casein-kinase-ii and 4-5-6-7-tetrabromobenzimidazole

The development of selective cell-permeable inhibitors of protein kinase CK2 has represented an important advance in the field. However, it is important to not overlook the existence of discrete molecular forms of CK2 that arise from the presence of distinct isozymic forms, and the existence of the catalytic CK2 subunits as free subunits and in complexes with the regulatory CK2beta subunits and, possibly, other proteins. This review examines two recently developed, and presently widely applied, CK2 inhibitors, 4,5,6,7-tetrabromobenzotriazole (TBBt) and the related 4,5,6,7-tetrabromobenzimidazole (TBBz), the latter of which was previously shown to discriminate between different molecular forms of CK2 in yeast. We have shown, by spectrophotometric titration, that TBBt, with a pK(a) approximately 5, exists in solution at physiological pH almost exclusively (>99%) as the monoanion; whereas TBBz, with a pKa approximately 9, is predominantly (>95%) in the neutral form, both of obvious relevance to their modes of binding. In vitro, TBBt inhibits different forms of CK2 with Ki values ranging from 80 to 210 nM. TBBz better discriminates between CK2 forms, with Ki values ranging from 70 to 510 nM. Despite their general similar in vitro activities, TBBz is more effective than TBBt in inducing apoptosis and, to a lesser degree, necrosis, in transformed human cell lines. Finally, development of shRNA strategies for the selective knockdown of the CK2alpha and CK2alpha' isoforms reinforces the foregoing results, indicating that inhibition of CK2 leads to attenuation of proliferation.

Topics: Apoptosis; Benzimidazoles; Casein Kinase II; Cell Membrane Permeability; Cell Proliferation; Evaluation Studies as Topic; HeLa Cells; Humans; Protein Isoforms; Triazoles

Quinone reductase 2 (NQO2) exhibits off-target interactions with two protein kinase CK2 inhibitors, 4,5,6,7-1H-tetrabromobenzimidazole (TBBz) and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT). TBBz and DMAT induce apoptosis in cells expressing an inhibitor-resistant CK2, suggesting that the interaction with NQO2 may mediate some of their pharmacological effects. In this study, we have fully characterized the binding of TBBz and DMAT to NQO2. Fluorescence titrations showed that TBBz and DMAT bind oxidized NQO2 in the low nanomolar range; in the case of TBBz, the affinity for NQO2 was 40-fold greater than its affinity for CK2. A related CK2 inhibitor, 4,5,6,7-tetrabromobenzotriazole (TBB), which failed to cause apoptosis in cells expressing inhibitor-resistant CK2, binds NQO2 with an affinity 1000-fold lower than those of TBBz and DMAT. Kinetic analysis indicated that DMAT inhibits NQO2 by binding with similar affinities to the oxidized and reduced forms. Crystal structure analysis showed that DMAT binds reduced NQO2 in a manner different from that in the oxidized state. In oxidized NQO2, TBBz and DMAT are deeply buried in the active site and make direct hydrogen and halogen bonds to the enzyme. In reduced NQO2, DMAT occupies a more peripheral region and hydrogen and halogen bonds with the enzyme are mediated through three water molecules. Therefore, although TBB, TBBz, and DMAT are all potent inhibitors of CK2, they exhibit different activity profiles toward NQO2. We conclude that the active site of NQO2 is fundamentally different from the ATP binding site of CK2 and the inhibition of NQO2 by CK2 inhibitors is adventitious.

Topics: Benzimidazoles; Casein Kinase II; Crystallography, X-Ray; Dose-Response Relationship, Drug; Drug Delivery Systems; Humans; Protein Binding; Protein Kinase Inhibitors; Protein Structure, Secondary; Protein Structure, Tertiary; Quinone Reductases

