Protein kinase CK2 is an emerging target for therapeutic intervention in human diseases, particularly in cancer. Inhibitors of this enzyme are currently in clinical trials, indicating the druggability of human CK2. By virtual screening of the ZINC database, we found that the natural compound bikaverin can fit well in the ATP binding site of the target enzyme CK2. By further in vitro evaluation using CK2 holoenzyme, bikaverin turned to be a potent inhibitor with an IC₅₀ value of 1.24 µM. In this work, the cell permeability of bikaverin was determined using a Caco-2 cell permeability assay as a prerequisite for cellular evaluation and the compound turned out to be cell permeable with a P_app- value of 4.46 × 10⁻⁶ cm/s. Bikaverin was tested for its effect on cell viability using a MTT assay and cell proliferation using an EdU assay in different cancer cell lines (MCF7, A427 and A431 cells). Cell viability and cell proliferation were reduced dramatically after treatment with 10 µM bikaverin for 24 h. Additionally the IncuCyte^® live-cell imaging system was applied for monitoring the cytotoxicity of bikaverin in the three tested cancer cell lines. Finally, molecular dynamic studies were performed to clarify the ligand binding mode of bikaverin at the ATP binding site of CK2 and to identify the amino acids involved.

Protein kinase CK2, a member of the eukaryotic protein kinase superfamily, is associated with cancer and other human pathologies and thus an attractive drug target. The indeno[1,2-b]indole scaffold is a novel lead structure to develop ATP-competitive CK2 inhibitors. Some indeno[1,2-b]indole-based CK2 inhibitors additionally obstruct ABCG2, an ABC half transporter overexpressed in breast cancer and co-responsible for drug efflux and resistance. Comprehensive derivatization studies revealed substitutions of the indeno[1,2-b]indole framework that boost either the CK2 or the ABCG2 selectivity or even support the dual inhibition potential. The best indeno[1,2-b]indole-based CK2 inhibitor described yet (IC₅₀ = 25 nM) is 5-isopropyl-4-(3-methylbut-2-enyl-oxy)-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (4p). Herein, we demonstrate the membrane permeability of 4p and describe co-crystal structures of 4p with CK2α and CK2α′, the paralogs of human CK2 catalytic subunit. As expected, 4p occupies the narrow, hydrophobic ATP site of CK2α/CK2α′, but surprisingly with a unique orientation: its hydrophobic substituents point towards the solvent while its two oxo groups are hydrogen-bonded to a hidden water molecule. An equivalent water molecule was found in many CK2α structures, but never as a critical mediator of ligand binding. This unexpected binding mode is independent of the interdomain hinge/helix αD region conformation and of the salt content in the crystallization medium.

Protein kinase CK2 is involved in regulating cellular processes, such as cell cycle, proliferation, migration, and apoptosis, making it an attractive anticancer target. We previously described a prenyloxy-substituted indeno[1,2-b]indole (5-isopropyl-4-(3-methylbut-2-enyloxy)-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (4p)) as a very potent inhibitor of CK2 holoenzyme (IC₅₀ = 25 nM). Here, we report the broad-spectrum anticancer activity of 4p and provide substantial progress on its pharmacokinetic properties. Using a cell-based CK2 activity assay and live-cell imaging of cultured A431, A549, and LNCaP cancer cell lines, cellular CK2 target engagement was shown as well as strong antiproliferative, anti-migratory and apoptosis-inducing effects of 4p. Furthermore, evidence was found for the ability of 4p to disrupt A549 spheroid cohesion. A series of LC-MS/MS experiments revealed high and rapid cellular uptake (intracellular concentration is approximately 5 µM after 1 h incubation) and low metabolic stability of 4p. These results point to the value of 4p as a potent CK2 inhibitor with promising anticancer activities and should trigger future medicinal chemistry efforts to improve the drug-like properties of this compound.

