crossref PubMed Scopus (63) Google Scholar). Ongoing preclinical studies targeting serine/threonine kinases have been carried out demonstrating the potential of kinase inhibitors as anticancer therapeutics, including the study of protein kinase CK2. CK2 (formally known as casein kinase II) is a ubiquitously distributed, constitutively active serine/threonine kinase implicated in multiple cellular processes, including cell survival, protection of cells from apoptosis, and tumorigenesis (4Litchfield D.W. Protein kinase CK2: structure, regulation and role in cellular decisions of life and death.Biochem. J. 2003; 369: 1-15Crossref PubMed Scopus (1038) Google Scholar). Elevated CK2 activity has been documented in a number of cancers where high CK2 activity has been correlated with aggressive tumor behavior (5Faust R.A. Tawfic S. Davis A.T. Bubash K. Ahmed K. Antisense oligonucleotides against protein kinase CK2-α inhibit growth of squamous cell carcinoma of the head and neck in vitro.Head Neck. 2000; 22: 341-346Crossref PubMed Scopus (77) Google Scholar). Detailed studies have been carried out to elucidate the molecular mechanism of CK2 in tumorigenesis, demonstrating that increased CK2 activity promotes a number of pathways participating in the development of cancer (6Daya-Makin M. Sanghera J.S. Mogentale T. Lipp M. Parchomchuk J. Hogg J. Pelech S. Activation of a tumour-associated protein kinase (p40TAK) and casein kinase II in human squamous cell carcinomas and adenocarcinomas of the lung.Cancer Res. 1994; 54: 2262-2268PubMed Google Scholar, 7Landesman-Bollag E. Romieu-Mourez R. Song D.H. Sonenshein G.E. Cardiff R.D. Seldin D.C. Protein kinase CK2 in mammary gland tumorigenesis.Oncogene. 2001; 20: 3247-3257Crossref PubMed Scopus (269) Google Scholar). Proposed modes of action of CK2 in tumorigenesis are through the regulation of key oncogenes and tumor suppressor proteins within various prosurvival pathways including the Wnt (8Song D.H. Dominguez I. Mizuno J. Kaut M. Mohr S.C. Seldin D.C. CK2 phosphorylation of the armadillo repeat region of β-catenin potentiates Wnt signaling.J. Biol. Chem. 2003; 278: 24018-24025Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), nuclear factor-κB (9Barroga C.F. Stevenson J.F. Schwarz E.M. Verma I.M. Constitutive phosphorylation of I kappa B alpha by casein kinase II.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7637-7641Crossref PubMed Scopus (140) Google Scholar), and phosphatidylinositol 3-kinase pathways (10Di Maira G. Salvi M. Arrigoni G. Marin O. Sarno S. Brustolon F. Pinna L.A. Ruzzene M. Protein kinase CK2 phosphorylates and upregulates Akt/PKB.Cell Death Differ. 2005; 12: 668-677Crossref PubMed Scopus (269) Google Scholar). Regulation of oncogene and tumor suppressor proteins by CK2 phosphorylation has been shown to influence susceptibility to proteosome degradation, protect proteins from caspase-mediated cleavage, and alter the protein activity (11Duncan J.S. Litchfield D.W. Too much of a good thing: the role of protein kinase CK2 in tumorigenesis and prospects for therapeutic inhibition of CK2.Biochim. Biophys. Acta. 2008; 1784: 33-47Crossref PubMed Scopus (268) Google Scholar). Taken together, elevated CK2 activity promotes a prosurvival signal in cancer by facilitating the transcription, activation, or stability of oncogenes and antiapoptotic proteins while repressing tumor suppressor activity through increased proteosome degradation. Recently an antiapoptotic role of CK2 has emerged as a number of substrates were identified that exhibited protection from CK2 phosphorylation S. A. S. E. F. A. L. of by casein kinases I and II by 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, A. R. R. Targeting of the by an in J. 2001; PubMed Scopus (107) Google Scholar, M. Pinna L.A. Protein kinase CK2 inhibitor apoptosis and of protein in J. 2002; PubMed Scopus Google Scholar, J. A. J. of by and of Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S. The of by is by casein kinase Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Consequently CK2 has emerged as a promising for therapeutic in the treatment of of specific protein kinase inhibitors