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A convenient and simple method for the preparation of polybrominated benzimidazole derivatives has been described. Reaction of 4,5,6,7-tetrabromo-1-(3-chloropropyl)-1 H-benzimidazole, obtained by of 4,5,6,7-tetrabromo-1 H-benzimidazole (TBBi) with 1-bromo-3-chloropropane in the presence of KOH, with morpholine, aniline, benzylamine, N-methyl piperazine, and 2-arylpyrrolidines afforded new TBBi derivatives. [ABSTRACT FROM AUTHOR]

Abstract: Two series (para- and meta-substituted) of racemic methyl esters of 3-aryl-3-hydroxypropionic acid were prepared after which the enantiomers were separated by an enzyme-catalyzed transesterification. Several lipases were investigated as the catalyst. The influence of the enzyme pretreatment, as well as substrate concentration, reaction temperature, stirring manner, and substrate conversion on the stereochemical outcome of the biotransformation process were investigated in detail. The best results were achieved by using solvent-pretreated lipase from Pseudomonas fluorescens or Burkholderia cepacia suspended in toluene, and vinyl acetate as the acetyl group donor. [Copyright &y& Elsevier]

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 Ki values in the range 0.5–1 μM. 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 CH3 (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. [Copyright &y& Elsevier]

Abstract The examination of physicochemical properties of selected protein kinase CK2 inhibitors: 4,5,6,7-tetrabromo-1 H -benzotriazole (TBBt), 4,5,6,7-tetrabromo-1 H -benzimidazole (TBBi) and 4,5,6,7-tetrabromo-2-methyl-1 H -benzimidazole (2-Me-TBBi) was performed. The study includes modification of synthesis method and determination of compounds' thermal properties, the solid-liquid phase equilibria and pKa. Thermal properties (melting temperature, enthalpy of fusion, temperature and enthalpy of phase transitions) were determined by means of differential scanning calorimetry (DSC). For solid-liquid phase equilibria study, as a solvent, three liquids were used independently: water, ethanol and 1-octanol. In order to elicit the inhibitors' solubility with alcohols (ethanol and 1-octanol), the dynamic method was applied. In the case of poorly soluble inhibitors in water, equilibria were determined by spectrophotometric method. Based on a phase diagram obtained results were interpreted and correlated using three equations: Wilson equation, NRTL equation and UNIQUAC equation. pKa values of studied polybrominated benzoazoles were estimated by the spectrophotometric Bates–Schwarzenbach method at two constant temperatures: 298.15 K as a room temperature and 310.15 K as a human body temperature. Obtained results are very important for further design of CK2 inhibitors, because they provide information about the solubility of pharmacophore in binary systems: active compound (1) + solvent (2), that may be used in differentiating the way how to apply potential drug. Moreover, all values of dissociation constants bring valuable guidelines to establish the therapeutic dose of pharmaceutics. Graphical abstract Image 1 Highlights • Synthesis of protein kinase CK2 inhibitors. • Evaluation of physicochemical properties of synthesized CK2 inhibitors. • Thermodynamic description of solubility of inhibitors in water and alcohols. • Determination of pKa' values of inhibitors by the Bates-Schwarzenbach method. [ABSTRACT FROM AUTHOR]

Three out of 16 newly synthesized 1,3-dimethylxanthine derivatives (proxyphylline analogues) exhibited consistencies between antifungal and anticancer properties. Proxyphylline possessing 1-(10 H -phenothiazin-10-yl)propan-2-yl ( 6 ) and polybrominated benzimidazole ( 41 ) or benzotriazole moiety ( 42 ) remained selectively cidal against Candida albicans (lg R ≥ 3 at conc. of 31, 36 and 20  μ M, respectively) however not against normal mammalian Vero cell line in vitro (IC 50  ≥ 280  μ M) and Galleria mellonella in vivo . These compounds also displayed moderate antineoplastic activity against human breast adenocarcinoma (MCF-7) cell line (EC 50  = 80  μ M) and high against peripheral blood T lymphoblast (CCRF-CEM) (EC 50  = 6.3–6.5  μ M). In addition, 6 and 42 exerted: (1) dual activity against fungal adhesion and damage mature biofilm; (2) necrosis of planktonic cells due to loss of membrane function and of structural integrity; (3) biochemical (inhibition of sessile cell respiration) and morphological changes in cell wall polysaccharide contents. Therefore, leading proxyphylline derivatives can be employed to prevent cancer-associated biofilm Candida infections. [ABSTRACT FROM AUTHOR]