Glioblastoma (GBM) is the most common and fatal primary brain tumor in humans, and it is essential that new and better therapies are developed to treat this disease. Previous research suggests that casein kinase 2 (CK2) may be a promising therapeutic target for GBMs. CK2 has enhanced expression or activity in numerous cancers, including GBM, and it has been demonstrated that inhibitors of CK2 regressed tumor growth in GBM xenograft mouse models. Our studies demonstrate that the CK2 subunit, CK2α, is overexpressed in and has an important role in regulating brain tumor-initiating cells (BTIC) in GBM. Initial studies showed that two GBM cell lines (U87-MG and U138) transduced with CK2α had enhanced proliferation and anchorage-independent growth. Inhibition of CKα using siRNA or small-molecule inhibitors (TBBz, CX-4945) reduced cell growth, decreased tumor size, and increased survival rates in GBM xenograft mouse models. We also verified that inhibition of CK2α decreased the activity of a well-known GBM-initiating cell regulator, β-catenin. Loss of CK2α decreased two β-catenin-regulated genes that are involved in GBM-initiating cell growth, OCT4 and NANOG. To determine the importance of CK2α in GBM stem cell maintenance, we reduced CK2α activity in primary GBM samples and tumor spheres derived from GBM patients. We discovered that loss of CK2α activity reduced the sphere-forming capacity of BTIC and decreased numerous GBM stem cell markers, including CD133, CD90, CD49f and A2B5. Our study suggests that CK2α is involved in GBM tumorigenesis by maintaining BTIC through the regulation of β-catenin.

Topics: Animals; Benzimidazoles; beta Catenin; Brain Neoplasms; Casein Kinase II; Cell Line, Tumor; Cell Proliferation; Glioblastoma; Humans; Mice; Naphthyridines; Neoplasm Transplantation; Neoplastic Stem Cells; Phenazines; Prognosis; Signal Transduction; Survival Analysis

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.

Topics: Benzimidazoles; Binding Sites; Calorimetry; Calorimetry, Differential Scanning; Casein Kinase II; Drug Design; Enzyme Inhibitors; Humans; Hydrogen-Ion Concentration; Inhibitory Concentration 50; Ligands; Methylation; Microscopy, Fluorescence; Protein Binding; Static Electricity; Temperature; Thermodynamics; Triazoles

CK2 is a ubiquitous serine/threonine protein kinase, which has the potential to catalyze the generation of a large proportion of the human phosphoproteome. Due to its role in numerous cellular functions and general anti-apoptotic activity, CK2 is an important target of research with therapeutic potential. This emphasizes the need for cell-permeable highly potent and selective inhibitors and photoluminescence probes of CK2 for investigating the protein phosphorylation networks in living cells. Previously, we had developed bisubstrate inhibitors for CK2 (CK2-targeted ARCs) that showed remarkable affinity (KD < 1 nM) and selectivity, but lacked proteolytic stability and plasma membrane permeability. In this report, the structures of CK2-targeted ARCs were modified for the application in live cells. Based on structure-activity studies, proteolytically stable achiral oligoanionic peptoid conjugates of 4,5,6,7-tetrabromo-1H-benzimidazole (TBBz) were constructed. Affinity of the conjugates toward CK2 reached subnanomolar range. Acetoxymethyl (AM) prodrug strategy was applied for loading TBBz-peptoid conjugates into living cells. The uptake of inhibitors was visualized by live cell imaging and the reduction of the phosphorylation levels of two CK2-related phosphosites, Cdc37 pSer13 and NFκB pSer529, was demonstrated by Western blot analysis.

Topics: Benzimidazoles; Casein Kinase II; Cell Survival; Esterification; HeLa Cells; Humans; Models, Molecular; Peptoids; Protein Kinase Inhibitors

Human La protein (hLa) is a multifunctional RNA-binding protein involved in the regulation of hepatitis B virus (HBV) expression. Casein kinase II (CK2), a protein kinase, is known to activate hLa by phosphorylating Ser(366). Tetrabromobenzimidazole (TBBz) has been shown to be a specific inhibitor of CK2 activity, which suggests that TBBz may be useful for reducing HBV gene expression. The aim of our study was to determine whether inhibition of CK2 by TBBz and decreased phosphorylation of hLa Ser(366) (pLa) would reduce HBV gene expression. pLa and total La expression levels were evaluated by immunohistochemistry in human liver tissues with or without HBV infection. HepG2.2.15 cells (an HBV-expressing cell line) were treated with TBBz, and cell viability and pLa levels were evaluated. Knockdown of hLa and CK2 levels by specific siRNA and mutant hLa Ala(366) were utilized to establish the roles of pLa and CK2 in HBV gene expression. HBV DNA replication and HBsAg and HBeAg levels were analysed in HepG2.2.15 cell supernatants by standard methods. pLa was significantly overexpressed in HBV-infected human liver samples. TBBz decreased the phosphorylation of hLa, which coincided with decreased HBV expression. Mutant hLa Ala(366) had reduced viral expression compared with hLa Ser(366) treatment in hLa siRNA knockdown cells. Knockdown of CK2 also decreased the HBV parameters. hLa plays a key role in the regulation of HBV gene expression in a CK2-dependent mechanism via phosphorylation of hLa at Ser(366).

