Organic & Biomolecular Chemistry Dynamic Article Links Cite this: Org. Biomol. Chem., 2012, 10, 8645 www.rsc.org/obc PAPER A subnanomolar fluorescent probe for protein kinase CK2 interaction studies† Erki Enkvist, a Kaido Viht, a Nils Bischoff, b Jürgen Vahter, a Siiri Saaver, a Gerda Raidaru, a Olaf-Georg Issinger, c Karsten Niefind b and Asko Uri* a Received 25th May 2012, Accepted 4th September 2012 DOI: 10.1039/c2ob26022k Up-regulation of an acidophilic protein kinase, CK2, has been established in several types of cancer. This cognition has made CK2 an important target for drug development for cancer chemotherapy. The characterization of potential drug candidates, determination of the structure and clarification of the functions of CK2 could be facilitated by the application of small-molecule fluorescent probes that bind to the active site of the enzyme with high affinityand selectivity. We have used a bisubstrate approach for the development of a highly potent inhibitor of CK2. 4,5,6,7-Tetrabromo-1H-benzimidazole was conjugated with peptides containing multiple aspartate residues via different linkers. The design of the inhibitors was by crystallographic analysis of the complex of an inhibitor with the catalytic subunit of the enzyme (CK2α). The inhibitory potency of the synthesized compounds was established in a kinetic assay that used thin layer chromatography for the measurement of the rate of phosphorylation of fluorescently labelled peptide 5-TAMRA-RADDSDDDDD. The most potent inhibitor, ARC-1502 (K i = 0.5 nM), revealed high selectivity for CK2α in a panel of 140 protein kinases. Labelling of ARC-1502 with PromoFluor-647 gave the fluorescent probe ARC-1504 that possessed subnanomolar affinity towards both CK2α and the holoenzyme. The probe was used in a fluorescence anisotropy-based binding assay to measure the concentration of CK2α and characterize non-labelled ligands binding to the active site of CK2α. Introduction CK2 is a ubiquitous, highly conserved and pleiotropic serine/ threonine protein kinase (PK) with numerous physiological func- tions. 1 It has been estimated that up to 20% of the human phos- phoproteome may result from CK2-mediated phosphorylation of its substrate proteins. 2 Activity of CK2 is involved in cell growth, proliferation, angiogenesis, suppression of apoptosis, etc., making the kinase a potential target for cancer chemotherapy. 3 CK2 is constitutively active and does not require pre-phos- phorylation or the presence of cofactors for catalytic activity; still, multiple factors modulate its activity allowing specific regu- lation of cell functions. 1 In cells, CK2 is mostly present in the form of the holoenzyme, 4 a hetero-tetramer composed of two catalytic (α and/or α′) and two regulatory (β) subunits. 5 Both the α-subunit and the holoenzyme are catalytically active but their substrate specificity is different. 6 CK2 is an acidophilic protein kinase (PK) that prefers sub- strates containing multiple negatively charged amino acid resi- dues (Asp, Glu or phosphorylated residues) near the phospho- acceptor site, 7 although proteins possessing another kind of amino acid sequences near the phosphorylation site may also be targets of CK2. While numerous crystal structures are available for CK2α subunit, 8 CK2 holoenzyme 5 and complexes of both with various ATP competitive inhibitors, 9,10 the attempts to solve crystal structures of CK2 in complex with peptide substrates have not been successful. Thus the 3D positioning of the resi- dues engaged in binding of a substrate protein has not been established. 11,12 Several selective ATP-competitive inhibitors of CK2 have been developed in extensive studies. A highly potent and orally available inhibitor, CX-4945, is now in clinical trials for cancer treatment. 10 Non-ATP-competitive inhibitors of CK2 have found less attention 13,14 and only one attempt to design biligand inhibi- tors that resulted in compounds with micromolar inhibitory potency has been disclosed. 15 Bisubstrate inhibitors consist of two conjugated fragments, usually structurally related to the substrates (i.e., a phosphoryl † Electronic supplementary information (ESI) available: HPLC-MS data of synthesized compounds, selectivity table, X-ray diffraction data and copies of UV, 1 H NMR and 13 C NMR spectra of new compounds. See DOI: 10.1039/c2ob26022k a Institute of Chemistry, Universityof Tartu, 14A Ravila St., 50411 Tartu, Estonia. E-mail: asko.uri@ut.ee; Tel: +372 7375275 b Department für Chemie, Institut für Biochemie, Universität zu Köln, Otto-Fischer-Str. 12-14, D-50674 Köln, Germany c Institut for Biokemi og Molekylær Biologi, Syddansk Universitet, Campusvej 55, DK-5230 Odense, Denmark This journal is © The Royal Society of Chemistry 2012 Org. Biomol. Chem., 2012, 10, 8645–8653 | 8645 Downloaded by University of Cologne on 17 October 2012 Published on 04 September 2012 on http://pubs.rsc.org | doi:10.1039/C2OB26022K View Online / Journal Homepage / Table of Contents for this issue