Skip to main content

Identification of novel CK2 inhibitors with a benzofuran scaffold by novel non-radiometric in vitro assays

Molecular and Cellular Biochemistry volume 356, pages 83–90 (2011)Cite this article

Abstract

Protein kinase CK2 is emerging as a target in neoplastic diseases. Inhibition of CK2 by small compounds could lead to new therapies by counteracting the elevated CK2 activities found in a variety of tumors. Currently, CK2 inhibitors are primarily evaluated by a radiometric in vitro assay tracing the amount of transferred γ-32P from ATP to a substrate peptide. Here, we present two alternative assays abandoning radioisotopes. The first assay is based on Förster resonance energy transfer between the fluorescence donor EDANS and the acceptor molecule DABCYL within the CK2 substrate peptide [DABCYL]-RRRDDDSDDD-[EDANS]. This peptide comprises a cleavage site for pancreatic elastase, which is located next to the phosphate acceptor serine. Only the non-phosphorylated peptide can be cleaved by elastase, disrupting the FRET effect. Thus fluorescence intensity is inversely correlated with CK2 activity. The second non-radiometric assay deploys the changing of charge that occurs within the peptide substrate RRRDDDSDDD upon phosphorylation by CK2. Substrate and product of a CK2 reaction consequently show a difference in electrophoretic mobility and thus can be separated by capillary electrophoresis. Absorption detection enabled quantification of both peptide species and allowed the determination of IC50 values. This method facilitated the testing of a small compound library by which benzofuran derivatives were identified as potent CK2 inhibitors with IC50 values in the submicromolar range.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

39,95 €

Price includes VAT (Finland)

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100(1):57–70

    Article  PubMed  CAS  Google Scholar 

  2. Pagano MA, Cesaro L, Meggio F, Pinna LA (2006) Protein kinase CK2: a newcomer in the ‘druggable kinome’. Biochem Soc Trans 34(Pt 6):1303–1306. doi:10.1042/BST0341303

    PubMed  CAS  Google Scholar 

  3. Cozza G, Bortolato A, Moro S (2010) How druggable is protein kinase CK2? Med Res Rev 30(3):419–462. doi:10.1002/med.20164

    Article  PubMed  CAS  Google Scholar 

  4. Sarno S, Pinna LA (2008) Protein kinase CK2 as a druggable target. Mol BioSyst 4(9):889–894. doi:10.1039/b805534c

    Article  PubMed  CAS  Google Scholar 

  5. Guerra B, Issinger O-G (2008) Protein kinase CK2 in human diseases. Curr Med Chem 15(19):1870–1886

    Article  PubMed  CAS  Google Scholar 

  6. Daya-Makin M, Sanghera JS, Mogentale TL, Lipp M, Parchomchuk J, Hogg JC, Pelech SL (1994) Activation of a tumor-associated protein kinase (p40TAK) and casein kinase 2 in human squamous cell carcinomas and adenocarcinomas of the lung. Cancer Res 54(8):2262–2268

    PubMed  CAS  Google Scholar 

  7. Landesman-Bollag E, Romieu-Mourez R, Song DH, Sonenshein GE, Cardiff RD, Seldin DC (2001) Protein kinase CK2 in mammary gland tumorigenesis. Oncogene 20(25):3247–3257. doi:10.1038/sj.onc.1204411

    Article  PubMed  CAS  Google Scholar 

  8. Münstermann U, Fritz G, Seitz G, Lu YP, Schneider HR, Issinger OG (1990) Casein kinase II is elevated in solid human tumours and rapidly proliferating non-neoplastic tissue. Eur J Biochem 189(2):251–257

    Article  PubMed  Google Scholar 

  9. Stalter G, Siemer S, Becht E, Ziegler M, Remberger K, Issinger O-G (1994) Asymmetric expression of protein kinase CK2 subunits in human kidney tumors. Biochem Biophys Res Commun 202(1):141–147. doi:10.1006/bbrc.1994.1904

    Article  PubMed  CAS  Google Scholar 

  10. Yenice S, Davis AT, Goueli SA, Akdas A, Limas C, Ahmed K (1994) Nuclear casein kinase 2 (CK-2) activity in human normal, benign hyperplastic, and cancerous prostate. Prostate 24(1):11–16

