Development of screening assays for human protein kinase CK2 using the advantages of click chemistry.

Nienberg C, Retterath A, Becher K-S, Saenger T, Mootz HD, Jose J

Abstract

Human CK2 is a heterotetrameric constitutively active serine / threonine protein kinase and is an emerging target in current anti-cancer drug discovery [1]. The kinase is composed of twocatalytic CK2α subunits and two regulatory CK2β subunits. In order to establish an assay to identify protein-protein-interaction inhibitors (PPI) of the CK2α/ CK2β interface, a bioorthogonalclick reaction was used to modify the protein kinase α-subunit with a fluorophore. By expanding the genetic code, the unnatural amino acid para azidophenylalanine (pAzF) [2] could beincorporated into CK2α. Performing the SPAAC click reaction (Strain-Promoted Alkyne-Azide Cycloaddition) [3] by the use of a dibenzylcyclooctyne-fluorophore (DBCO-fluorophore) led to aspecifically labeled human protein kinase CK2α. This site specific labeling does not impair the phosphorylation activity of CK2, which was evaluated by capillary electrophoresis. Furthermorea dissociation constant (KD) of 631 ± 86.2 nM was determined for the substrate αS1-casein towards CK2α. The innovatively labeled CK2α in combination with Autodisplay [5] could be anadvancement for flow cytometry based screening assays to identify inhibitors of the CK2α/CK2β interaction.