1,2,3-Triazole-totarol conjugates as potent PIP5K1α lipid kinase inhibitors.

The human phosphatidylinositol 4-phosphate 5-kinase type I α (hPIP5K1α) plays a key role in the development of prostate cancer. In this work, seventeen derivatives of the natural diterpene totarol were prepared by copper(I)-catalysed Huisgen 1,3-dipolar cycloaddition reaction of the correspondingO-propargylated totarol with aryl or alkyl azides and screened for their inhibitory activities toward hPIP5K1α. Five compounds, 3a, 3e, 3f, 3i, and 3r, strongly inhibited the enzyme activity with IC50 values of 1.44, 0.46, 1.02, 0.79, and 3.65 µM, respectively, with the most potent inhibitor 3e 13-[(1-(3-nitrophenyl)triazol-4yl)methoxy]-totara-8,11,13-triene). These compounds were evaluated on their antiproliferative effects in a panel of prostate cancer cell lines. Compound 3r inhibited the proliferation of LNCaP, PC3 and DU145 cells at 20 µM, strongly, but also has strong cytotoxic effects on all tested cells.

4,5,7-Trisubstituted indeno[1,2-b]indole inhibits CK2 activity in tumor cells equivalent to CX-4945 and shows strong anti-migratory effects.

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.

Natural Compounds Isolated from Stachybotrys chartarum Are Potent Inhibitors of Human Protein Kinase CK2.

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.

Broad-Spectrum Anticancer Activity and Pharmacokinetic Properties of a Prenyloxy-Substituted Indeno[1,2-b]indole Derivative, Discovered as CK2 Inhibitor.

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.

In Silico and In Vitro Studies of Natural Compounds as Human CK2 Inhibitors.

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, the inhibition of CK2 can induce the physiological process of apoptosis leading to tumor cell death. OBJECTIVES: Selecting natural inhibitors toward the target enzyme using database mining. METHODS: 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. RESULTS: Three compounds were selected as candidates and evaluated in vitro using CK2 holoenzyme, their effect on three cancer cell lines was determined. The selected candidates were weak inhibitors toward the target enzyme, only one compound showed moderate effect on cell viability. CONCLUSION: Several natural databases were screened, compounds were selected and tested in vitro. Despite the unexpected low inhibitory activity of the tested compounds, this study can help in directing the search of potent CK2 inhibitors and better understand the binding requirements of the ATP competitive inhibitors.

Design, synthesis and biological evaluation of new embelin derivatives as CK2 inhibitors.

A new series of furan embelin derivatives was synthesized and characterized as ATP-competitive CK2 inhibitors. The new compounds were efficiently synthesized using a multicomponent approach from embelin (1), aldehydes and isonitriles through a Knoevenagel condensation/Michael addition/heterocyclization. Several compounds with inhibitory activities in the low micromolar or even submicromolar were identified. The most active derivative was compound 4l (2-(tert-butylamino)-3-(furan-3-yl)-5-hydroxy-6-undecylbenzofuran-4,7-dione) with an IC50 value of 0.63 µM. It turned out to be an ATP competitive CK2 inhibitor with a Ki value determined to be 0.48 µM. Docking studies allowed the identification of key ligand-CK2 interactions, which could help to further optimize this family of compounds as CK2 inhibitors.

In Vitro and in Silico Evaluation of Bikaverin as a Potent Inhibitor of Human Protein Kinase CK2.

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 Papp- 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.

QSAR Model of Indeno[1,2-b]indole Derivatives and Identification of N-isopentyl-2-methyl-4,9-dioxo-4,9-Dihydronaphtho[2,3-b]furan-3-carboxamide as a Potent CK2 Inhibitor.

