Thermodynamic parameters for binding of some halogenated inhibitors of human protein kinase CK2

Abstract

The interaction of human CK2α with a series of tetrabromobenzotriazole (TBBt) and tetrabromobenzimidazole (TBBz) analogs, in which one of the bromine atoms proximal to the triazole/imidazole ring is replaced by a methyl group, was studied by biochemical (IC50) and biophysical methods (thermal stability of protein-ligand complex monitored by DSC and fluorescence). Two newly synthesized tri-bromo derivatives display inhibitory activity comparable to that of the reference compounds, TBBt and TBBz, respectively. DSC analysis of the stability of protein-ligand complexes shows that the heat of ligand binding (Hbind) is driven by intermolecular electrostatic interactions involving the triazole/imidazole ring, as indicated by a strong correlation between Hbind and ligand pKa. Screening, based on fluorescence-monitored thermal unfolding of protein-ligand complexes, gave comparable results, clearly identifying ligands that most strongly bind to the protein. Overall results, additionally supported by molecular modeling, confirm that a balance of hydrophobic and electrostatic interactions contribute predominantly, relative to possible intermolecular halogen bonding, in binding of the ligands to the CK2α ATP-binding site.

5,6-diiodo-1H-benzotriazole: new TBBt analogue that minutely affects mitochondrial activity

“…3 ). This common trend reaffirms our interpretation that the binding of halogenated benzotriazoles to the catalytic subunit of protein kinase CK2 is predominately driven by hydrophobic effect 16 , 18 , 20 , 30 , 31 . In this view, the excess heat of binding of BIIB at the ATP binding site (− 8 kJ/mol) may be assigned to the contribution of halogen bond(s), which are putatively formed between two iodine atoms of BIIB and two backbone carbonyl groups of residues located in the hinge region, resembling a virtual interaction that has been recently identified in the crystal structure of TBBt at hCK2α ATP binding site 32 .…”

5,6-diiodo-1H-benzotriazole: new TBBt analogue that minutely affects mitochondrial activity

“…3 ). This common trend reaffirms our interpretation that the binding of halogenated benzotriazoles to the catalytic subunit of protein kinase CK2 is predominately driven by hydrophobic effect 16 , 18 , 20 , 30 , 31 . In this view, the excess heat of binding of BIIB at the ATP binding site (− 8 kJ/mol) may be assigned to the contribution of halogen bond(s), which are putatively formed between two iodine atoms of BIIB and two backbone carbonyl groups of residues located in the hinge region, resembling a virtual interaction that has been recently identified in the crystal structure of TBBt at hCK2α ATP binding site 32 .…”

Thermodynamics parameters for binding of halogenated benzotriazole inhibitors of human protein kinase CK2α

“…Both effects indicate that all three bind to hCK2α more weakly than any from the previous group. An analogous trend in fluorescence quenching has been recently observed for TBBt and TBBz, which bind to maize CK2α in different orientations (see structures based on PDB: 1j91 [57] and PDB: 2oxy [91] in Figures 6A and 6B, respectively), and also for their derivatives with bromine at C4 replaced by a methyl group [63]. It may thus be hypothesized that 4,5-Br 2 Bt, 4,6-Br 2 Bt, and most likely 4,5,7-Br 3 Bt, bind to CK2α in different orientations than TBBt.…”

Rational drug-design approach supported with thermodynamic studies — a peptide leader for the efficient bi-substrate inhibitor of protein kinase CK2

“…IC 50 values were determined using three independent experiments, 7 inhibitor concentrations in the range of 1nM-1mM each, by global fitting of the sigmoidal dose-response equation implemented in Origin 9.0 package (www.originlab.com). Single experiment was analyzed for TBBt to confirm agreement with already published data 42,46,47 .…”

5,6-diiodo-1H-benzotriazole: new TBBt analogue that minutely affects mitochondrial activity

“…3 ). This common trend reaffirms our interpretation that the binding of halogenated benzotriazoles to the catalytic subunit of protein kinase CK2 is predominately driven by hydrophobic effect 16 , 18 , 20 , 30 , 31 . In this view, the excess heat of binding of BIIB at the ATP binding site (− 8 kJ/mol) may be assigned to the contribution of halogen bond(s), which are putatively formed between two iodine atoms of BIIB and two backbone carbonyl groups of residues located in the hinge region, resembling a virtual interaction that has been recently identified in the crystal structure of TBBt at hCK2α ATP binding site 32 .…”

Thermodynamics parameters for binding of halogenated benzotriazole inhibitors of human protein kinase CK2α

“…Both effects indicate that all three bind to hCK2α more weakly than any from the previous group. An analogous trend in fluorescence quenching has been recently observed for TBBt and TBBz, which bind to maize CK2α in different orientations (see structures based on PDB: 1j91 [57] and PDB: 2oxy [91] in Figures 6A and 6B, respectively), and also for their derivatives with bromine at C4 replaced by a methyl group [63]. It may thus be hypothesized that 4,5-Br 2 Bt, 4,6-Br 2 Bt, and most likely 4,5,7-Br 3 Bt, bind to CK2α in different orientations than TBBt.…”

Rational drug-design approach supported with thermodynamic studies — a peptide leader for the efficient bi-substrate inhibitor of protein kinase CK2

“…IC 50 values were determined using three independent experiments, 7 inhibitor concentrations in the range of 1nM-1mM each, by global fitting of the sigmoidal dose-response equation implemented in Origin 9.0 package (www.originlab.com). Single experiment was analyzed for TBBt to confirm agreement with already published data 42,46,47 .…”

5,6-diiodo-1H-benzotriazole: new TBBt analogue that minutely affects mitochondrial activity

“…3 ). This common trend reaffirms our interpretation that the binding of halogenated benzotriazoles to the catalytic subunit of protein kinase CK2 is predominately driven by hydrophobic effect 16 , 18 , 20 , 30 , 31 . In this view, the excess heat of binding of BIIB at the ATP binding site (− 8 kJ/mol) may be assigned to the contribution of halogen bond(s), which are putatively formed between two iodine atoms of BIIB and two backbone carbonyl groups of residues located in the hinge region, resembling a virtual interaction that has been recently identified in the crystal structure of TBBt at hCK2α ATP binding site 32 .…”

Thermodynamics parameters for binding of halogenated benzotriazole inhibitors of human protein kinase CK2α

“…Both effects indicate that all three bind to hCK2α more weakly than any from the previous group. An analogous trend in fluorescence quenching has been recently observed for TBBt and TBBz, which bind to maize CK2α in different orientations (see structures based on PDB: 1j91 [57] and PDB: 2oxy [91] in Figures 6A and 6B, respectively), and also for their derivatives with bromine at C4 replaced by a methyl group [63]. It may thus be hypothesized that 4,5-Br 2 Bt, 4,6-Br 2 Bt, and most likely 4,5,7-Br 3 Bt, bind to CK2α in different orientations than TBBt.…”

Rational drug-design approach supported with thermodynamic studies — a peptide leader for the efficient bi-substrate inhibitor of protein kinase CK2

“…IC 50 values were determined using three independent experiments, 7 inhibitor concentrations in the range of 1nM-1mM each, by global fitting of the sigmoidal dose-response equation implemented in Origin 9.0 package (www.originlab.com). Single experiment was analyzed for TBBt to confirm agreement with already published data 42,46,47 .…”