title: A subnanomolar fluorescent probe for protein kinase CK2 interaction studies.
abstract: 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 affinity and 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.
authors.affiliation: Institute of Chemistry, University of Tartu, 14A Ravila St., 50411 Tartu, Estonia.
authors.forename: Erki
authors.initials: E
authors.lastname: Enkvist
authors.forename: Kaido
authors.initials: K
authors.lastname: Viht
authors.forename: Nils
authors.initials: N
authors.lastname: Bischoff
authors.forename: Jürgen
authors.initials: J
authors.lastname: Vahter
authors.forename: Siiri
authors.initials: S
authors.lastname: Saaver
authors.forename: Gerda
authors.initials: G
authors.lastname: Raidaru
authors.forename: Olaf-Georg
authors.initials: OG
authors.lastname: Issinger
authors.forename: Karsten
authors.initials: K
authors.lastname: Niefind
authors.forename: Asko
authors.initials: A
authors.lastname: Uri
mesh_terms: D001562:Benzimidazoles / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D047390:Casein Kinase II / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain / Q000187:drug effects; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D047428:Protein Kinase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: D001562:Benzimidazoles; D005456:Fluorescent Dyes; D047428:Protein Kinase Inhibitors; D047390:Casein Kinase II