title: Toward selective CK2alpha and CK2alpha' inhibitors: Development of a novel whole-cell kinase assay by Autodisplay of catalytic CK2alpha'.
abstract: 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 α- and/or α'-subunits, bound to a dimer of the regulatory β-subunit. Inhibitors targeting one of the two isoforms of the catalytically active CK2-subunit (α- and α') 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 CK2α'-subunit, the paralogous isoform of CK2α. Expression on the cell surface of E. coli of CK2α' alone and in combination with the regulatory CK2β-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 3.06×10(-6) μmol/min for CK2α' alone and 1.02×10(-5) μmol/min when expressed in combination with CK2β. The comparison of the influence of NaCl on activity of the α'-subunit alone and in combination with the non-catalytically active β-subunit indicated interaction of both subunits on the cell surface. TMCB (4,5,6,7-tetrabromo-2-(dimethylamino)-1H-benzo[d]imidazol-1-yl)acetic acid), a known CK2 inhibitor described with distinct Ki values of 83 nM and 21 nM for the two different catalytic CK2 subunits α and α' was used for testing. First, inhibition of TMCB toward the purified CK2 holoenzyme CK2α2β2 was determined and resulted in a Ki value of 10.1 nM. Second, Ki values were determined with the surface displayed isoform CK2 holoenzymes and turned out to be of 31.1 nM for CK2α2β2 and 19.6 nM for CK2α'2β2. The inhibition data as obtained represented the distinct affinities of TMCB toward the two isoform holoenzymes. This indicated, that the surface display of CKα and CK2α', 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.
authors.affiliation: Institut für Pharmazeutische und Medizinische Chemie, PharmaCampus, Westfälische Wilhelms-Universität Münster, Corrensstrasse 48, 48149 Münster, Germany.
authors.forename: Andre
authors.initials: A
authors.lastname: Bollacke
authors.affiliation: Institut für Pharmazeutische und Medizinische Chemie, PharmaCampus, Westfälische Wilhelms-Universität Münster, Corrensstrasse 48, 48149 Münster, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Nienberg
authors.affiliation: Université de Lyon, Université Lyon 1, Faculté de Pharmacie-ISPB, EA 4446 Biomolécules Cancer et Chimiorésistances, SFR Santé Lyon-Est CNRS UMS3453-INSERM US7, 8 Avenue Rockefeller, F-69373 Lyon Cedex 8, France.
authors.forename: Marc Le
authors.initials: ML
authors.lastname: Borgne
authors.affiliation: Institut für Pharmazeutische und Medizinische Chemie, PharmaCampus, Westfälische Wilhelms-Universität Münster, Corrensstrasse 48, 48149 Münster, Germany. Electronic address: joachim.jose@uni-muenster.de.
authors.forename: Joachim
authors.initials: J
authors.lastname: Jose
mesh_terms: D000595:Amino Acid Sequence; D001681:Biological Assay / Q000379:methods*; D047390:Casein Kinase II / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*
chemical_list: D020033:Protein Isoforms; D047428:Protein Kinase Inhibitors; D047390:Casein Kinase II
keywords: Autodisplay; CE; CK2; Inhibitor testing; Kinase; Selectivity