Halogen Atoms in the Protein-Ligand System. Structural and Thermodynamic Studies of the Binding of Bromobenzotriazoles by the Catalytic Subunit of Human Protein Kinase CK2.

Honorata Czapinska,Matthias Bochtler,Jarosław Poznański,Maria Winiewska-Szajewska,Anna Szymaniec-Rutkowska,Anna Piasecka

doi:10.1021/acs.jpcb.0c10264

Abstract

Binding of a family of brominated benzotriazoles to the catalytic subunit of human protein kinase CK2 (hCK2α) was used as a model system to assess the contribution of halogen bonding to protein–ligand interaction. CK2 is a constitutively active pleiotropic serine/threonine protein kinase that belongs to the CMGC group of eukaryotic protein kinases (EPKs). Due to the addiction of some cancer cells, CK2 is an attractive and well-characterized drug target. Halogenated benzotriazoles act as ATP-competitive inhibitors with unexpectedly good selectivity for CK2 over other EPKs. We have characterized the interaction of bromobenzotriazoles with hCK2α by X-ray crystallography, low-volume differential scanning fluorimetry, and isothermal titration calorimetry. Properties of free ligands in solution were additionally characterized by volumetric and RT-HPLC measurements. Thermodynamic data indicate that the affinity increases with bromo substitution, with greater contributions from 5- and 6-substituents than 4- and 7-substituents. Except for 4,7-disubstituted compounds, the bromobenzotriazoles adopt a canonical pose with the triazole close to lysine 68, which precludes halogen bonding. More highly substituted benzotriazoles adopt many additional noncanonical poses, presumably driven by a large hydrophobic contribution to binding. Some noncanonical ligand orientations allow the formation of halogen bonds with the hinge region. Consistent with a predominantly hydrophobic interaction, the isobaric heat capacity decreases upon ligand binding, the more so the higher the substitution.

Highlights

CK2, formerly designated as casein kinase II, is a pleiotropic serine/threonine kinase found in all eukaryotes.[1]
The results clearly demonstrated that a balance of hydrophobic and electrostatic interactions has predominant contribution to the binding affinity.[32−34] The structures of hCK2α complexes shown above as well as the published values of IC50 (Table 2)[32] were determined at pH 7.5, while the previous thermodynamic studies were performed at pH 8.0.33,35 This prompted us to measure the thermal stabilities of hCK2α and its complexes at pH 7.5 by fluorescence-monitored thermal denaturation (Table 2)
In the canonical hCK2α bromobenzotriazole complex structures, the distances between the backbone carbonyl oxygen atoms of E114 from the hinge region of hCK2α and the bromine atoms are longer than the 3.4 Å sum of van der Waals radii of oxygen and bromine.[70]

Summary

Introduction

CK2, formerly designated as casein kinase II, is a pleiotropic serine/threonine kinase found in all eukaryotes.[1] The holoenzyme is a heterotetramer, consisting of two catalytic α- and/or α′-subunits and two regulatory β-subunits.[2] Unlike other kinases, in particular signaling cascade ones, CK2 is a constitutively active enzyme. It phosphorylates substrates with serine/threonine in regions enriched in acidic residues. The enzyme favors targets with an acid or a phosphorylated serine residue at the +3 position relative to the substrate S/T.1 CK2 belongs to (or according to some classifications, is closely related to) CMGC kinases that in addition to CK2 comprise cyclin-dependent kinases (CDKs), mitogen-activated protein kinase (MAPK), glycogen synthase kinase (GSK), and cyclin-dependent kinase-like kinases (CDK-like kinases).[3]

Methods

Results

Discussion

Conclusion