Abstract: Since protein kinases have been implicated in numerous human diseases, kinase inhibitors have emerged as promising therapeutic agents. Despite this promise, there has been a relative lag in the development of unbiased strategies to validate both inhibitor specificity and the ability to inhibit target activity within living cells. To overcome these limitations, our efforts have been focused on the development of systematic strategies that employ chemical and functional proteomics. We utilized these strategies to evaluate small molecule inhibitors of protein kinase CK2, a constitutively active kinase that has recently emerged as target for anti-cancer therapy in clinical trials. Our chemical proteomics strategies used ATP or CK2 inhibitors immobilized on sepharose beads together with mass spectrometry to capture and identify binding partners from cell extracts. These studies have verified that interactions between CK2 and its inhibitors occur in complex mixtures. However, in the case of CK2 inhibitors related to 4,5,6,7-tetrabromo-1H-benzotriazole (TBB), our work has also revealed off-targets for the inhibitors. To complement these studies, we devised functional proteomics approaches to identify proteins that exhibit decreases in phosphorylation when cells are treated with CK2 inhibitors. To identify and validate those proteins that are direct substrates for CK2, we have also employed mutants of CK2 with decreased inhibitor sensitivity. Overall, our studies have yielded systematic platforms for studying CK2 inhibitors which we believe will foster efforts to define the biological functions of CK2 and to rigorously investigate its potential as a candidate for molecular-targeted therapy. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012). [Copyright &y& Elsevier]

Abstract: CK2 is a constitutively active protein kinase with key regulatory roles in many cellular signaling events which has been implicated in several human diseases. To investigate its roles in biological events and potential as a therapeutic target, several potent CK2 inhibitors have been developed including TBB and its derivatives that have been employed in many studies. Despite the utility of these compounds, a precise understanding of their mode of action within cells remains incomplete. In fact, cells are typically treated with inhibitor concentrations (>5μM) that are orders of magnitude higher than their in vitro inhibitory constants (<0.05μM). Accordingly, we hypothesized that CK2 inhibitors could have off-target effects that are not recognized when inhibitors are profiled using panels of recombinant protein kinases. To address this issue, we combined structural modeling with inhibitor-affinity purification and proteomics to test the specificity of derivatives of TBB using whole cell lysates of HeLa cells. While these studies confirmed that CK2 does bind to the immobilized inhibitor, several other abundant ATP/GTP-binding proteins were also identified and confirmed. These results suggest that highly abundant nucleotide binding proteins may limit the bioavailability of the free inhibitor and interactions with CK2 in the cellular environment. This article is part of a Special Issue entitled: From protein structures to clinical applications. [Copyright &y& Elsevier]

Abstract: An efficient and convenient approach to synthesis of new, enantiopure hydroxyalkyl derivatives of 4,5,6,7-tetrabromo-1H-benzotriazole (TBBt) has been developed. Prochiral ketones synthesized through alkylation of TBBt were reduced to racemic secondary alcohols, which were separated by kinetic resolution carried out by lipase-catalyzed transesterification. After optimization of solvent, lipase and acyl donor group, the highest enantioselectivity factor (E >200) and optical purity of the products (>99%) was achieved with lipase Amano AK from Pseudomonas fluorescens in tert-butyl methyl ether. [Copyright &y& Elsevier]

Formylation of ansa[4]-ferrocene, obtained through the ruthenium-catalysed olefin metathesis, yields two separable, planar chiral 1,3- and 1,2-ansa-ferrocene aldehydes. Single-crystal X-ray structure analysis reveals that both regioisomers crystallize with spontaneous resolution of the racemate in the chiral P212121 space group with one molecule in the asymmetric unit. The major 1,3-isomer was further transformed into a conjugate with 1,2,3-triazole and uracil using "click" chemistry as the key synthetic step. This inorganic–organic hybrid displays anticancer activity (MCF-7, A549, MDA-MB-231 cell lines) with EC50 values comparable to those for cisplatin. [ABSTRACT FROM AUTHOR]