has been as cellular proteins as a of unique of kinases has provided an avenue to inhibitors. number of inhibitors have been developed to CK2 the structure, unique that CK2 from the of kinases S. Ruzzene M. M.A. Meggio F. A. M. Maira Pinna L.A. and of CK2 2005; PubMed Scopus Google Scholar). CK2 was to have a with protein to the of that are for Exploitation of these within the has provided to CK2 inhibitors. the of a known CK2 a novel 4,5,6,7-tetrabromo-1H-benzotriazole TBB, TBBz, with with of cell quinone reductase was M.A. M. Ruzzene M. Sarno S. Cesaro L. J. M. Meggio F. Pinna L.A. of protein kinase CK2 inhibitors from Chem. PubMed Scopus Google that a high for CK2 to and with the in the exhibited high for CK2 in a of were a of kinases that were to the including specificity kinase was to the as kinase and kinase were to a strategies to the of the inhibitors were compounds that had with the unique Utilizing the as a next generation were 4,5,6,7-tetrabromo-1H-benzimidazole and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) M.A. Meggio F. Ruzzene M. M. Pinna L.A. a novel and inhibitor of protein kinase Biophys. Res. PubMed Scopus Google Scholar, M. K. R. of as an inhibitor of protein kinase CK2 from various Biophys. Res. 2003; PubMed Scopus Google Scholar). In studies the of DMAT that of the and the of in a and CK2 with a of In study, an unbiased evaluation of and related CK2 inhibitors utilizing chemoproteomics was carried out to the potential of the inhibitors as anticancer study of the specificity of the CK2 inhibitors TBB, TBBz, and DMAT for CK2 as well as the cellular effects associated with inhibitor was carried inhibitors in the of apoptosis were observed treatment of cells with TBB, TBBz, or indicating that the inhibitors have unique biological as well as the potential for off-target effects. rescue experiments utilizing inhibitor-resistant CK2 mutants were to the specificity of the the apoptosis associated with drug treatment was The to rescue and apoptosis with the mutants a of inhibitor protein targets utilizing a chemoproteomics and cells were in and or and cells were with inhibitor-resistant or in to with a of were with TBBz or DMAT a of for and were an a cells were in and were to and for in chemoproteomics CK2 mutants were a II was to the are shown in the and the was to the the and the cells were treatment with or CK2 inhibitors. were in and a cell to cell were by the of and were in and the was with and in the for were with to and by and representing the of in cell were and cells were from and in and were and protein were by were by by to were with or for by an with the in or were with against the and against the were with or and with for and for and CK2α′ for and for and were with or and by was developed and to a a were in cells were with or inhibitor-resistant and with a of cells were with and TBB, TBBz, or DMAT for the of to rescue cells were with or and and with or DMAT for were in and and the protein was by of protein were with of protein and of a protein were by and were in for and to were by and to a by were with and cells in were in and and for were and to for of were with and TBBz or TBB, TBBz, or DMAT for 2 with described for the of protein to of protein kinase J. PubMed Scopus Google were to the the inhibitors and for to for were by and with of by a with and were with A. were in for with Protein were by of protein from treatment were by and inhibitor treatment, cells were from and by were via to and Protein from with proteins were in 2 and Protein were by Protein were with 2 and and to or in The was carried out the II the for the for for for for for for and for The was for the for for 2 for for for for for and for The were in by The were to a with and for the were protein were and with were and the in protein were utilizing and from treatment were into and were The were into for of differences and was to to for and with a of of were and the were with a where of with and of differences were from or by the and in and was the were in with in and by the was carried out with an of and of with a of was in conjunction with an with a of a of to and a of with more of