Topics: Adolescent; Adult; Aged; Benzimidazoles; Casein Kinase II; Cell Line, Tumor; Dose-Response Relationship, Drug; Down-Regulation; Female; Gene Expression Regulation, Viral; Hepatitis B; Hepatitis B Antigens; Hepatitis B virus; Humans; Liver; Male; Middle Aged; Mutation, Missense; Phosphoproteins; Phosphorylation; Serine; Virus Replication; Young Adult

Advantage has been taken of the relative promiscuity of commonly used inhibitors of protein kinase CK2 to develop compounds that can be exploited for the selective inhibition of druggable kinases other than CK2 itself. Here we summarize data obtained by altering the scaffold of CK2 inhibitors to give rise to novel selective inhibitors of DYRK1A and to a powerful cell permeable dual inhibitor of PIM1 and CK2. In the former case one of the new compounds, C624 (naphto [1,2-b]benzofuran-5,9-diol) displays a potency comparable to that of the first-in-class DYRK1A inhibitor, harmine, lacking however the drawback of drastically inhibiting monoamine oxidase-A (MAO-A) as harmine does. On the other hand the promiscuous CK2 inhibitor 4,5,6,7-tetrabromo-1H-benzimidazole (TBI,TBBz) has been derivatized with a sugar moiety to generate a 1-(β-D-2'-deoxyribofuranosyl)-4,5,6,7-tetrabromo-1H-benzimidazole (TDB) compound which inhibits PIM1 and CK2 with comparably high efficacy (IC50 values<100nM) and remarkable selectivity. TDB, unlike other dual PIM1/CK2 inhibitors described in the literature is readily cell permeable and displays a cytotoxic effect on cancer cells consistent with concomitant inhibition of both its onco-kinase targets. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

Topics: Adenosine Triphosphate; Adipocytes; Benzimidazoles; Binding Sites; Casein Kinase II; Cells, Cultured; Dose-Response Relationship, Drug; Dyrk Kinases; Harmine; Humans; Kinetics; Models, Molecular; Molecular Structure; Monoamine Oxidase; Monoamine Oxidase Inhibitors; Phosphorylation; Protein Binding; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-pim-1

The goal of this study was to test the inhibitory activity of a series of tetrahalogenobenzimidazoles, including a number of novel derivatives, on individual catalytic subunits of human CK2. 4,5,6,7-tetrabromo- and 4,5,6,7-tetraiodo-1H-benzimidazoles and their newly obtained N(1)- and 2-S-carboxyalkyl derivatives showed potent inhibitory activity against both these subunits. CK2α' was up to 6 times more sensitive to the studied compounds than CK2α. The investigated iododerivatives showed, in most cases, stronger inhibitory properties than the respective brominated congeners, but the differences showed considerable dependence on the protein substrate used.

Topics: Benzimidazoles; Casein Kinase II; Catalytic Domain; Humans; Protein Kinase Inhibitors; Protein Subunits