A series of chlorine‐substituted benzotriazole derivatives, representing all possible substitution patterns of halogen atoms attached to the benzotriazole benzene ring, were synthetized as potential inhibitors of human protein kinase CK2. Basic ADME parameters for the free solutes (hydrophobicity, electronic properties) together with their binding affinity to the catalytic subunit of protein kinase CK2 were determined with reverse‐phase HPLC, spectrophotometric titration, and Thermal Shift Assay Method, respectively. The analysis of position‐dependent thermodynamic contribution of a chlorine atom attached to the benzotriazole ring confirmed the previous observation for brominated benzotriazoles, in which substitution at positions 5 and 6 with bromine was found crucial for ligand binding. In all tested halogenated benzotriazoles the replacement of Br with Cl decreases the hydrophobicity, while the electronic properties remain virtually unaffected. Supramolecular architecture identified in the just resolved crystal structures of three of the four possible dichloro‐benzotriazoles shows how substitution distant from the triazole ring affects the pattern of intermolecular interactions. Summarizing, the benzotriazole benzene ring substitution pattern has been identified as the main driver of ligand binding, predominating the non‐specific hydrophobic effect. [ABSTRACT FROM AUTHOR]

The ring-expanded (“fat”) nucleoside, 4,8-diamino-6-imino-6H-1-β-D-ribo- furanosylimidazo[4,5-e][1,3]diazepine (1) and its 2′,3′,5′-tri-O-benzoyl derivative (2) exhibited potent broad spectrum anticancer activities in vitro against a wide variety of human tumor cell lines. The tribenzoyl derivative 2 was found to be considerably more active than the parent nucleoside 1. Further studies using human prostate cancer cells PC-3 and DU-145 suggest that the treatment of exponentially growing culture cells with 1 and 2 leads to marked loss of cell viability in a dose-dependent manner. [ABSTRACT FROM AUTHOR]

Dihydrofolate reductase (DHFR) is a prominent molecular target in antitumor, antibacterial, antiprotozoan, and immunosuppressive chemotherapies, and CK2 protein kinase is an ubiquitous enzyme involved in many processes, such as tRNA and rRNA synthesis, apoptosis, cell cycle or oncogenic transformation. We show for the first time that CK2α subunit strongly interacted with and phosphorylated DHFR in vitro. Using quartz crystal microbalance with dissipation monitoring (QCM-D) we determined DHFR-CK2α binding kinetic parameters (K d below 0.5 μM, k on = 10.31 × 104 M−1s−1 and k off = 1.40 × 10−3s−1) and calculated Gibbs free energy (−36.4 kJ/mol). In order to identify phosphorylation site(s) we used site-directed mutagenesis to obtain several DHFR mutants with predicted CK2-phosphorylable serine or threonine residues substituted with alanines. All enzyme forms were subjected to CK2α subunit catalytic activity and the results pointed to serine 168 as a phosphorylation site. Mass spectrometry analyses confirmed the presence of phosphoserine 168 and revealed additionally the presence of phosphoserine 145, although the latter phosphorylation was on a very low level. • Dihydrofolate reductase and CK2 kinase are molecular targets in chemotherapies. • CK2 strongly interacts with and phosphorylates dihydrofolate reductase. • Serines 168 and 145 were identified as CK2 phosphorylation sites. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract The 1 H -1,2,3-triazole-originated derivatives of willardiine were obtained by: (i) construction of the 1 H -1,2,3-triazole ring in 1,3-dipolar cycloaddition of the uracil-derived azides and the carboxylate-bearing alkynes or α-acylphosphorus ylide, or (ii) N -alkylation of the uracil derivative with the 1 H -1,2,3-triazole-4-carboxylate-derived mesylate. The latter method offered: (i) reproducible results, (ii) a significant reduction of amounts of auxiliary materials, (iii) reduction in wastes and (iv) reduction in a number of manual operations required for obtaining the reaction product. Compound 6a exhibited significant binding affinity to hHS1S2I ligand-binding domain of GluR2 receptor (EC 50 = 2.90 µM) and decreased viability of human astrocytoma MOG-G-CCM cells in higher extent than known AMPA antagonist GYKI 52466. [ABSTRACT FROM AUTHOR]

