Click chemistry and bacterial surface display: Identification of new potent allosteric inhibitors of human protein kinase CK2

Nienberg C, Garmann C, Saenger T, Bollacke A, Gratz A, Jose J

Abstract

Human CK2 is a heterotetrameric constitutively active serine/threonine protein kinase, phosphorylating ahuge number of substrates and regulating a variety of cellular processes. CK2 is related to severalhuman diseases and represents an emerging target in today's cancer research [1]. Consequentlyscreening- and protein-protein interaction (PPI) assays are required for the identification andcharacterization of new inhibitors or interaction partners of CK2.In this study the catalytic subunit CK2α was used for the incorporation of the unnatural amino acid paraazidophenylalanine (pAzF) followed by a SPAAC click reaction with a dibenzylcyclooctyne-fluorophore.By this labeling strategy CK2 maintained its functionality and phosphorylation activity contrary tocommercially CK2-labeling by fluorescein isothiocyanate [2]. By Autodisplay [3] fluorescently labeledCK2 could be used for bacterial surface display library screening. Thereby a potent allosteric peptidicinhibitor, called B2, was identified. B2 inhibited the CK2 holoenzyme as well as the CK2α subunit with anIC50 value of 0.8 μM and was neither ATP- nor substrate competitive [4]. Furthermore binding studies ofspecifically labeled CK2 and interaction partners were performed by microscale thermophoresis.Unknown binding affinities were clarified and the KD value of CK2α and B2 was determined to be2.16 μM [4]. The incorporation of pAzF was also performed with bacterial cell surface translocated CK2subunits and demonstrated the click reaction with a membrane anchored protein [2]. Finally these resultsillustrate that the combination of click chemistry and surface display is promising for drug discovery ofhuman protein kinase CK2.