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Hochscherf J.,University of Cologne | Hochscherf J.,KinaseDetect Aps | Lindenblatt D.,University of Cologne | Steinkruger M.,University of Cologne | And 10 more authors.
Analytical Biochemistry | Year: 2014

Increased activity of protein kinase CK2 is associated with various types of cancer, neurodegenerative diseases, and chronic inflammation. In the search for CK2 inhibitors, attention has expanded toward compounds disturbing the interaction between CK2α and CK2β in addition to established active site-directed approaches. The current article describes the development of a fluorescence anisotropy-based assay that mimics the principle of CK2 subunit interaction by using CK2α1-335 and the fluorescent probe CF-Ahx-Pc as a CK2β analog. In addition, we identified new inhibitors able to displace the fluorescent probe from the subunit interface on CK2α1-335. Both CF-Ahx-Pc and the inhibitors I-Pc and Cl-Pc were derived from the cyclic peptide Pc, a mimetic of the C-terminal CK2α-binding motif of CK2β. The design of the two inhibitors was based on docking studies using the known crystal structure of the Pc/CK2α1-335 complex. The dissociation constants obtained in the fluorescence anisotropy assay for binding of all compounds to human CK2α1-335 were validated by isothermal titration calorimetry. I-Pc was identified as the tightest binding ligand with a KD value of 240 nM and was shown to inhibit the CK2 holoenzyme-dependent phosphorylation of PDX-1, a substrate requiring the presence of CK2β, with an IC50 value of 92 μM. © 2014 Elsevier Inc. All rights reserved.

Viht K.,University of Tartu | Saaver S.,University of Tartu | Vahter J.,University of Tartu | Enkvist E.,University of Tartu | And 7 more authors.
Bioconjugate Chemistry | Year: 2015

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 NFkB pSer529, was demonstrated by Western blot analysis. © 2015 American Chemical Society.

Guerra B.,University of Southern Denmark | Fischer M.,University of Southern Denmark | Schaefer S.,University of Southern Denmark | Issinger O.-G.,KinaseDetect Aps
Journal of Experimental and Clinical Cancer Research | Year: 2015

Background: Multi-drug resistance and predisposition to metastasize are major clinical problems in cancer treatment. Malignant primary brain tumor and pancreatic cancer are two well-known examples of malignant tumors resistant to conventional therapies where aberrant EGFR-mediated and NF-κB signal transduction pathways are likely to play an important role. We have recently identified 1,3-Dichloro-6-[(E)-((4-methoxyphenyl)imino)methyl] diben-zo(b,d) furan-2,7-diol (D11) as a potent and selective inhibitor of CK2 a serine/threonine protein kinase that modulates the aforementioned signaling cascades. Methods: Human cancer cell lines (glioblastoma and pancreatic adenocarcinoma) resistant to conventional chemotherapeutic agents were incubated with increasing concentrations of D11 for variable amounts of time. Cell viability, cell death and effects on major signal transduction pathways deregulated in cancer cells were analyzed by ELISA, FACS and Western blot-based assays, respectively. Moreover, effects on cell migration and in cell protein-protein association were investigated by wound-healing and in situ proximity ligation assays, respectively. Results: We show here, that D11 treatment leads to i) significant caspase-mediated apoptotic cell death, ii) down-regulation of EGFR expression and iii) inhibition of NF-κB transcriptional activity. Furthermore, cell exposure to D11 results in impaired cell migration and correlates with reduced expression of the ion co-transporter and cell volume regulator Na+-K+-2Cl- (NKCC1). Conclusions: Data reported here underline the therapeutic potential of D11 with respect to certain types of cancer that carry aberrant intracellular signaling cascades and/or exhibit sustained cell migration and suggest a new therapeutic strategy against chemotherapy resistance. © 2015 Guerra et al.

Guerra B.,University of Southern Denmark | Rasmussen T.D.L.,University of Southern Denmark | Schnitzler A.,University of Cologne | Jensen H.H.,University of Southern Denmark | And 6 more authors.
Cancer Letters | Year: 2015

Screening for protein kinase CK2 inhibitors of the structural diversity compound library (DTP NCI/NIH) led to the discovery of 4-[(E)-(fluoren-9-ylidenehydrazinylidene)-methyl]benzoic acid (E9).E9 induces apoptotic cell death in various cancer cell lines and upon hypoxia, the compound suppresses CK2-catalyzed HSP90/Cdc37 phosphorylation and induces HIF-1α degradation.Furthermore, E9 exerts a strong anti-tumour activity by inducing necrosis in murine xenograft models underlining its potential to be used for cancer treatment in future clinical studies.Crystal structure analysis of human and maize CK2α in complex with E9 reveals unique binding properties of the inhibitor to the enzyme, accounting for its affinity and selectivity. © 2014 Elsevier Ireland Ltd.

Guerra B.,University of Southern Denmark | Hochscherf J.,KinaseDetect Aps | Jensen N.B.,KinaseDetect Aps | Issinger O.-G.,University of Southern Denmark | Issinger O.-G.,KinaseDetect Aps
Molecular and Cellular Biochemistry | Year: 2015

The anti-apoptotic protein kinase CK2 increasingly becomes an attractive target in cancer research with great therapeutic potential. Here, we have performed an in vitro screening of the Diversity Set III of the DTP program from the NCI/NIH, comprising 1600 compounds. We have identified 1,3-Dichloro-6-[(E)-((4-methoxyphenyl)imino)methyl] dibenzo(b,d) furan-2,7-diol (referred to as D11) to be a potent and selective inhibitor of protein kinase CK2. The D11 compound was tested against 354 eukaryotic protein kinases. By setting the threshold for inhibition to <2 % remaining kinase activity, only DYRK1B, IRAK1 and PIM3 were inhibited to an extent as the tetrameric CK2 holoenzyme and its catalytic subunits α and α′. The IC50 values for the CK2α and CK2α′ were on average 1–2 nM in comparison to the DYRK1B, IRAK1 and PIM3 kinases, which ranged from 18 to 49 nM. Cell permeability and efficacy of D11 were tested with cells in culture. In MIA PaCa-2 cells (human pancreatic carcinoma cell line), the phosphorylation of the CK2 biomarker CDC37 at S13 was almost completely inhibited in the presence of D11. This was observed both under normoxia and hypoxia. In the case of the human non-small cell lung carcinoma cell line, H1299, increasing amounts of D11 led to an inhibition of S380/T382/383 phosphorylation in PTEN, another biomarker for CK2 activity. © 2015, Springer Science+Business Media New York.

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