Protein kinase 2 (CK2), a member of the serine/threonine kinase family, has been established as a promising target in anticancer therapy. New derivatives of known CK2 inhibitors 4,5,6,7-tetrabromo-1 H -benzimidazole (TBBi) and 4,5,6,7-tetrabromo-1 H -benzotriazole (TBBt) bearing azide or substituted triazole groups were synthesized. Their influence on the activity of human recombinant CK2α and cytotoxicity against normal and cancer cell lines were evaluated. TBBi derivatives with triazole substituted with carboxyl substituent ( 7 and 10 ) exhibited the most potent inhibitory activity against CK2 with K i value in the range of 1.96–0.91 μM, respectively. New TBBi derivatives 2 , 3 , 5 and 9 have demonstrated the EC 50 , in the range of 12–25 μM and 13–29 μM respectively towards CCRF-CEM and MCF-7 cells. Derivatives TBBi decreased viability of cancer cells more efficiently than BALB cells and the biggest differences were observed for the azide substituted compounds 3 and 5. The effect of the most active compounds on the activity of eight off-target kinases was evaluated. Inhibitory efficiency of CK2-mediated p65 phosphorylation was demonstrated for the TBBi and compound 12 . [ABSTRACT FROM AUTHOR]

A series of new N-substituted benzimidazole derivatives was synthesized and their antifungal activity against Candida albicans was evaluated. The chemical step included synthesis of appropriate ketones containing benzimidazole ring, to the racemic alcohols, and to the esters. All benzimidazole derivatives were obtained with satisfactory yields and in relatively short times. All synthesized compounds exhibit significant antifungal activity against Candida albicans 900028 ATCC (% cell inhibition at 0.25 μg concentration > 98%). Additionally, racemic mixtures of alcohols were separated by lipase-catalyzed kinetic resolution. In the enzymatic step a reaction was applied and the influence of a lipase type and solvent on the of the reaction was studied. The most selective enzymes were Novozyme SP 435 and lipase Amano AK from Pseudomonas fluorescens (E > 100). Chirality 28:347-354, 2016. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The efficient method for the synthesis of novel cell permeable inhibitors of protein kinase CK2 with anticancer and proapoptotic activity has been developed. A series of polybrominated benzimiadazole derivatives substituted by various cyanoalkyl groups have been synthesized. Cyanoethyl derivatives were obtained by Michael type addition of 4,5,6,7-tetrabromo-1 H -benzimidazole (TBBi) and 4,5,6,7-tetrabromo-2-methyl-1 H -benzimidazole to acrylonitrile, whilst cyanomethyl, cyanopropyl and cyanobutyl analogs by N-alkylation of 4,5,6,7-tetrabromo-1 H -benzimidazole and 4,5,6,7-tetrabromo-2-methyl-1 H -benzimidazole with appropriate cyanoalkyl halides. The inhibitory activity against protein kinase rhCK2α catalytic subunit and cytotoxicity against two human cancer cell lines: acute lymphocytic leukemia (CCRF-CEM) and breast (MCF-7) were evaluated for all newly synthesized compounds. Additionally, the proapoptotic activity toward leukemia cells and intracellular inhibition of CK2 for the most cytotoxic derivatives have been performed, demonstrating 4,5,6,7-tetrabromo-2-methyl-1 H -benzimidazole as a new selective inhibitor of rhCK2 with twenty-fold better proapoptotic activity than parental compound (TBBi). [ABSTRACT FROM AUTHOR]

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 (IC 50 ) 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 ( H bind ) is driven by intermolecular electrostatic interactions involving the triazole/imidazole ring, as indicated by a strong correlation between H bind and ligand p K a . 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. [ABSTRACT FROM AUTHOR]

The efficient method for the synthesis of novel 4,5,6,7-tetrabromo-1H-benzotriazole (TBBt) derivatives bearing a single stereogenic center has been developed. New compounds with a variety of substituents at the meta- and para-position of the phenyl ring are reported. All of the presented compounds were obtained using classical synthetic methods, such as bromination of benzotriazole, and its subsequent alkylation by monotosylated arylpropane-1,3-diols, which in turn have been synthesized through reduction of the corresponding prochiral β-keto esters, and the selective monotosylation of the primary hydroxyl group. The influence of the new and previously reported N-hydroxyalkyl TBBt derivatives on the activity of human protein kinase CK2α catalytic subunit was examined. The most active were derivatives with N-hydroxyalkyl substituents (IC50 in 0.80-7.35 µM range). A binding mode of (R)-1-(4,5,6,7-tetrabromo-2H-benzotriazol-2-yl)butan-3-ol 7b to hCK2α has been proposed based on in silico docking studies. Additionally, MTT-based cytotoxicity tests demonstrated high activities of novel 1-aryl-3-TBBt-propan-1-ol and 3-TBBt-propan-1,2-diol derivatives against human peripheral blood T lymphoblast (CCRF-CEM), and moderate anti-tumor activities against human breast adenocarcinoma (MCF7) cell lines. [ABSTRACT FROM AUTHOR]