Highly pleiotropic and constitutively active protein kinase CK2 is a key target in cancer therapy, but only one small‐molecule inhibitor has reached clinical trials—CX‐4945. In this study, we present the indeno[1,2‐b]indole derivative 5‐isopropyl‐4‐methoxy‐7‐methyl‐5,6,7,8‐tetrahydroindeno[1,2‐b]indole‐9,10‐dione (5a‐2) that decreased the intracellular CK2 activity in A431, A549, and LNCaP tumor cell lines analogous to CX‐4945 (> 75% inhibition at 20 µm) and similarly blocked CK2‐specific Akt phosphorylation in LNCaP cells. Cellular uptake analysis demonstrated higher intracellular concentrations of 5a‐2 (408.3 nm) compared with CX‐4945 (119.3 nm). This finding clarifies the comparable effects of both compounds on the intracellular CK2 activity despite their different inhibitory potency in vitro [IC₅₀ = 25 nm (5a‐2) and 3.7 nm (CX‐4945)]. Examination of the effects of both CK2 inhibitors on cancer cells using live‐cell imaging revealed notable differences. Whereas CX‐4945 showed a stronger pro‐apoptotic effect on tumor cells, 5a‐2 was more effective in inhibiting tumor cell migration. Our results showed that 49% of intracellular CX‐4945 was localized in the nuclear fraction, whereas 71% of 5a‐2 was detectable in the cytoplasm. The different subcellular distribution, and thus the site of CK2 inhibition, provides a possible explanation for the different cellular effects. Our study indicates that investigating CK2 inhibition‐mediated cellular effects in relation to the subcellular sites of CK2 inhibition may help to improve our understanding of the preferential roles of CK2 within different cancer cell compartments.

A large number of secondary metabolites have been isolated from the filamentous fungus Stachybotrys chartarum and have been described before. Fourteen of these natural compounds were evaluated in vitro in the present study for their inhibitory activity towards the cancer target CK2. Among these compounds, stachybotrychromene C, stachybotrydial acetate and acetoxystachybotrydial acetate turned out to be potent inhibitors with IC₅₀ values of 0.32 µM, 0.69 µM and 1.86 µM, respectively. The effects of these three compounds on cell proliferation, growth and viability of MCF7 cells, representing human breast adenocarcinoma as well as A427 (human lung carcinoma) and A431 (human epidermoid carcinoma) cells, were tested using EdU assay, IncuCyte^® live-cell imaging and MTT assay. The most active compound in inhibiting MCF7 cell proliferation was acetoxystachybotrydial acetate with an EC₅₀ value of 0.39 µM. In addition, acetoxystachybotrydial acetate turned out to inhibit the growth of all three cell lines completely at a concentration of 1 µM. In contrast, cell viability was impaired only moderately, to 37%, 14% and 23% in MCF7, A427 and A431 cells, respectively.

Aims: Selecting natural inhibitors toward CK2 enzyme using database mining. Background: Casein Kinase 2 (CK2) is a ubiquitous cellular serine-threonine kinase with broad spectrum of substrates. This enzyme is widely expressed in eukaryotic cells and is overexpressed in different human cancers. Thus, inhibition of CK2 can induce the physiological process of apoptosis leading to tumor cell death. Objective: Selecting natural inhibitors toward the target enzyme using database mining. Method: With our continuous effort to discover new compounds with CK2 inhibitory effect, several commercial natural databases were searched using molecular modeling approach and the selected compounds were evaluated in vitro. Result: Three compounds were selected as candidates and evaluated in vitro using holoenzyme and their effect on three cancer cell lines was performed. Unfortunately, the selected candidates were weak inhibitors toward the target enzyme, and only one compound showed moderate cell viability effect on cancer cells. Conclusion: Several natural databases were screened and compounds were selected and tested in vitro, despite of the unexpected low activity of the compounds, this study can help in directing the search of potent CK2 inhibitors and better understand the binding requirements of the ATP competitive inhibitors.