Increased glutamatergic input in the paraventricular nucleus (PVN) is important for high sympathetic outflow in hypertension, but the associated molecular mechanisms remain unclear. Here, we determined the role of protein kinase CK2 (formerly casein kinase II) in increased N-methyl-d-aspartate receptor (NMDAR) activity in spinally projecting PVN neurons and sympathetic vasomotor tone in spontaneously hypertensive rats (SHRs). The selective CK2 inhibitors 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) or 4,5,6,7-tetrabromobenzotriazole (TBB) significantly decreased the frequency of miniature EPSCs (mEPSCs) of labeled PVN neurons in SHRs but not in Wistar-Kyoto (WKY) normotensive rats. Also, DRB abolished the inhibitory effect of the NMDAR antagonist AP5 on the frequency of mEPSCs in SHRs. Treatment with DRB or TBB significantly reduced the amplitude of evoked NMDA-EPSCs but not AMPA-EPSCs in SHRs. Furthermore, DRB significantly decreased the firing activity of PVN neurons in SHRs but not in WKY rats. The membrane protein level of CK2α in the PVN, but not brainstem and prefrontal cortex, was significantly higher in SHRs than in WKY rats. Lowering blood pressure with celiac ganglionectomy in SHRs did not alter the increased CK2α level and the effects of DRB on mEPSCs and NMDA-EPSCs. In addition, intracerebroventricular injection of DRB not only significantly reduced blood pressure and lumbar sympathetic nerve discharges but also eliminated the inhibitory effect of AP5 microinjected into the PVN on sympathetic nerve activity in SHRs. Our findings suggest that augmented CK2 activity critically contributes to increased presynaptic and postsynaptic NMDAR activity in the PVN and elevated sympathetic vasomotor tone in essential hypertension.

Topics: Animals; Benzimidazoles; Blood Pressure; Brain Stem; Casein Kinase II; Dichlororibofuranosylbenzimidazole; Excitatory Postsynaptic Potentials; Ganglionectomy; Heart Rate; Hypertension; In Vitro Techniques; Injections, Intraventricular; Male; Microinjections; Paraventricular Hypothalamic Nucleus; Prefrontal Cortex; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Sympathetic Nervous System; Valine

A series of new polybrominated benzimidazoles and benzotriazoles has been synthesized and their influence on the activity of protein kinase CK2 was evaluated. It was revealed that the most active inhibitors are those with methyl or ethyl substituent at benzene ring, namely 5,6,7-tribromo-4-methyl-1H-benzotriazole (38, IC(50) 0.51 μM) and 5,6,7-tribromo-4-ethyl-1H-benzotriazole (40, IC(50) 0.16 μM). The derivatives with large aromatic or heterocyclic substituents connected to benzimidazole or benzotriazole scaffold appeared to be less potent inhibitors.

Topics: Benzimidazoles; Casein Kinase II; Drug Design; Humans; Inhibitory Concentration 50; Protein Kinase Inhibitors; Triazoles

Aberrant activation of protein kinases is one of the essential oncogenic driving forces inherent to the process of tumorigenesis. The protein kinase CK2 plays an important role in diverse biological processes, including cell growth and proliferation as well as in the governing and transduction of prosurvival signals. Increased expression of CK2 is a hallmark of some cancers, hence its antiapoptotic properties may be relevant to cancer onset. Thus, the designing and synthesis of the CK2 inhibitors has become an important pursuit in the search for cancer therapies.. Using a high-throughput microarray approach, we demonstrate that two potent inhibitors of CK2, 4,5,6,7-tetrabromo-benzimidazole (TBBz) and 2-Dimethyloamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), blocked mitogen induced mRNA expression of immediate early genes. Given the impact of these inhibitors on the process of transcription, we investigated their effects on RNA Polymerase II (RNAPII) elongation along the mitogen inducible gene, EGR1 (early growth response 1), using chromatin immunoprecipitation (ChIP) assay. ChIP analysis demonstrated that both drugs arrest RNAPII elongation. Finally, we show that CDK9 kinase activity, essential for the triggering of RNAPII elongation, was blocked by TBBz and to lesser degree by DMAT.. Our approach revealed that small molecules derived from halogenated imidazole compounds may decrease cell proliferation, in part, by inhibiting pathways that regulate transcription elongation.

Topics: Antineoplastic Agents; Benzimidazoles; Casein Kinase II; Cell Proliferation; Chromatin Immunoprecipitation; Cyclin-Dependent Kinase 9; Early Growth Response Protein 1; Gene Expression Profiling; Halogenation; HeLa Cells; Humans; Protein Kinase Inhibitors; RNA Polymerase II; Transcription, Genetic

Several studies indicate the involvement of protein kinases in the progression of various malignancies. Kinase inhibitors are therefore becoming important anticancer drugs. CK2 kinase (casein kinase-2) has been suggested to be a constituent of a neoplastic milleu, and its inhibition might represent a new approach to cancer therapy. Adrenocortical carcinomas (ACCs) are highly malignant neoplasms with poor overall prognosis. We have examined the effects of 2-dimethylamino-4,5,6,7-tetrabromobenzimidazole (DMAT), a potent CK2 inhibitor, on the H295R human adrenocortical cancer cell line. Treatment with DMAT decreases the secretion of aldosterone, dehydroepiandrosterone sulfate, and androstendione and results in an accumulation of 17-OH-progesterone. Cell growth as measured by the MTT and 5-bromo-2'-deoxyuridine incorporation assays is inhibited, and cell cycle analysis has revealed a slight induction of apoptosis. Thus, CK2 kinase activity is probably involved in human ACC endocrine activity and growth.