Maf1 protein is a global negative regulator of RNA polymerase (Pol) III transcription conserved from yeast to man. We report that phosphorylation of Maf1 by casein kinase II (CK2), a highly evolutionarily conserved eukaryotic kinase, is required for efficient Pol III transcription. Both recombinant human and yeast CK2 were able to phosphorylate purified human or yeast Maf1, indicating that Maf1 can be a direct substrate of CK2. Upon transfer of Saccharomyces cerevisiae from repressive to favorable growth conditions, CK2 activity is required for the release of Maf1 from Pol III bound to a tRNA gene and for subsequent activation of tRNA transcription. In a yeast strain lacking Maf1, CK2 inhibition showed no effect on tRNA synthesis, confirming that CK2 activates Pol III via Maf1. Additionally, CK2 was found to associate with tRNA genes, and this association is enhanced in absence of Maf1, especially under repressive conditions. These results corroborate the previously reported TFIIIB-CK2 interaction and indicate an important role of CK2-mediated Maf1 phosphorylation in triggering Pol III activation. [ABSTRACT FROM AUTHOR]

Abstract: Protein kinase CK2 (formerly “casein kinase 2”) is composed of a central dimer of noncatalytic subunits (CK2β) binding two catalytic subunits. In humans, there are two isoforms of the catalytic subunit (and an additional splicing variant), one of which (CK2α) is well characterized. To supplement the limited biochemical knowledge about the second paralog (CK2α′), we developed a well-soluble catalytically active full-length mutant of human CK2α′, characterized it by Michaelis–Menten kinetics and isothermal titration calorimetry, and determined its crystal structure to a resolution of 2 Å. The affinity of CK2α′ for CK2β is about 12 times lower than that of CK2α and is less driven by enthalpy. This result fits the observation that the β4/β5 loop, a key element of the CK2α/CK2β interface, adopts an open conformation in CK2α′, while in CK2α, it opens only after assembly with CK2β. The open β4/β5 loop in CK2α′ is stabilized by two elements that are absent in CK2α: (1) the extension of the N-terminal β-sheet by an additional β-strand, and (2) the filling of a conserved hydrophobic cavity between the β4/β5 loop and helix αC by a tryptophan residue. Moreover, the interdomain hinge region of CK2α′ adopts a fully functional conformation, while unbound CK2α is often found with a nonproductive hinge conformation that is overcome only by CK2β binding. Taken together, CK2α′ exhibits a significantly lower affinity for CK2β than CK2α; moreover, in functionally critical regions, it is less dependent on CK2β to obtain a fully functional conformation. [Copyright &y& Elsevier]

Abstract: We synthesized new tropolone derivatives substituted with cyclic amines: piperidine, piperazine or pyrrolidine. The most active anti-helicase compound (IC50 =3.4μM), 3,5,7-tri[(4′-methylpiperazin-1′-yl)methyl]tropolone (2), inhibited RNA replication by 50% at 46.9μM (EC50) and exhibited the lowest cytotoxicity (CC50) >1mM resulting in a selectivity index (SI=CC50/EC50) >21. The most efficient replication inhibitor, 3,5,7-tri[(4′-methylpiperidin-1′-yl)methyl]tropolone (6), inhibited RNA replication with an EC50 of 32.0μM and a SI value of 17.4, whereas 3,5,7-tri[(3′-methylpiperidin-1′-yl)methyl]tropolone (7) exhibited a slightly lower activity with an EC50 of 35.6μM and a SI of 9.8. [Copyright &y& Elsevier]

Abstract: New derivatives of 4,5,6,7-tetrabromo-1H-1,2,3-benzotriazole (TBBt), 4,5,6,7-tetrabromo-1H-benzimidazole (TBBi), and N-substituted tetrabromophthalimides were synthesized and their effect on the activity of human protein kinase CK2 was examined. The most active were derivatives with N-hydroxypropyl substituents (IC50 in 0.32–0.54μM range) whereas derivatives of phthalimide were almost ineffective. [Copyright &y& Elsevier]