and the of and were against the human 2007; with as a inhibition of CK2 has been shown to various cancer cell to apoptosis as well as apoptosis M.A. Meggio F. Ruzzene M. M. Pinna L.A. a novel and inhibitor of protein kinase Biophys. Res. PubMed Scopus Google Scholar, K. L. A. of casein kinase II in tumor apoptosis in human Res. PubMed Scopus Google Scholar, K. L. A. kinase II activity in apoptosis in human cancer cell 2005; PubMed Scopus Google Scholar, J.S. J. M. Litchfield D.W. and as inhibitors of protein kinase CK2: evaluation of effects cells and molecular of human CK2.Biochim. Biophys. Acta. 2005; PubMed Scopus Google Scholar). number of specific compounds have been developed to CK2 in cells including and TBBz and the of these inhibitors cancer cell viability, an evaluation of apoptosis was utilizing cells The of was as a biological of apoptosis and a known was as a the drug for the inhibitors was as a of apoptosis was by the of of treatment with TBB, TBBz, and the of apoptosis was observed of treatment with TBBz and DMAT not treatment with the of that the have to apoptosis the where TBBz and DMAT were the most inhibitors. The of apoptosis by TBBz and DMAT was a in drug in a more as is by an of the biological of cells with CK2 inhibitors TBB, TBBz, and in protein cells were with TBB, TBBz, or for and to and the were with differences were identified from the and to for protein 2 and were identified in the TBBz and DMAT (but not in or as by The were with demonstrating a apoptosis where was to by and into with molecular of and F. R. M. of and promotes Death Differ. 2001; PubMed Scopus Google Scholar). was identified as a present in the (but not or by The of by and apoptosis has been shown in studies and results in the generation of including a of and of cell Biol. PubMed Scopus Google Scholar). an of the of and cells provided for apoptosis as well as that the apoptosis by TBBz and DMAT was The of apoptosis TBBz and DMAT treatment a unique mechanism of action to The of a number of CK2 inhibitors to apoptosis in various cancer cells promising for targeting CK2 for in utilizing CK2 inhibitors have high CK2 kinase However, the specificity and mechanism of action of these inhibitors have not been in an unbiased the of TBB, TBBz, and DMAT to inhibit CK2 activity in the of by was The of has been shown to for stability and to a of of CK2 in with in the Litchfield D.W. of protein kinase CK2: of and mutant J. 2001; PubMed Scopus Google Scholar, K. Litchfield D.W. The of protein kinase CK2 of CK2 Biol. Chem. 2000; Full Text Full Text PDF Scopus Google Scholar, S. T. of protein kinase CK2 as 2005; PubMed Scopus Google Scholar, G. Litchfield D.W. the stability of the 2002; PubMed Scopus Google Scholar). In studies the of CK2 inhibitor-resistant mutants have been and that the mutants are and to TBB, TBBz, and DMAT S. Ruzzene M. M.A. Meggio F. A. M. Maira Pinna L.A. and of CK2 2005; PubMed Scopus Google Scholar, J.S. J. M. Litchfield D.W. and as inhibitors of protein kinase CK2: evaluation of effects cells and molecular of human CK2.Biochim. Biophys. Acta. 2005; PubMed Scopus Google Scholar). the of the inhibitors and to drug cells or in to were with TBBz, or as well as as a for The mutant was to CK2 Litchfield D.W. of a of the that cell Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). CK2α and were from and the of were with treatment of cells CK2 in the of the CK2 inhibitors in the of as with treatment with the drug as was by the CK2α and the of the inhibitor-resistant CK2 the of as was by of and increased CK2α and of the inhibitor-resistant the of CK2α and in the indicating that of CK2α kinase activity was of CK2α′ mutants were shown to of as well as the of of associated with inhibitor treatment the treatment of cells with CK2 inhibitors TBB, TBBz, and DMAT in the inhibition of of by The rescue of the of by that the inhibitors CK2 kinase activity in cells that by of CK2. number of inhibitors of CK2 including TBB, TBBz, and DMAT have been shown to the of various cancer cell M.A. Meggio F. Ruzzene M. M. Pinna L.A. a novel and inhibitor of protein kinase Biophys. Res. PubMed Scopus Google