Maria Bretner

Inhibition of protein kinase CK2 suppresses angiogenesis and hematopoietic stem cell recruitment to retinal neovascularization sites

Ubiquitous protein kinase CK2 participates in a variety of key cellular functions. We have explored CK2 involvement in angiogenesis. As shown previously, CK2 inhibition reduced endothelial cell proliferation, survival and migration, tube formation, and secondary sprouting on Matrigel. Intraperitoneally administered CK2 inhibitors significantly reduced preretinal neovascularization in a mouse model of proliferative retinopathy. In this model, CK2 inhibitors had an additive effect with somatostatin analog, octreotide, resulting in marked dose reduction for the drug to achieve the same effect. CK2 inhibitors may thus emerge as potent future drugs aimed at inhibiting pathological angiogenesis. Immunostaining of the retina revealed predominant CK2 expression in astrocytes. In human diabetic retinas, mRNA levels of all CK2 subunits decreased, consistent with increased apoptosis. Importantly, a specific CK2 inhibitor prevented recruitment of bone marrow-derived hematopoietic stem cells to areas of retinal neovascularization. This may provide a novel mechanism of action of CK2 inhibitors on newly forming vessels.

Casein kinase II-mediated phosphorylation of general repressor Maf1 triggers RNA polymerase III activation

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.

Synthesis of new analogs of benzotriazole, benzimidazole and phthalimide—potential inhibitors of human protein kinase CK2

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 (IC(50) in 0.32-0.54 microM range) whereas derivatives of phthalimide were almost ineffective.

Structure of the Human Protein Kinase CK2 Catalytic Subunit CK2α′ and Interaction Thermodynamics with the Regulatory Subunit CK2β

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 2xa0Å. 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.

Studies on the anti-hepatitis C virus activity of newly synthesized tropolone derivatives: Identification of NS3 helicase inhibitors that specifically inhibit subgenomic HCV replication

We synthesized new tropolone derivatives substituted with cyclic amines: piperidine, piperazine or pyrrolidine. The most active anti-helicase compound (IC50=3.4 microM), 3,5,7-tri[(4-methylpiperazin-1-yl)methyl]tropolone (2), inhibited RNA replication by 50% at 46.9 microM (EC50) and exhibited the lowest cytotoxicity (CC50)>1 mM 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 32.0 microM 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 35.6 microM and a SI of 9.8.

New inhibitors of protein kinase CK2, analogues of benzimidazole and benzotriazole

Derivatives of 4,5,6,7-tetrabromobenzotriazole (TBBt) and 4,5,6,7-tetrabromobenzimidazole (TBBi) with IC50 in the low micromolar range and with high selectivity belong to the most promising inhibitors of protein kinase CK2 (casein kinase 2). Treatment of various cell lines with TBBt, TBBi or 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) affected cell viability with simultaneous induction of apoptosis. The inhibitory activity of newly synthesized hydroxyalkyl derivatives of TBBi and TBBt depends on the length of the alkyl chain. The hydroxypropyl substituted derivatives show higher or similar inhibitory activity than the parent compounds when tested with human protein kinase CK2. To test the distribution of this class of compounds in mammals, [14C] TBBi was synthesized.

Enzymatic activity with an incomplete catalytic spine: insights from a comparative structural analysis of human CK2α and its paralogous isoform CK2α′

Eukaryotic protein kinases are fundamental factors for cellular regulation and therefore subject of strict control mechanisms. For full activity a kinase molecule must be penetrated by two stacks of hydrophobic residues, the regulatory and the catalytic spine that are normally well conserved among active protein kinases. We apply this novel spine concept here on CK2α, the catalytic subunit of protein kinase CK2. Homo sapiens disposes of two paralog isoforms of CK2α (hsCK2α and hsCK2α′). We describe two new structures of hsCK2α constructs one of which in complex with the ATP-analog adenylyl imidodiphosphate and the other with the ATP-competitive inhibitor 3-(4,5,6,7-tetrabromo-1H-benzotriazol-1-yl)propan-1-ol. The former is the first hsCK2α structure with a well defined cosubstrate/magnesium complex and the second with an open β4/β5-loop. Comparisons of these structures with existing CK2α/CK2α′ and cAMP-dependent protein kinase (PKA) structures reveal: in hsCK2α′ an open conformation of the interdomain hinge/helix αD region that is critical for ATP-binding is found corresponding to an incomplete catalytic spine. In contrast hsCK2α often adopts the canonical, PKA-like version of the catalytic spine which correlates with a closed conformation of the hinge region. HsCK2α can switch to the incomplete, non-canonical, hsCK2α′-like state of the catalytic spine, but this transition apparently depends on binding of either ATP or of the regulatory subunit CK2β. Thus, ATP looks like an activator of hsCK2α rather than a pure cosubstrate.

Treatment of cultured human astrocytes and vascular endothelial cells with protein kinase CK2 inhibitors induces early changes in cell shape and cytoskeleton

Ubiquitous protein kinase CK2 is a key regulator of cell migration, proliferation and tumor growth. CK2 is abundant in retinal astrocytes, and its inhibition suppresses retinal neovascularization in a mouse retinopathy model. In human astrocytes, CK2 co-distributes with GFAP-containing intermediate filaments, which implies its association with cytoskeleton. Contrary to astrocytes, CK2 is co-localized in microvascular endothelial cells (HBMVEC) with microtubules and actin stress fibers, but not with vimentin-containing intermediate filaments. Specific CK2 inhibitors (TBB, TBI, TBCA and DMAT) and nine novel CK2 inhibiting compounds (TID43, TID46, Quinolone-7, Quinolone-39, FNH28, FNH62, FNH64, FNH68 and FNH74) were tested at 10–200xa0μM for their ability to induce morphological alterations in cultured human astrocytes (HAST-40), and HBMVEC (For explanation of the inhibitor names, see “Methods” section). CK2 inhibitors caused dramatic changes in shape of cultured cells with effective inhibitor concentrations between 50 and 100xa0μM. Attached cells retracted, acquired shortened processes, and eventually rounded up and detached. CK2 inhibitor-induced morphological alterations were completely reversible and were not blocked by caspase inhibition. However, longer treatment or higher inhibitor concentration did cause apoptosis. The speed and potency of the CK2 inhibitors effects on cell shape and adhesion were inversely correlated with serum concentration. Western analyses showed that TBB and TBCA elicited a significant (about twofold) increase in the activation of p38 and ERK1/2 MAP kinases that may be involved in cytoskeleton regulation. This novel early biological cell response to CK2 inhibition may underlie the anti-angiogenic effect of CK2 suppression in the retina.

Synthesis of novel polybrominated benzimidazole derivatives-potential CK2 inhibitors with anticancer and proapoptotic activity.

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-1H-benzimidazole (TBBi) and 4,5,6,7-tetrabromo-2-methyl-1H-benzimidazole to acrylonitrile, whilst cyanomethyl, cyanopropyl and cyanobutyl analogs by N-alkylation of 4,5,6,7-tetrabromo-1H-benzimidazole and 4,5,6,7-tetrabromo-2-methyl-1H-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-1H-benzimidazole as a new selective inhibitor of rhCK2 with twenty-fold better proapoptotic activity than parental compound (TBBi).

Functional proteomics strategy for validation of protein kinase inhibitors reveals new targets for a TBB-derived inhibitor of protein kinase CK2.

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 (>5 μM) that are orders of magnitude higher than their in vitro inhibitory constants (<0.05 μM). 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.