    Article  PubMed  CAS  Google Scholar 

  11. Laramas M, Pasquier D, Filhol O, Ringeisen F, Descotes J-L, Cochet C (2007) Nuclear localization of protein kinase CK2 catalytic subunit (CK2alpha) is associated with poor prognostic factors in human prostate cancer. Eur J Cancer 43(5):928–934. doi:10.1016/j.ejca.2006.11.021

    Article  PubMed  CAS  Google Scholar 

  12. Llobet D, Eritja N, Encinas M, Llecha N, Yeramian A, Pallares J, Sorolla A, Gonzalez-Tallada FJ, Matias-Guiu X, Dolcet X (2008) CK2 controls TRAIL and Fas sensitivity by regulating FLIP levels in endometrial carcinoma cells. Oncogene 27(18):2513–2524. doi:10.1038/sj.onc.1210924

    Article  PubMed  CAS  Google Scholar 

  13. Roig J, Krehan A, Colomer D, Pyerin W, Itarte E, Plana M (1999) Multiple forms of protein kinase CK2 present in leukemic cells: in vitro study of its origin by proteolysis. Mol Cell Biochem 191(1–2):229–234

    Article  PubMed  CAS  Google Scholar 

  14. Unger G, Davis AT, Slaton JW, Ahmed K (2004) Protein kinase CK2 as regulator of cell survival: implications for cancer therapy. Curr Cancer Drug Targets 4(1):77–84

    Article  PubMed  Google Scholar 

  15. Tawfic S, Yu S, Wang H, Faust R, Davis A, Ahmed K (2001) Protein kinase CK2 signal in neoplasia. Histol Histopathol 16(2):573–582

    PubMed  CAS  Google Scholar 

  16. Trembley JH, Wang G, Unger G, Slaton J, Ahmed K (2009) Protein kinase CK2 in health and disease: CK2: a key player in cancer biology. Cell Mol Life Sci 66(11–12):1858–1867. doi:10.1007/s00018-009-9154-y

    Article  PubMed  CAS  Google Scholar 

  17. Ruzzene M, Pinna LA (2010) Addiction to protein kinase CK2: a common denominator of diverse cancer cells? Biochim Biophys Acta 1804(3):499–504. doi:10.1016/j.bbapap.2009.07.018

    PubMed  CAS  Google Scholar 

  18. Meggio F, Pinna LA (2003) One-thousand-and-one substrates of protein kinase CK2? FASEB J 17(3):349–368. doi:10.1096/fj.02-0473rev

    Article  PubMed  CAS  Google Scholar 

  19. Salvi M, Sarno S, Cesaro L, Nakamura H, Pinna LA (2009) Extraordinary pleiotropy of protein kinase CK2 revealed by weblogo phosphoproteome analysis. Biochim Biophys Acta 1793(5):847–859. doi:10.1016/j.bbamcr.2009.01.013

    Article  PubMed  CAS  Google Scholar 

  20. Guerra B (2006) Protein kinase CK2 subunits are positive regulators of AKT kinase. Int J Oncol 28(3):685–693

    PubMed  CAS  Google Scholar 

  21. Seldin DC, Landesman-Bollag E, Farago M, Currier N, Lou D, Dominguez I (2005) CK2 as a positive regulator of Wnt signalling and tumourigenesis. Mol Cell Biochem 274(1–2):63–67

    Article  PubMed  CAS  Google Scholar 

  22. Di Maira G, Brustolon F, Pinna LA, Ruzzene M (2009) Dephosphorylation and inactivation of Akt/PKB is counteracted by protein kinase CK2 in HEK 293T cells. Cell Mol Life Sci 66(20):3363–3373. doi:10.1007/s00018-009-0108-1

    Article  PubMed  Google Scholar 

  23. Dominguez I, Sonenshein GE, Seldin DC (2009) Protein kinase CK2 in health and disease: CK2 and its role in Wnt and NF-kappaB signaling: linking development and cancer. Cell Mol Life Sci 66(11–12):1850–1857. doi:10.1007/s00018-009-9153-z

    Article  PubMed  CAS  Google Scholar 

  24. Allende-Vega N, Dias S, Milne D, Meek D (2005) Phosphorylation of the acidic domain of Mdm2 by protein kinase CK2. Mol Cell Biochem 274(1–2):85–90