Scholar, J.S. J. M. Litchfield D.W. and as inhibitors of protein kinase CK2: evaluation of effects cells and molecular of human CK2.Biochim. Biophys. Acta. 2005; PubMed Scopus Google Scholar, S. Meggio F. Ruzzene M. A. Pinna L.A. of an inhibitor of protein kinase CK2 2001; PubMed Scopus Google Scholar). was observed in the present study in treatment of cells with TBBz, and to a apoptosis in a and In rescue it was shown that TBB, TBBz, and DMAT the of in cells that by inhibitor-resistant CK2 the associated with TBB, and TBBz was to inhibition of CK2 rescue experiments utilizing inhibitor-resistant CK2 were carried cells and were with or TBB, or TBBz for and apoptosis was by for by and by the of cells in the via cells the inhibitor-resistant of CK2 of apoptosis treatment with TBBz or as by the of as well as a of cells in the with apoptosis was observed in cells with or of CK2. that the apoptosis associated with TBBz and DMAT was not to of CK2 activity as via of inhibitor-resistant mutants was not of cells from treatment of cells with in a of CK2 activity as was by the of of The of TBBz and DMAT in a apoptosis, drug the of off-target drug effects and In that the associated with TBBz and DMAT not to the inhibition of CK2 the inhibition of proteins that are for cell CK2 as an inhibitor target, a chemoproteomics was a approach was to the specificity of TBB, TBBz, and DMAT for CK2. inhibitor was of of cell with or TBB, or TBBz by the of the inhibitor with an protein with it that protein from to the CK2 was a bona fide of TBB, or TBBz cellular a of the proteins to the inhibitor treatment was via for CK2α and CK2α′ of cell with TBB, or TBBz CK2α and CK2α′ from to the to the the in CK2α′ observed in the of TBBz to by the inhibitor to indicating a was as a was by the inhibitor Taken together, CK2α and CK2α′ were to with TBB, and TBBz CK2 as a bona fide of and the of CK2 as a of the a more of inhibitor protein was utilizing the chemoproteomics The inhibitor as described was in with and to potential drug of the proteins to the was utilizing and the protein were number of putative inhibitor drug targets were identified by as present the and the CK2 including B protein and the protein quinone reductase 2 and was identified as present in the and not in the and that an inhibitor for a novel TBBz and DMAT drug The were by to differences and treatment were a from the and was to a with a of was carried out the to protein The were by and was identified with a of The of a chemoproteomics to TBB, TBBz, and DMAT protein targets revealed that CK2 is a for the discovery of novel targets that with TBBz and DMAT the of the specificity of these CK2 inhibitors. The role of CK2 in the of tumorigenesis has become as the of a number of pathways has been to high of CK2 Consequently preclinical studies CK2 as a molecular therapeutic have been resulting in the development of various inhibitors including the structurally related compounds TBB, TBBz, and DMAT However, the molecular mechanism by these in cells has not been unbiased of CK2 inhibitors was to CK2 as a anticancer as well as to the potential of the inhibitors to study CK2 The inhibitors to apoptosis in cells suggesting a mechanism of of the of the inhibitor to CK2 activity in cells was by the of of by that was in cells inhibitor-resistant CK2 Rescue experiments inhibitor specificity were carried out and that the apoptosis observed DMAT and TBBz treatment not by of CK2. CK2 as an inhibitor target, a chemoproteomics approach was carried out that that CK2α and CK2α′ were TBB, TBBz, and DMAT a number of putative off-target drug were including a novel TBBz and DMAT target, The discovery of as a putative CK2 inhibitor drug for the effects associated with the inhibitors as well as the of a therapeutic potential for TBBz and DMAT in diseases. The unbiased evaluation of CK2 inhibitors that TBBz and DMAT have potential off-target to the role of CK2 in tumorigenesis, inhibition of kinase a promising anticancer therapeutic The for kinase inhibitor specificity has been the of in a number of kinases from are for an M.A. Cesaro L. Meggio F. Pinna L.A. Protein kinase CK2: a newcomer in the ‘druggable kinome'.Biochem. Soc. Trans. 