Toward selective CK2alpha and CK2alpha' inhibitors: Development of a novel whole-cell kinase assay by Autodisplay of catalytic CK2alpha'

Human protein kinase CK2 is an emerging target for the development of novel anti-cancer therapeutics. CK2 is a tetramer composed of two catalytically active alpha- and/or alpha'-subunits, bound to a dimer of the regulatory beta-subunit. Inhibitors targeting one of the two isoforms of the catalytically active CK2-subunit (alpha- and alpha') are important to study the distinct functions of these isoforms toward different CK2 associated pathologies. The present study for the first time describes the successful Autodisplay of the CK2 alpha'-subunit, the paralogous isoform of CK2 alpha. Expression on the cell surface of E. coli of CK2 alpha' alone and in combination with the regulatory CK2 beta-subunit was confirmed by outer membrane isolation and protease accessibility test. Kinase activity of surface displayed CK2 could be detected with a CE-based assay and was found to be 3.06 x 10(-6) mu mol/min for CK2 alpha' alone and 1.02 x 10(-5) mu mol/min when expressed in combination with CK2 beta. The comparison of the influence of NaCI on activity of the alpha'-subunit alone and in combination with the non-catalytically active beta-subunit indicated interaction of both subunits on the cell surface. TMCB (4,5,6,7-tetrabromo-2-(dimethylamino)-1H-benzo[d]imidazol-1-ypacetic acid), a known CK2 inhibitor described with distinct K-i values of 83 nM and 21 nM for the two different catalytic CK2 subunits alpha and alpha' was used for testing. First, inhibition of TMCB toward the purified CK2 holoenzyme CK2 alpha(2)beta(2) was determined and resulted in a K-i value of 10.1 nM. Second, K-i values were determined with the surface displayed isoform CK2 holoenzymes and turned out to be of 31.1 nM for CK2 alpha(2)beta(2) and 19.6 nM for CK2 alpha'(2)beta(2). The inhibition data as obtained represented the distinct affinities of TMCB toward the two isoform holoenzymes. This indicated, that the surface display of CK alpha and CK2 alpha', in the context of the corresponding holoenzymes, can be used to identify selective compounds. A set of twelve ATP competitive CK2 inhibitors with an indeno[1,2-b]indole scaffold was tested in order to demonstrate suitability for this application. (C) 2016 Elsevier B.V. All rights reserved.