    Article  PubMed  CAS  Google Scholar 

  25. Scaglioni PP, Yung TM, Choi SC, Baldini C, Konstantinidou G, Pandolfi PP (2008) CK2 mediates phosphorylation and ubiquitin-mediated degradation of the PML tumor suppressor. Mol Cell Biochem 316(1–2):149–154. doi:10.1007/s11010-008-9812-7

    Article  PubMed  CAS  Google Scholar 

  26. Druker BJ (2009) Perspectives on the development of imatinib and the future of cancer research. Nat Med 15(10):1149–1152. doi:10.1038/nm1009-1149

    Article  PubMed  CAS  Google Scholar 

  27. Cohen P (2002) Protein kinases—the major drug targets of the twenty-first century? Nat Rev Drug Discov 1(4):309–315

    Article  PubMed  CAS  Google Scholar 

  28. Eglen RM, Reisine T (2009) The current status of drug discovery against the human kinome. Assay Drug Dev Technol 7(1):22–43. doi:10.1089/adt.2008.164

    Article  PubMed  CAS  Google Scholar 

  29. Prudent R, Moucadel V, López-Ramos M, Aci S, Laudet B, Mouawad L, Barette C, Einhorn J, Einhorn C, Denis J-N, Bisson G, Schmidt F, Roy S, Lafanechere L, Florent J-C, Cochet C (2008) Expanding the chemical diversity of CK2 inhibitors. Mol Cell Biochem 316(1–2):71–85. doi:10.1007/s11010-008-9828-z

    Article  PubMed  CAS  Google Scholar 

  30. Laudet B, Moucadel V, Prudent R, Filhol O, Wong Y-S, Royer D, Cochet C (2008) Identification of chemical inhibitors of protein-kinase CK2 subunit interaction. Mol Cell Biochem 316(1–2):63–69. doi:10.1007/s11010-008-9821-6

    Article  PubMed  CAS  Google Scholar 

  31. Siddiqui-Jain A, Drygin D, Streiner N, Chua P, Pierre F, O’Brien SE, Bliesath J, Omori M, Huser N, Ho C, Proffitt C, Schwaebe MK, Ryckman DM, Rice WG, Anderes K (2010) CX-4945, an orally bioavailable selective inhibitor of protein kinase CK2, inhibits prosurvival and angiogenic signaling and exhibits antitumor efficacy. Cancer Res 70(24):10288–10298. doi:10.1158/0008-5472.CAN-10-1893

    Article  PubMed  CAS  Google Scholar 

  32. Pierre F, Chua PC, O’Brien SE, Siddiqui-Jain A, Bourbon P, Haddach M, Michaux J, Nagasawa J, Schwaebe MK, Stefan E, Vialettes A, Whitten JP, Chen TK, Darjania L, Stansfield R, Anderes K, Bliesath J, Drygin D, Ho C, Omori M, Proffitt C, Streiner N, Trent K, Rice WG, Ryckman DM (2011) Discovery and SAR of 5-(3-chlorophenylamino)benzo[c][2,6]naphthyridine-8-carboxylic acid (CX-4945), the first clinical stage inhibitor of protein kinase CK2 for the treatment of cancer. J Med Chem 54(2):635–654. doi:10.1021/jm101251q

    Article  PubMed  CAS  Google Scholar 

  33. Hastie CJ, McLauchlan HJ, Cohen P (2006) Assay of protein kinases using radiolabeled ATP: a protocol. Nat Protoc 1(2):968–971. doi:10.1038/nprot.2006.149

    Article  PubMed  CAS  Google Scholar 

  34. Jia Y, Quinn CM, Kwak S, Talanian RV (2008) Current in vitro kinase assay technologies: the quest for a universal format. Curr Drug Discov Technol 5(1):59–69

    Article  PubMed  CAS  Google Scholar 

  35. Hung M-S, Xu Z, Lin Y-C, Mao J-H, Yang C-T, Chang P-J, Jablons DM, You L (2009) Identification of hematein as a novel inhibitor of protein kinase CK2 from a natural product library. BMC Cancer 9:135 9:135. doi:10.1186/1471-2407-9-135

  36. Ferguson AD, Sheth PR, Basso AD, Paliwal S, Gray K, Fischmann TO, Le HV (2010) Structural basis of CX-4945 binding to human protein kinase CK2. FEBS Lett. doi:10.1016/j.febslet.2010.11.019