2006; 34: 1303-1306Crossref PubMed Scopus (63) Google Scholar). The to has become as the are not and to the of including key and proteins. The chemoproteomics approach an unbiased of proteins through in conjunction with and R. K. M. of novel targets of in the human 2002; PubMed Scopus Google Scholar). chemoproteomics approach revealed that the CK2 inhibitors CK2α and CK2α′ from to in a that CK2 was a of TBB, TBBz, and the that TBBz was not a to inhibit CK2α′ from to the an to apoptosis in cells that the apoptosis was not to the inhibition of CK2. was to off-target that provide an for the of inhibitor-resistant CK2 mutants to rescue the apoptosis associated with TBBz and of the proteins with CK2 inhibitors revealed a number of proteins in cell survival, and drug detoxification of putative off-target CK2 inhibitor B by protein by by Protein by Protein by in a of from to was observed with TBBz and DMAT not TBB, indicating drug differences inhibitor has a role in the detoxification of the of by the of or F. G. reductase an enzyme of and 2005; PubMed Scopus Google Scholar). of in in an increased susceptibility to inhibition of in of cells by the of the cell R. K. M. of novel targets of in the human 2002; PubMed Scopus Google Scholar, J. mechanism of quinone 2 and inhibition by the 20: Scopus Google Scholar). Recently a role for in the tumor has emerged in were shown to have and increased tumor apoptosis G. of 2 in of apoptosis with of cell Res. 2007; PubMed Scopus Google Scholar). studies the mechanism of action of protein kinase and protein kinase kinase inhibitors as a novel drug target, suggesting a potential role for in the drug K. J. of cellular targets by protein kinase Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. M. S. T. J. M. G. A. T. U. G. J. G. of action of clinical kinase 2007; PubMed Scopus Google Scholar, U. O. G. M. I. J. T. G. of the inhibitors and novel kinase and 2007; PubMed Scopus Google Scholar). In the present study, the discovery of as a novel TBBz and DMAT drug to the mechanism by cell is treatment with CK2 inhibitors. a and known inhibitor of CK2 with an has been described as a inhibitor of with a of L. M. of quinone reductase 2 in with PubMed Scopus Google Scholar, S. Salvi M. R. G. Pinna L.A. and of CK2 Biophys. Acta. 2005; PubMed Scopus Google Scholar). The of with the inhibitor has been and that the active was of a the of L. M. of quinone reductase 2 in with PubMed Scopus Google Scholar). The of the of CK2 inhibitors that were to with the within the CK2 The of to inhibitors for CK2 in an as well as the of to the with the of CK2 M.A. Meggio F. Ruzzene M. M. Pinna L.A. a novel and inhibitor of protein kinase Biophys. Res. PubMed Scopus Google Scholar). the and of TBBz and DMAT into the or to studies have shown that has the potential to into a indicating the that TBBz and DMAT L. of quinone reductase 2 in with cancer Biophys. Res. 2005; PubMed Scopus Google Scholar). The of to to an as to TBBz and DMAT are more TBBz and DMAT to and inhibit the of the protein to in or by into more compounds facilitating an off-target However, studies are to the of TBBz and DMAT to inhibit in as well as in will of to the of TBBz and DMAT to inhibit as in the study of such as The of the inhibitors to study the role of CK2 in will and the of rescue experiments to the The of a number of putative that the study of inhibitors such as CK2 inhibitors will to the specificity of the inhibitors in cells. the of the drug within various cells will in the specificity of inhibitors. cellular targets that are to the inhibitors or by the to that of the strategies to inhibit CK2 to including compounds that inhibit CK2 via of the The of inhibitors specific for CK2 has been a promising avenue of as a number of have developed and the inhibition of CK2 in cells. of CK2 a in the of CK2 activity and of tumor growth in a CK2 as a promising drug O. A. R. A. O. O. of inhibitors of protein kinase CK2 J. 2007; PubMed Scopus Google Scholar). and for of the within the of and the of as well as for into the of and from the the of for in the and of as well as for in the of with