Bioorthogonal click reaction of human CK2 subunits in combination with surface display for drug discovery appliations

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

Abstract

Central aspects for the identification and characterization of new inhibitors or interaction partners of human protein kinase CK2 are screening- and protein-protein interaction (PPI) assays. This often requires themodification of the target enzyme by a fluorophore. In case of labeling CK2 by commercially available fluorescein isothiocyanate (FITC) a loss of phosphorylation activity was observed. Accordingly, abioorthogonal click reaction was used to modify the CK2α subunit with a fluorophore. For this purpose the DNA codon (TAT) encoding Y239 was replaced by the amber stop codon TAG. By suppression of themutation with an amber suppressor tRNA, the unnatural amino acid para azidophenylalanine (pAzF) could be incorporated at this position [1]. Performing the SPAAC click reaction (Strain-Promoted Alkyne-AzideCycloaddition) [2] by the use of a dibenzylcyclooctyne-fluorophore (DBCO fluorophore) led to a specifically labeled human protein kinase CK2α. This site specific labeling did not impair the phosphorylationactivity of CK2, which was confirmed by capillary electrophoresis [3]. Additionally a dissociation constant (KD) of 631 ± 86.2 nM was determined for the substrate αS1 casein [4] towards CK2α by microscalethermophoresis. In combination with Autodisplay [5] this innovative modification of CK2α could be a significant advancement for screening assays by flow cytometry and for CK2α/CK2β interaction studies. Thislabeling strategy was also applied to surface displayed CK2β on Escherichia coli, which could replace the need for purifying CK2β and could enable measurements with whole cells [6].