  37. Kaessler A, Olgen S, Jose J (2011) Autodisplay of catalytically active human hyaluronidase hPH-20 and testing of enzyme inhibitors. Eur J Pharm Sci 42(1–2):138–147. doi:10.1016/j.ejps.2010.11.004

    Article  PubMed  CAS  Google Scholar 

  38. Jose J (2009) Bioanalytics in medicinal chemistry: from assay development to evolutive drug design. Ann Pharm Fr 67(6):399–407. doi:10.1016/j.pharma.2009.07.002

    PubMed  CAS  Google Scholar 

  39. Jose J, Betscheider D, Zangen D (2005) Bacterial surface display library screening by target enzyme labeling: identification of new human cathepsin G inhibitors. Anal Biochem 346(2):258–267. doi:10.1016/j.ab.2005.08.019

    Article  PubMed  CAS  Google Scholar 

  40. Reichert W, Michel A, Hartmann RW, Jose J (2001) Stable expression of human 5alpha-reductase type II in COS1 cells due to chromosomal gene integration: a novel tool for inhibitor identification. J Steroid Biochem Mol Biol 78(3):275–284

    Article  PubMed  CAS  Google Scholar 

  41. Hartmann RW, Hector M, Haidar S, Ehmer PB, Reichert W, Jose J (2000) Synthesis and evaluation of novel steroidal oxime inhibitors of P450 17 (17 alpha-hydroxylase/C17–20-lyase) and 5 alpha-reductase types 1 and 2. J Med Chem 43(22):4266–4277

    Article  PubMed  CAS  Google Scholar 

  42. Ehmer PB, Jose J, Hartmann RW (2000) Development of a simple and rapid assay for the evaluation of inhibitors of human 17alpha-hydroxylase-C(17, 20)-lyase (P450cl7) by coexpression of P450cl7 with NADPH-cytochrome-P450-reductase in Escherichia coli. J Steroid Biochem Mol Biol 75(1):57–63

    Article  PubMed  CAS  Google Scholar 

  43. Rodems SM, Hamman BD, Lin C, Zhao J, Shah S, Heidary D, Makings L, Stack JH, Pollok BA (2002) A FRET-based assay platform for ultra-high density drug screening of protein kinases and phosphatases. Assay Drug Dev Technol 1(1 pt 1):9–19. doi:10.1089/154065802761001266

    Article  PubMed  CAS  Google Scholar 

  44. Merrifield RB (1963) Solid phase peptide synthesis I. The synthesis of a tetrapeptide. J Am Chem Soc 85(14):2149–2154

    Article  CAS  Google Scholar 

  45. Chang CD, Meienhofer J (1978) Solid-phase peptide synthesis using mild base cleavage of N alpha-fluorenylmethyloxycarbonylamino acids, exemplified by a synthesis of dihydrosomatostatin. Int J Pept Protein Res 11(3):246–249

    Article  PubMed  CAS  Google Scholar 

  46. Beythien J, White PD (2005) A solid phase linker strategy for the direct synthesis of EDANS-labelled peptide substrates. Tetrahedron Lett 46(1):101–104

    Article  CAS  Google Scholar 

  47. Kucklaender U, Bollig R, Frank W, Gratz A, Jose J (2011) A novel application of DDQ as electrophile in the Nenitzescu reaction. Bioorg Med Chem 19(8):2666–2674. doi:10.1016/j.bmc.2011.03.006

    Article  PubMed  CAS  Google Scholar 

  48. Grankowski N, Boldyreff B, Issinger OG (1991) Isolation and characterization of recombinant human casein kinase II subunits alpha and beta from bacteria. Eur J Biochem 198(1):25–30

    Article  PubMed  CAS  Google Scholar 

  49. Guerra B, Götz C, Wagner P, Montenarh M, Issinger OG (1997) The carboxy terminus of p53 mimics the polylysine effect of protein kinase CK2-catalyzed MDM2 phosphorylation. Oncogene 14(22):2683–2688. doi:10.1038/sj.onc.1201112

    Article  PubMed  CAS  Google Scholar 

  50. Schneider CC, Hessenauer A, Götz C, Montenarh M (2009) DMAT, an inhibitor of protein kinase CK2 induces reactive oxygen species and DNA double strand breaks. Oncol Rep 21(6):1593–1597

