Development of a high-throughput screening-compatible assay to identify inhibitors of the CK2alpha/CK2beta interaction

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  • Jennifer Hochscherf
  • Dirk Lindenblatt
  • Michaela Steinkrueger
  • Eungyoung Yoo
  • Oezlem Ulucan
  • Stefan Herzig
  • Olaf-Georg Issinger
  • Volkhard Helms
  • Claudia Goetz
  • Ines Neundorf
  • Karsten Niefind
  • Markus Pietsch

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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 CK2alpha and CK2beta 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 CK2alpha1-335 and the fluorescent probe CF-Ahx-Pc as a CK2beta analog. In addition, we identified new inhibitors able to displace the fluorescent probe from the subunit interface on CK2alpha1-335. 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 CK2alpha-binding motif of CK2beta. The design of the two inhibitors was based on docking studies using the known crystal structure of the Pc/CK2alpha1-335 complex. The dissociation constants obtained in the fluorescence anisotropy assay for binding of all compounds to human CK2alpha1-335 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 CK2beta, with an IC50 value of 92muM.
TidsskriftAnalytical Biochemistry
Sidetal (fra-til)4-14
StatusUdgivet - 2015