Development Of CK2 Inhibitors Research Articles

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

4,5,6,7-Tetrabromo-1H-benzotriazole is widely used as the reference ATP-competitive inhibitor of protein kinase CK2. Herein, we study its new analogs: 5,6-diiodo- and 5,6-diiodo-4,7-dibromo-1H-benzotriazole. We used biophysical (MST, ITC) and biochemical (enzymatic assay) methods to describe the interactions of halogenated benzotriazoles with the catalytic subunit of human protein kinase CK2 (hCK2α). To trace the biological activity, we measured their cytotoxicity against four reference cancer cell lines and the effect on the mitochondrial inner membrane potential. The results obtained lead to the conclusion that iodinated compounds are an attractive alternative to brominated ones. One of them retains the cytotoxicity against selected cancer cell lines of the reference TBBt with a smaller side effect on mitochondrial activity. Both iodinated compounds are candidate leaders in the further development of CK2 inhibitors.

Design of CK2β-Mimicking Peptides as Tools To Study the CK2α/CK2β Interaction in Cancer Cells.

The ubiquitously expressed Ser/Thr kinase CK2 is a key regulator in a variety of key processes in normal and malignant cells. Due to its distinctive anti-apoptotic and tumor-driving properties, elevated levels of CK2 have frequently been found in tumors of different origin. In recent years, development of CK2 inhibitors has largely been focused on ATP-competitive compounds; however, targeting the CK2α/CK2β interface has emerged as a further concept that might avoid selectivity issues. To address the CK2 subunit interaction site, we have synthesized halogenated CK2β-mimicking cyclic peptides modified with the cell-penetrating peptide sC18 to mediate cellular uptake. We investigated the binding of the resulting chimeric peptides to recombinant human CK2α using a recently developed fluorescence anisotropy assay. The iodinated peptide sC18-I-Pc was identified as a potent CK2α ligand (Ki =0.622 μm). It was internalized in cells to a high extent and exhibited significant cytotoxicity toward cancerous HeLa cells (IC50 =37 μm) in contrast to non-cancerous HEK-293 cells. The attractive features and functionalities of sC18-I-Pc offer the opportunity for further improvement.

The Development of CK2 Inhibitors: From Traditional Pharmacology to in Silico Rational Drug Design.

Casein kinase II (CK2) is an ubiquitous and pleiotropic serine/threonine protein kinase able to phosphorylate hundreds of substrates. Being implicated in several human diseases, from neurodegeneration to cancer, the biological roles of CK2 have been intensively studied. Upregulation of CK2 has been shown to be critical to tumor progression, making this kinase an attractive target for cancer therapy. Several CK2 inhibitors have been developed so far, the first being discovered by “trial and error testing”. In the last decade, the development of in silico rational drug design has prompted the discovery, de novo design and optimization of several CK2 inhibitors, active in the low nanomolar range. The screening of big chemical libraries and the optimization of hit compounds by Structure Based Drug Design (SBDD) provide telling examples of a fruitful application of rational drug design to the development of CK2 inhibitors. Ligand Based Drug Design (LBDD) models have been also applied to CK2 drug discovery, however they were mainly focused on methodology improvements rather than being critical for de novo design and optimization. This manuscript provides detailed description of in silico methodologies whose applications to the design and development of CK2 inhibitors proved successful and promising.

An Unbiased Evaluation of CK2 Inhibitors by Chemoproteomics: Characterization of Inhibitor Effects on CK2 and Identification of Novel Inhibitor Targets

Recently protein kinases have emerged as some of the most promising drug targets; and therefore, pharmaceutical strategies have been developed to inhibit kinases in the treatment of a variety of diseases. CK2 is a serine/threonine-protein kinase that has been implicated in a number of cellular processes, including maintenance of cell viability, protection of cells from apoptosis, and tumorigenesis. Elevated CK2 activity has been established in a number of cancers where it was shown to promote tumorigenesis via the regulation of the activity of various oncogenes and tumor suppressor proteins. Consequently the development of CK2 inhibitors has been ongoing in preclinical studies, resulting in the generation of a number of CK2-directed compounds. In the present study, an unbiased evaluation of CK2 inhibitors 4,5,6,7-tetrabromo-1H-benzotriazole (TBB), 4,5,6,7-tetrabromo-1H-benzimidazole (TBBz), and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) was carried out to elucidate the mechanism of action as well as inhibitor specificity of these compounds. Utilizing a chemoproteomics approach in conjunction with inhibitor-resistant mutant studies, CK2alpha and CK2alpha' were identified as bona fide targets of TBB, TBBz, and DMAT in cells. However, inhibitor-specific cellular effects were observed indicating that the structurally related compounds had unique biological properties, suggesting differences in inhibitor specificity. Rescue experiments utilizing inhibitor-resistant CK2 mutants were unable to rescue the apoptosis associated with TBBz and DMAT treatment, suggesting the inhibitors had off-target effects. Exploitation of an unbiased chemoproteomics approach revealed a number of putative off-target inhibitor interactions, including the discovery of a novel TBBz and DMAT (but not TBB) target, the detoxification enzyme quinone reductase 2 (QR2). The results described in the present study provide insight into the molecular mechanism of action of the inhibitors as well as drug specificity that will assist in the development of more specific next generation CK2 inhibitors.