    PubMed  CAS  Google Scholar 

  51. Narayanan AS, Anwar RA (1969) The specificity of purified porcine pancreatic elastase. Biochem J 114(1):11–17

    PubMed  CAS  Google Scholar 

  52. Naughton MA, Sanger F (1961) Purification and specificity of pancreatic elastase. Biochem J 78:156–163

    PubMed  CAS  Google Scholar 

  53. Gratz A, Götz C, Jose J (2010) A FRET-based microplate assay for human protein kinase CK2, a target in neoplastic disease. J Enzyme Inhib Med Chem 25(2):234–239. doi:10.3109/14756360903170038

    Article  PubMed  CAS  Google Scholar 

  54. Gratz A, Götz C, Jose J (2010) A CE-based assay for human protein kinase CK2 activity measurement and inhibitor screening. Electrophoresis 31(4):634–640. doi:10.1002/elps.200900514

    Article  PubMed  CAS  Google Scholar 

  55. Kuenzel EA, Mulligan JA, Sommercorn J, Krebs EG (1987) Substrate specificity determinants for casein kinase II as deduced from studies with synthetic peptides. J Biol Chem 262(19):9136–9140

    PubMed  CAS  Google Scholar 

  56. Sarno S, Reddy H, Meggio F, Ruzzene M, Davies SP, Donella-Deana A, Shugar D, Pinna LA (2001) Selectivity of 4, 5, 6, 7-tetrabromobenzotriazole, an ATP site-directed inhibitor of protein kinase CK2 (‘casein kinase-2’). FEBS Lett 496(1):44–48

    Article  PubMed  CAS  Google Scholar 

  57. Sarno S, Ruzzene M, Frascella P, Pagano MA, Meggio F, Zambon A, Mazzorana M, Di Maira G, Lucchini V, Pinna LA (2005) Development and exploitation of CK2 inhibitors. Mol Cell Biochem 274(1–2):69–76

    Article  PubMed  CAS  Google Scholar 

  58. Sarno S, Moro S, Meggio F, Zagotto G, Dal Ben D, Ghisellini P, Battistutta R, Zanotti G, Pinna LA (2002) Toward the rational design of protein kinase casein kinase-2 inhibitors. Pharmacol Ther 93(2–3):159–168

    Article  PubMed  CAS  Google Scholar 

  59. Sarno S, De Moliner E, Ruzzene M, Pagano MA, Battistutta R, Bain J, Fabbro D, Schoepfer J, Elliott M, Furet P, Meggio F, Zanotti G, Pinna LA (2003) Biochemical and three-dimensional-structural study of the specific inhibition of protein kinase CK2 by [5-oxo-5,6-dihydroindolo-(1, 2-a)quinazolin-7-yl]acetic acid (IQA). Biochem J 374(Pt 3):639–646

    Article  PubMed  CAS  Google Scholar 

  60. Yim H, Lee YH, Lee CH, Lee SK (1999) Emodin, an anthraquinone derivative isolated from the rhizomes of Rheum palmatum, selectively inhibits the activity of casein kinase II as a competitive inhibitor. Planta Med 65(1):9–13. doi:10.1055/s-1999-13953

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University Düsseldorf, Universitätsstr. 1, 40225, Düsseldorf, Germany

    Andreas Gratz, Uwe Kuckländer, Ricardo Bollig & Joachim Jose

  2. Medicinal Biochemistry and Molecular Biology, Saarland University, 66424, Homburg-Saarbrücken, Germany

    Claudia Götz

Authors
  1. Andreas Gratz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Uwe Kuckländer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ricardo Bollig
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Claudia Götz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Joachim Jose
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Joachim Jose.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Gratz, A., Kuckländer, U., Bollig, R. et al. Identification of novel CK2 inhibitors with a benzofuran scaffold by novel non-radiometric in vitro assays. Mol Cell Biochem 356, 83–90 (2011). https://doi.org/10.1007/s11010-011-0957-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-011-0957-4

Keywords

  • Protein kinase
  • CK2 inhibition
  • Assay
  • Non-radiometric
  • Benzofuran derivatives
  • Capillary electrophoresis
  • FRET