Ribosomal S6 kinases (S6Ks) are principal players in the regulation of cell growth and energy metabolism. Signaling via phosphatidylinositol 3-kinase and mammalian target of rapamycin pathways mediates the activation of S6K in response to various mitogenic stimuli. The family of S6Ks consists of two forms, S6K1 and -2, that have cytoplasmic and nuclear splicing variants, S6K1 II and S6K1 I, respectively. Nuclear-cytoplasmic shuttling of both isoforms induced by mitogenic stimuli has been reported recently. Here we present the identification of protein kinase CK2 (CK2) as a novel binding and regulatory partner for S6K1 II. The interaction between S6K1 II and CK213 regulatory subunit was initially identified in a yeast two-hybrid screen and further confirmed by co-immunoprecipitation of transiently expressed and endogenous proteins. The interaction between S6K1 II and CK2 was found to occur in serum-starved and serum-stimulated cells. In addition, we found that S6K1 II is a substrate for CK2. The localization of the CK2 phosphorylation site was narrowed down to Ser-17 in S6K1 II. Mutational analysis and the use of phosphospecific antibody indicate that Ser-17 is a major in vitro and in vivo phosphorylation site for CK2. Functional studies reveal that, in contrast to the wild type kinase, the phosphorylation-mimicking mutant of S6K1 II (S17E) retains its cytoplasmic localization in serum-stimulated cells. Treatment of cells with the nuclear export inhibitor leptomycin B revealed that the S17E mutant accumulates in the nucleus to the same extent as S6K1 II wild type. These results indicate that nuclear import of the S17E mutant is not affected, although the export is significantly enhanced. We also provide evidence that nuclear export of 56K1 is mediated by a CRM1-dependent mechanism. Taken together, this study establishes a functional link between S6K1 II and CK2 signaling, which involves the regulation of S6K1 II nuclear export by CK2-mediated phosphorylation of Ser-17. [ABSTRACT FROM AUTHOR]

Abstract: Hepatitis C virus (HCV) is considered one of the most dangerous pathogens since about 3% of the world population is HCV-infected and the virus is a major cause of hepatitis, cirrhosis, and liver carcinoma. A need for a more efficient therapy prompted us to investigate new class of compounds, such as tropolone derivatives that possess antiviral, antibacterial, and antifungal activities. To synthesize bromo- and morpholinomethyl-analogues of tropolone, the previously reported methods were modified. The influence of new derivatives on the activity of the helicase and NTP-ase of HCV was investigated. The most potent inhibitory effect in the fluorometric helicase assay was exerted by 3,7-dibromo-5-morpholinomethyltropolone, for which the IC50 value was at low micromolar range. All the morpholino-derivatives had inhibitory activities higher than those of the non-modified analogues. Low toxicity in a yeast-based toxicity assay indicates that these compounds could be further modified to develop potent inhibitors of the HCV helicase and of viral replication. [Copyright &y& Elsevier]

Abstract: 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 CK2β 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 ≈5, exists in solution at physiological pH almost exclusively (>99%) as the monoanion; whereas TBBz, with a pK a ≈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 K i values ranging from 80 to 210 nM. TBBz better discriminates between CK2 forms, with K i 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 CK2α and CK2α′ isoforms reinforces the foregoing results, indicating that inhibition of CK2 leads to attenuation of proliferation. [Copyright &y& Elsevier]

Chloro-, bromo- and methyl- analogues of 1H-benzimidazole and 1H-benzotriazole and their N-alkyl derivatives have been synthesized and tested in vitro against the protozoa Acanthamoeba castellanii. The results indicate that 5,6-dimethyl-1H-benzotriazole (11) and 5,6-dibromo-1H-benzotriazole (14) have higher efficacy than the antiprotozoal agent chlorohexidine. [Copyright &y& Elsevier]

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 CK2α′. 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 α′ subunit of CK2 in phosphorylation of ribosomal proteins. It occurs that CK2α′ but not CK2 holoenzyme is responsible for phosphorylation of P-proteins in vivo. Moreover, elimination of both forms of protein kinase CK2 (hCK2 and CK2α′) activity in living cells led to dramatic loss of the translational activity of the ribosome. [Copyright &y& Elsevier]