Topics: 17-alpha-Hydroxyprogesterone; Adrenal Cortex Hormones; Adrenal Cortex Neoplasms; Adrenocortical Carcinoma; Aldosterone; Antineoplastic Agents; Benzimidazoles; Casein Kinase II; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dehydroepiandrosterone Sulfate; Drug Screening Assays, Antitumor; Humans; Hydrocortisone; Protein Kinase Inhibitors

N-methyl-D-aspartate (NMDA) receptors (NMDARs) play a central role in development, synaptic plasticity, and neurological disease. NMDAR subunit composition defines their biophysical properties and downstream signaling. Casein kinase 2 (CK2) phosphorylates the NR2B subunit within its PDZ-binding domain; however, the consequences for NMDAR localization and function are unclear. Here we show that CK2 phosphorylation of NR2B regulates synaptic NR2B and NR2A in response to activity. We find that CK2 phosphorylates NR2B, but not NR2A, to drive NR2B-endocytosis and remove NR2B from synapses resulting in an increase in synaptic NR2A expression. During development there is an activity-dependent switch from NR2B to NR2A at cortical synapses. We observe an increase in CK2 expression and NR2B phosphorylation over this same critical period and show that the acute activity-dependent switch in NR2 subunit composition at developing hippocampal synapses requires CK2 activity. Thus, CK2 plays a central role in determining the NR2 subunit content of synaptic NMDARs.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Animals; Animals, Newborn; Benzimidazoles; Biotinylation; Casein Kinase II; Cells, Cultured; Cerebral Cortex; Disks Large Homolog 4 Protein; Embryo, Mammalian; Endocytosis; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Gene Expression Regulation; Green Fluorescent Proteins; Hippocampus; Humans; Immunoprecipitation; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Models, Biological; Neurons; Patch-Clamp Techniques; PDZ Domains; Phosphorus Isotopes; Phosphorylation; Piperidines; Protein Subunits; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Sodium Channel Blockers; Subcellular Fractions; Synapses; Synaptophysin; Tetrodotoxin; Transfection; Tyrosine

We present an adaptation of the MM-PB/SA method for the estimation of binding free energies in protein-ligand complexes simulated with QM/MM molecular dynamics. The method is applied to understand the binding of a set of tetrabromobenzimidazole inhibitors to the CK2 protein. We find that the QM/MM interaction energy alone cannot always be used as a predictor of the binding affinity, and the inclusion of solvation effects via the PB/SA method is essential in getting reliable results. In agreement with experimental observations, we show that the van der Waals interactions are the driving force for the binding, while the electrostatic interactions orient these inhibitors in the CK2 active site. Additionally a per-residue energy decomposition analysis was applied to determine the individual contributions to the protein-inhibitor interaction. Based on these results, we hypothesize that the inclusion of a sufficiently large polar group on the tetrabromobenzimidazole skeleton could increase the binding affinity. The results show that the QM/MM-PB/SA method can be successfully employed to understand complicated structure-activity relationships and to design new inhibitors.

Topics: Algorithms; Amino Acids; Benzimidazoles; Casein Kinase II; Computer Simulation; Databases, Factual; Energy Transfer; Enzyme Inhibitors; Models, Molecular; Protein Binding; Protein Conformation; Structure-Activity Relationship

Topics: Animals; Benzimidazoles; Casein Kinase II; Cell Line, Tumor; Cell Survival; Fusion Proteins, bcr-abl; Humans; Leukemia, Lymphoid; Mice

CK2 is a highly pleiotropic Ser/Thr protein kinase that is able to promote cell survival and enhance the tumour phenotype under specific circumstances. We have determined the crystal structure of three new complexes with tetrabromobenzimidazole derivatives that display K(i) values between 0.15 and 0.30 microM. A comparative analysis of these data with those of four other inhibitors of the same family revealed the presence of some highly conserved water molecules in the ATP-binding site. These waters reside near Lys68, in an area with a positive electrostatic potential that is able to attract and orient negatively charged ligands. The presence of this positive region and two unique bulky residues that are typical of CK2, Ile66 and Ile174, play a critical role in determining the ligand orientation and binding selectivity.

