Research Article A CE-based assay for human protein kinase CK2 activity measurement and inhibitor screening A new assay for protein kinase CK2 activity determination based on the quantification of a phosphorylated substrate was developed. The common CK2 substrate peptide RRRDDDSDDD, conjugated with the fluorophore 5-[(2-aminoethyl)amino]naphthalene- 1-sulfonic acid at the C-terminus served as the analyte. By means of CZE using 2 mol/L acetic acid as electrolyte and UV detection at 214 nm, the non-phosphorylated and the phosphorylated peptide variants could be resolved within 6 min from a complex assay mixture. By this means, activity of human CK2 could be monitored by a kinetic, as well as an endpoint, method. Inhibition of human recombinant CK2 holoenzyme by 6-methyl- 1,3,8-trihydroxyanthraquinone and 4,5,6,7-tetrabromobenzotriazole resulted in IC 50 values of 1.33 and 0.27 mM, respectively, which were similar to those obtained with the standard radiometric assay. These results suggest that the CE/UV strategy described here is a straightforward assay for CK2 inhibitor testing. Keywords: CE / CK2 / Kinase inhibitor / Mobility shift / Protein kinase DOI 10.1002/elps.200900514 1 Introduction Since the early 1980s, when the first protein kinase inhibitor was identified [1], the number of reports on compounds with kinase inhibiting activity has steadily increased. One highlight during this period was the successful development of imatinib, an inhibitor of Bcr-Abl. The corresponding oncogene arises from a chromosome translocation between the genes of bcr and c-abl. Compared with the unmutated tyrosine kinase c-Abl, Bcr-Abl is not regulated appropriately and has an increased kinase activity, which causes cancer. The drug imatinib inhibits Bcr-Abl and was approved by the FDA for the treatment of chronic myelogenous leukemia in 2001 [2] and the treatment of gastrointestinal stromal tumor in 2002 [3]. Today, protein kinases are regarded as major drug targets and it is estimated that about 30% of all drug discovery projects in the pharmaceutical industry are currently developing protein kinase inhibitors [4, 5]. An upcoming tumor target in the human kinome is the constitutively active protein kinase CK2. Elevated CK2 activity in different tumors has been reported [6] and several natural CK2 substrates, such as p53, PTEN and PML, play major roles in cancer development [7]. In cancer cells, increased CK2 activity prevents apoptosis [8], whereas downregulation of CK2 leads to apoptosis [9]. Therefore, CK2 inhibitors could provide a new access to therapeutic anticancer approaches. At present, several ATP-competitive CK2 inhibitors are known, which belong to different chemical groups [10, 11]. Typical representatives are the anthraquinone derivative 6-methyl-1,3,8-trihydroxyan- thraquinone (emodin) and the benzoimidazole derivative 4,5,6,7-tetrabromobenzotriazole (TBB) each with IC 50 values in the low micromolar range (Fig. 1). Other potent inhibi- tors have a pyrazolo-triazine scaffold with IC 50 values in the nanomolar range [12]. More recently, Cylene pharmaceu- ticals (San Diego, CA, USA) reported that its CK2 inhibitor CX-4945, which is based on a 2,6-phenanthroline scaffold, is currently entering phase 1 clinical trials [13] (Fig. 1). However, despite the attractiveness of CK2 as a druggable target in neoplastic diseases, the number of published potent CK2 inhibitors is rather limited. Two bottlenecks in the development of CK2 inhibitors are the availability of the human target enzyme for testing and an inhibitor assay for efficient throughput. In principle, the available protein kinase assays can be divided into three categories: (i) radiometric assays, (ii) antibody-dependent spectroscopic assays and (iii) antibody- independent spectroscopic assays [14]. The radiometric Andreas Gratz 1 Claudia Go ¨ tz 2 Joachim Jose 1 1 Bioanalytics, Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine- University Du ¨ sseldorf, Du ¨ sseldorf, Germany 2 Medicinal Biochemistry and Molecular Biology, Saarland University, Homburg- Saarbru ¨ cken, Germany Received August 28, 2009 Revised October 21, 2009 Accepted October 23, 2009 Abbreviations: AB, assay buffer; ADP, adenosine-5 0 - diphosphate; ATP, adenosine-5 0 -triphosphate; EDANS, 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid; emodin, 56-methyl-1,3,8-trihydroxyanthraquinone; TBB, 4,5,6,7- tetrabromobenzotriazole Correspondence: Professor Joachim Jose, Bioanalytics, Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine- University Du ¨ sseldorf, Universita ¨ tsstr. 1, D-40225 Du ¨ sseldorf, Germany E-mail: joachim.jose@uni-duesseldorf.de Fax: 149-211-8113847 & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com Electrophoresis 2010, 31, 634–640 634