Related Experiment Video
Updated: Apr 23, 2026
Identification of Kinase-substrate Pairs Using High Throughput Screening
Published on: August 29, 2015
Development of a high-throughput screening-compatible assay to identify inhibitors of the CK2α/CK2β interaction
Jennifer Hochscherf1, Dirk Lindenblatt2, Michaela Steinkrüger3
1Institute of Biochemistry, Department of Chemistry, University of Cologne, D-50674 Cologne, Germany; KinaseDetect ApS, DK-5220 Odense, Denmark.
Abstract:
Increased activity of protein kinase CK2 is associated with various types of cancer, neurodegenerative diseases, and chronic inflammation. In the search for CK2 inhibitors, attention has expanded toward compounds disturbing the interaction between CK2α and CK2β in addition to established active site-directed approaches. The current article describes the development of a fluorescence anisotropy-based assay that mimics the principle of CK2 subunit interaction by using CK2α(1-335) and the fluorescent probe CF-Ahx-Pc as a CK2β analog. In addition, we identified new inhibitors able to displace the fluorescent probe from the subunit interface on CK2α(1-335). Both CF-Ahx-Pc and the inhibitors I-Pc and Cl-Pc were derived from the cyclic peptide Pc, a mimetic of the C-terminal CK2α-binding motif of CK2β. The design of the two inhibitors was based on docking studies using the known crystal structure of the Pc/CK2α(1-335) complex. The dissociation constants obtained in the fluorescence anisotropy assay for binding of all compounds to human CK2α(1-335) were validated by isothermal titration calorimetry. I-Pc was identified as the tightest binding ligand with a KD value of 240nM and was shown to inhibit the CK2 holoenzyme-dependent phosphorylation of PDX-1, a substrate requiring the presence of CK2β, with an IC50 value of 92μM.
Insights
Researchers developed a new assay to find inhibitors of protein kinase CK2 (CK2) that disrupt its subunit interactions. They identified novel compounds, including I-Pc, which effectively inhibit CK2 activity by targeting the CK2α/CK2β interface.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Protein kinase CK2 (CK2) hyperactivity is linked to cancer, neurodegenerative diseases, and inflammation.
- Inhibiting CK2 is a therapeutic strategy, with research expanding beyond active site inhibitors to those disrupting CK2α and CK2β subunit interactions.
Purpose of the Study:
- To develop a fluorescence anisotropy-based assay to study CK2 subunit interactions.
- To identify novel CK2 inhibitors that target the CK2α/CK2β interface.
Main Methods:
- Development of a fluorescence anisotropy assay using CK2α(1-335) and a CK2β analog (CF-Ahx-Pc).
- Design of inhibitors (I-Pc, Cl-Pc) based on docking studies of the cyclic peptide Pc.
- Validation of binding affinities using isothermal titration calorimetry.
Main Results:
- Identification of new inhibitors, I-Pc and Cl-Pc, that displace the fluorescent probe from CK2α(1-335).
- I-Pc demonstrated the tightest binding to CK2α(1-335) with a dissociation constant (KD) of 240 nM.
- I-Pc inhibited holoenzyme-dependent PDX-1 phosphorylation with an IC50 of 92 μM.
Conclusions:
- A novel fluorescence anisotropy assay effectively monitors CK2 subunit interactions.
- The identified inhibitors, particularly I-Pc, represent promising leads for targeting CK2 via subunit interface disruption.
- These findings offer new avenues for developing therapeutics for CK2-associated diseases.
