title: Thermodynamics parameters for binding of halogenated benzotriazole inhibitors of human protein kinase CK2α.
abstract: The interaction of human CK2α (hCK2α) with nine halogenated benzotriazoles, TBBt and its analogues representing all possible patterns of halogenation on the benzene ring of benzotriazole, was studied by biophysical methods. Thermal stability of protein-ligand complexes, monitored by calorimetric (DSC) and optical (DSF) methods, showed that the increase in the mid-point temperature for unfolding of protein-ligand complexes (i.e. potency of ligand binding to hCK2α) follow the inhibitory activities determined by biochemical assays. The dissociation constant for the ATP-hCK2α complex was estimated with the aid of microscale thermophoresis (MST) as 4.3±1.8 μM, and MST-derived dissociation constants determined for halogenated benzotriazoles, when converted according to known ATP concentrations, perfectly reconstruct IC50 values determined by the biochemical assays. Ligand-dependent quenching of tyrosine fluorescence, together with molecular modeling and DSC-derived heats of unfolding, support the hypothesis that halogenated benzotriazoles bind in at least two alternative orientations, and those that are efficient hCK2α inhibitors bind in the orientation which TBBt adopts in its complex with maize CK2α. DSC-derived apparent heat for ligand binding (ΔΔHbind) is driven by intermolecular electrostatic interactions between Lys68 and the triazole ring of the ligand, as indicated by a good correlation between ΔΔHbind and ligand pKa. Overall results, additionally supported by molecular modeling, confirm that a balance of hydrophobic and electrostatic interactions contribute predominantly (~40 kJ/mol), relative to possible intermolecular halogen/hydrogen bonding (less than 10 kJ/mol), in binding of halogenated benzotriazoles to the ATP-binding site of hCK2α. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.
authors.affiliation: Institute of Biochemistry and Biophysics PAS, Pawińskiego 5a, 02-106 Warszawa, Poland.
authors.forename: Maria
authors.initials: M
authors.lastname: Winiewska
authors.affiliation: Institute of Biochemistry and Biophysics PAS, Pawińskiego 5a, 02-106 Warszawa, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Kucińska
authors.affiliation: Institute of Biochemistry and Biophysics PAS, Pawińskiego 5a, 02-106 Warszawa, Poland.
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Makowska
authors.affiliation: Institute of Biochemistry and Biophysics PAS, Pawińskiego 5a, 02-106 Warszawa, Poland. Electronic address: jarek@ibb.waw.pl.
authors.forename: Jarosław
authors.initials: J
authors.lastname: Poznański
authors.affiliation: Institute of Biochemistry and Biophysics PAS, Pawińskiego 5a, 02-106 Warszawa, Poland. Electronic address: shugar@ibb.waw.pl.
authors.forename: David
authors.initials: D
authors.lastname: Shugar
mesh_terms: D001665:Binding Sites; D002151:Calorimetry; D047390:Casein Kinase II / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D054879:Halogenation; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D008958:Models, Molecular; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000378:metabolism; D055672:Static Electricity; D013816:Thermodynamics; D014230:Triazoles / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008024:Ligands; D047428:Protein Kinase Inhibitors; D014230:Triazoles; C012771:benzotriazole; C570325:CSNK2A1 protein, human; D047390:Casein Kinase II
keywords: Bromobenzotriazoles; CK2a; Halogenated ligands; Ligand binding; Protein kinase CK2; Thermodynamics