Casein kinase II (CK2) is an intensively studied enzyme, involved in different diseases, cancer in particular. Different scaffolds were used to develop inhibitors of this enzyme. Here, we report on the synthesis and biological evaluation of twenty phenolic, ketonic, and para-quinonic indeno[1,2-b]indole derivatives as CK2 inhibitors. The most active compounds were 5-isopropyl-1-methyl-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione 4h and 1,3-dibromo-5-isopropyl-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione 4w with identical IC50 values of 0.11 µM. Furthermore, the development of a QSAR model based on the structure of indeno[1,2-b]indoles was performed. This model was used to predict the activity of 25 compounds with naphtho[2,3-b]furan-4,9-dione derivatives, which were previously predicted as CK2 inhibitors via a molecular modeling approach. The activities of four naphtho[2,3-b]furan-4,9-dione derivatives were determined in vitro and one of them (N-isopentyl-2-methyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-3-carboxamide) turned out to inhibit CK2 with an IC50 value of 2.33 µM. All four candidates were able to reduce the cell viability by more than 60% after 24 h of incubation using 10 µM.

A new family of densely functionalized fused-benzoquinones as potent human protein kinase CK2 inhibitors.

A new series of 2-amino-4-phenyl-6-hydroxy-7-alkyl-pyranobenzoquinones was synthesized as ATP-competitive CK2 inhibitors. They were readily synthesized through a three-component Knoevenagel condensation-Michael addition-heterocyclization reaction from aldehydes, malononitrile, and 3-alkyl-2,5-dihydroxybenzoquinones. Some of the synthesized compounds presented interesting inhibitory activity with IC50 values in the submicromolar range. A structure-activity relationship study was carried out and the mode of binding was analysed by docking studies and supported by ATP competition assays.

Unexpected Binding Mode of a Potent Indeno[1,2-b]indole-Type Inhibitor of Protein Kinase CK2 Revealed by Complex Structures with the Catalytic Subunit CK2α and Its Paralog CK2α'.

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 hydro-gen-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.

Converting potent indeno[1,2-b]indole inhibitors of protein kinase CK2 into selective inhibitors of the breast cancer resistance protein ABCG2.

A series of indeno[1,2-b]indole-9,10-dione derivatives were synthesized as human casein kinase II (CK2) inhibitors. The most potent inhibitors contained a N(5)-isopropyl substituent on the C-ring. The same series of compounds was found to also inhibit the breast cancer resistance protein ABCG2 but with totally different structure-activity relationships: a N(5)-phenethyl substituent was critical, and additional hydrophobic substituents at position 7 or 8 of the D-ring or a methoxy at phenethyl position ortho or meta also contributed to inhibition. The best ABCG2 inhibitors, such as 4c, 4h, 4i, 4j, and 4k, behaved as very weak inhibitors of CK2, whereas the most potent CK2 inhibitors, such as 4a, 4p, and 4e, displayed limited interaction with ABCG2. It was therefore possible to convert, through suitable substitutions of the indeno[1,2-b]indole-9,10-dione scaffold, potent CK2 inhibitors into selective ABCG2 inhibitors and vice versa. In addition, some of the best ABCG2 inhibitors, which displayed a very low cytotoxicity, thus giving a high therapeutic ratio, and appeared not to be transported, constitute promising candidates for further investigations.

Protein kinase CK2 phosphorylates BAD at threonine-117.

Reversible phosphorylation of the 22 kDa BAD protein is crucial for cell survival. Five phosphorylation sites, all serines, had been identified. Here we report on number six. It is threonine-117 phosphorylated by the constitutively active kinase, CK2. Phosphoamino acid analysis and phospho-specific antibodies confirmed Thr117 as additional phosphorylation site. Immunoprecipitation furthermore revealed that BAD is phosphorylated at Thr117 in cultured cortical neurons. PP1, PP2A and PP2C dephosphorylated BAD at Thr117, but PP2B did not. The discovery of the constitutively active CK2 phosphorylating BAD is shedding an unexpected light in the otherwise strictly signal-regulated phosphorylation events on BAD.