Topics: Adenosine Triphosphate; Benzimidazoles; Binding Sites; Casein Kinase II; Humans; Isoleucine; Ligands; Lysine; Protein Conformation; Static Electricity; Water

CK2 is a very pleiotropic protein kinase whose high constitutive activity is suspected to cooperate to neoplasia. Here, the crystal structure of the complexes between CK2 and three selective tetrabromo-benzimidazole derivatives inhibiting CK2 with Ki values between 40 and 400 nM are presented. The ligands bind to the CK2 active site in a different way with respect to the parent compound TBB. They enter more deeply into the cavity, establishing halogen bonds with the backbone of Glu114 and Val116 in the hinge region. A detailed analysis of the interactions highlights a major role of the hydrophobic effect in establishing the rank of potency within this class of inhibitors and shows that polar interactions are responsible for the different orientation of the molecules in the active site.

Topics: Benzimidazoles; Bromine; Casein Kinase II; Humans; Hydrophobic and Hydrophilic Interactions; Inhibitory Concentration 50; Models, Molecular; Molecular Structure; Mutation; Phosphorylation; Protein Kinase Inhibitors; Structure-Activity Relationship

Casein kinase 2 (CK2) is a ubiquitous, essential, and highly pleiotropic protein kinase whose abnormally high constitutive activity is suspected to underlie its pathogenic potential in neoplasia and infective diseases. Thus, CK2 inhibitors designed to dissect the signaling pathways affected by this kinase, in perspective, may give rise to pharmacological tools. One of the most successful CK2 inhibitors is TBB (4,5,6,7-tetrabromobenzotriazole). Here we show that its inhibitory properties can be markedly improved by generating adducts in which N(2) is replaced by a carbon atom bound to a variety of polar functions. The most efficient inhibitor is 4,5,6,7-tetrabromo-2-(dimethylamino)benzimidazole (2c) followed by the methylsulfanyl (8), isopropylamino (2e), and amino (2a) congeners. All these compounds display K(i) values <100 nM (40 nM in the case of 2c). 2c induces apoptosis of Jurkat cells more readily than TBB (DC(50) value 2.7 vs 17 microM) and, unlike TBB, it does not display any side effect on mitochondria polarization up to 10 microM concentration. Molecular modeling of the CK2-2c complex, based on the crystal structure of the CK2-TBB complex suggests that a number of additional apolar contacts between its two methyl groups and hydrophobic residues nearby could account for its superior inhibitory properties. Consequently, 2c is even more susceptible than TBB to mutations of the unique hydrophobic residues V66 and/or I174 to alanine. We propose to adopt 2c as first choice CK2 inhibitor instead of TBB, especially for in cell studies.

Topics: Animals; Apoptosis; Benzimidazoles; Casein Kinase II; Catalytic Domain; Humans; Jurkat Cells; Kinetics; Models, Molecular; Molecular Structure; Mutation; Phosphorylation; Rats; Structure-Activity Relationship

Like the previously reported 4,5,6,7-tetrabromobenzotriazole (TBBt), the structurally related 4,5,6,7-tetrabromobenzimidazole (TBBz) is a selective ATP-competitive inhibitor of protein kinase CK2 from such divergent sources as yeast, rat liver, Neurospora crassa and Candida tropicalis, with K(i) values in the range 0.5-1 microM. It is virtually inactive vs. PKA, PKC, and a very weak inhibitor of protein kinase CK1. The corresponding tetrachlorobenzimidazole (TCBz) is a much weaker inhibitor of CK2, like tetrachlorobenzotriazole (TCBt) relative to TBBt. Bearing in mind the similarity of the van der Waals radii of Br (1.95 A) and CH(3) (2.0 A), the corresponding much less hydrophobic 4,5,6,7-tetramethylbenzotriazole (TMeBt) was prepared and found to be a very weak inhibitor of CK2, as well as of CK1. An unexpected, and significant, difference between TBBt and TBBz are their inhibitory activities vs. the yeast protein kinase PK60S, which phosphorylates, both in vitro and in intact yeast cells, three of the five pp13 kDa ribosomal surface acidic proteins in yeast cells. TBBt was previously noted to be a more effective inhibitor of PK60S than of yeast CK2; by contrast, TBBz is a relatively feeble inhibitor of PK60S, hence more selective than TBBt vs. CK2 in yeast cells. TMeBt was virtually inactive vs PK60S. Like TBBt, TBBz is an additional lead compound for development of more potent inhibitors of CK2.

