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Updated: May 18, 2026
Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach
Published on: February 21, 2019
A subnanomolar fluorescent probe for protein kinase CK2 interaction studies
Erki Enkvist1, Kaido Viht, Nils Bischoff
1Institute of Chemistry, University of Tartu, 14A Ravila St., 50411 Tartu, Estonia.
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.
Insights
Researchers developed a potent, selective small-molecule fluorescent probe (ARC-1504) targeting the cancer-related protein kinase CK2. This probe aids in characterizing CK2 inhibitors and understanding its role in cancer.
Area of Science:
- Biochemistry
- Chemical Biology
- Drug Discovery
Background:
- Protein kinase CK2 (casein kinase 2) is upregulated in various cancers, making it a key target for cancer chemotherapy.
- Developing selective small-molecule fluorescent probes is crucial for characterizing drug candidates, enzyme structure, and function.
Purpose of the Study:
- To design and synthesize a highly potent and selective small-molecule inhibitor and fluorescent probe for CK2.
- To utilize these tools for characterizing CK2 and its interactions.
Main Methods:
- A bisubstrate approach was used to develop CK2 inhibitors by conjugating 4,5,6,7-tetrabromo-1H-benzimidazole with aspartate-rich peptides.
- Crystallographic analysis guided inhibitor design.
- Inhibitory potency was assessed using kinetic assays with a fluorescently labeled peptide substrate.
- A fluorescent probe (ARC-1504) was generated by labeling the lead inhibitor (ARC-1502) with a fluorophore.
Main Results:
- The most potent inhibitor, ARC-1502, exhibited a K(i) of 0.5 nM and high selectivity for CK2α against 140 other kinases.
- The fluorescent probe ARC-1504 demonstrated subnanomolar affinity for both CK2α and the CK2 holoenzyme.
- ARC-1504 was successfully employed in fluorescence anisotropy assays to quantify CK2α and analyze ligand binding.
Conclusions:
- A novel, highly potent, and selective CK2 inhibitor (ARC-1502) and fluorescent probe (ARC-1504) were developed.
- These tools are valuable for drug discovery, structural biology, and functional studies of CK2 in cancer research.