A search has been initiated for lead inhibitors of the nonstructural protein 3 (NS3)-associated NTPase/helicase activities of hepatitis C virus, the related West Nile virus, Japanese encephalitis virus and the human mitochondrial Suv3 enzyme. Random screening of a broad range of unrelated low-molecular mass compounds, employing both RNA and DNA substrates, revealed that 4,5,6,7-tetrabromobenzotriazole (TBBT) hitherto known as a potent highly selective inhibitor of protein kinase 2, is a good inhibitor of the helicase, but not NTPase, activity of hepatitis C virus NTPase/helicase. The IC50 is approximately 20 µm with a DNA substrate, but only 60 µm with an RNA substrate. Several related analogues of TBBT were enzyme- and/or substrate-specific inhibitors. For example, 5,6-dichloro-1-(β-d-ribofuranosyl)benzotriazole (DRBT) was a good, and selective, inhibitor of the West Nile virus enzyme with an RNA substrate (IC50 ≈ 0.3 µm), but much weaker with a DNA substrate (IC50 ≈ 3 µm). Preincubation of the enzymes, but not substrates, with DRBT enhanced inhibitory potency, e.g. the IC50 vs the hepatitis C virus helicase activity was reduced from 1.5 to 0.1 µm. No effect of preincubation was noted with TBBT, suggesting a different mode of interaction with the enzyme. The tetrachloro congener of TBBT, 4,5,6,7,-tetrachlorobenzotriazole (TCBT; a much weaker inhibitor of casein kinase 2) is also a much weaker inhibitor than TBBT of all four helicases. Kinetic studies, supplemented by comparison of ATP-binding sites, indicated that, unlike the case with casein kinase 2, the mode of action of the inhibitors vs the helicases is not by interaction with the catalytic ATP-binding site, but rather by occupation of an allosteric nucleoside/nucleotide binding site. The halogeno benzimidazoles and benzotriazoles included in this study are excellent lead compounds for the development of more potent... [ABSTRACT FROM AUTHOR]

Protein kinase CK2 has been considered as an attractive drug target for anti-cancer therapy. The synthesis of N-hydroxypropyl TBBi and 2MeTBBi derivatives as well as their respective esters was carried out by using chemoenzymatic methods. Concomitantly with kinetic studies toward recombinant CK2, the influence of the obtained compounds on the viability of two human breast carcinoma cell lines (MCF-7 and MDA-MB-231) was evaluated using MTT assay. Additionally, an intracellular inhibition of CK2 as well as an induction of apoptosis in the examined cells after the treatment with the most active compounds were studied by Western blot analysis, phase-contrast microscopy and flow cytometry method. The results of the MTT test revealed potent cytotoxic activities for most of the newly synthesized compounds (EC50 4.90 to 32.77 µM), corresponding to their solubility in biological media. We concluded that derivatives with the methyl group decrease the viability of both cell lines more efficiently than their non-methylated analogs. Furthermore, inhibition of CK2 in breast cancer cells treated with the tested compounds at the concentrations equal to their EC50 values correlates well with their lipophilicity since derivatives with higher values of logP are more potent intracellular inhibitors of CK2 with better proapoptotic properties than their parental hydroxyl compounds. [ABSTRACT FROM AUTHOR]

• Serine sulfamidate - pyrimidine nuclebase coupling provides β-pyrimidine alanines. • β-Cytosine alanine is active against human brain astrocytoma MOG-G-CCM cells. • GluR2 receptor features ability to bind willardiine esters. Three series of the β-pyrimidine alanines, including willardiine - a naturally occurring amino acid, were prepared from the l -serine-derived sulfamidates. Compounds 3b , 4a and 4b demonstrated antiproliferative activity toward the studied cancer cell lines, albeit the effect of these compounds on human brain astrocytoma MOG-G-CCM cells was more significant than on human neuroblastoma SK- N -AS cells. The cytosine analog of willardiine, compound 4b , reduced viability of MOG-G-CCM cells with EC 50 = 36 ± 2 μM, more effectively than AMPA antagonist GYKI 52466. Willardiine showed possible capability of affecting invasiveness of glioblastoma U251 MG cells with no effect on their viability and morphology. Compound 3d , the ethyl ester of willardiine, featured activity toward binding domain hHS1S2I of the GluR2 receptor. Docking analysis revealed that the location mode of compound 3d at the S1S2 domain of hGluR2 (PDB ID: 3R7X) might differ from that of willardiine. [ABSTRACT FROM AUTHOR]