Topics: Adenosine Triphosphate; Animals; Benzimidazoles; Binding, Competitive; Candida tropicalis; Casein Kinase II; Enzyme Inhibitors; In Vitro Techniques; Kinetics; Liver; Neurospora crassa; Protein Serine-Threonine Kinases; Rats; Saccharomyces cerevisiae; Triazoles

Protein kinases are targets for therapeutic agents designed to intervene in signaling processes in the diseased state. Most kinase inhibitors are directed towards the conserved ATP binding site. Because the essential features of this site are conserved in all eukaryotic protein kinases, it is generally assumed that the same compound will bind in a similar manner to different protein kinases. The inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) is a selective inhibitor for the protein kinase CK2 (IC50 1.6 micro m) (Sarno et al. (2001) FEBS Letts.496, 44-48). Three other kinases [cyclin-dependent protein kinase 2 (CDK2), phosphorylase kinase and glycogen synthase kinase 3beta] exhibit approximately 10-fold weaker affinity for TBB than CK2. We report the crystal structure of TBB in complex with phospho-CDK2-cyclin A at 2.2 A resolution and compare the interactions with those observed for TBB bound to CK2. TBB binds at the ATP binding site of both kinases. In CDK2, each of the four bromine atoms makes polar contacts either to main chain oxygens in the hinge region of the kinase or to water molecules, in addition to several van der Waals contacts. The mode of binding of TBB to CDK2 is different from that to CK2. TBB in CDK2 is displaced more towards the hinge region between the N- and C-terminal lobes and rotated relative to TBB in CK2. The ATP binding pocket is wider in CDK2 than in CK2 resulting in fewer van der Waals contacts but TBB in CK2 does not contact the hinge. The structures show that, despite the conservation of the ATP binding pocket, the inhibitor is able to exploit different recognition features so that the same compound can bind in different ways to the two different kinases.

Topics: Adenosine Triphosphate; Benzimidazoles; Binding Sites; Casein Kinase II; CDC2-CDC28 Kinases; Cyclin A; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; Enzyme Inhibitors; Humans; Macromolecular Substances; Models, Molecular; Protein Binding; Protein Serine-Threonine Kinases

Two ATP-competitive inhibitors-4,5,6,7-tetrabromo-benzotriazole (TBBt) and 4,5,6,7-tetrabromo-benzimidazole (TBBz) have been shown to decrease activity of CK2 holoenzyme. Surprisingly it occurs that TBBz contrary to TBBt does not inhibit free catalytic subunit CK2 [Formula: see text]. Both inhibitors are virtually inactive against RAP protein kinase. The above-mentioned protein kinases phosphorylate in vitro a set of acidic ribosomal P-proteins of the 60S ribosomal subunit. Such a modification is one of the mechanisms regulating translational activity of ribosomes in vivo. Application of these two very selective inhibitors allows us to define the role of free catalytic [Formula: see text] subunit of CK2 in phosphorylation of ribosomal proteins. It occurs that CK2 [Formula: see text] but not CK2 holoenzyme is responsible for phosphorylation of P-proteins in vivo. Moreover, elimination of both forms of protein kinase CK2 (hCK2 and CK2 [Formula: see text] ) activity in living cells led to dramatic loss of the translational activity of the ribosome.

Topics: Benzimidazoles; Binding, Competitive; Casein Kinase II; Cell Division; Dose-Response Relationship, Drug; Enzyme Activation; Protein Biosynthesis; Protein Serine-Threonine Kinases; Protozoan Proteins; Ribosomal Proteins; Saccharomyces cerevisiae; Structure-Activity Relationship; Triazoles