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  • Flavonoids are most common and


    Flavonoids are most common and widely distributed group of natural phenolic compounds synthesized by plants that have varied effects on mammalian cell systems (Kumar and Pandey, 2013). Several studies demonstrate that flavonoids might be potent inhibitors of several protein kinases involved in various cellular responses including regulation of cell growth and proliferation (Batra and Sharma, 2013, Kumar and Pandey, 2013, Ravishankar et al., 2013). It has been shown that natural flavones and flavonols - including apigenin, luteolin, fisetin, kaempferol, quercetin, myricetin and coumestan derivative coumestrol – can inhibit the CK2 holoenzyme with Ki‘s in the sub-micromolar range (Liu et al., 2013, Lolli et al., 2012, McCarty, 2015).
    Results and discussion Twenty eight natural compounds were tested according their influence on human protein kinase CK2 (Fig. 1, Fig. 2). Among them were twenty four aglycones and glycosides of flavonoids belonging to various classes - flavones, flavonols, flavanones and aurones. Apart from them we determined the activity of two pyromeconic acid, one caffeoylquinic NPS-2143 and one phenylpropanoid derivatives. In former studies we had described differences in the inhibition data using either catalytic subunit CK2α or CK2α′ (Janeczko et al., 2011, Janeczko et al., 2012). In present study we investigated 4 already described CK2 holoenzyme inhibitors (apigenin (4), luteolin (5), kaempferol (7), and quercetin (8)) and 24 previously undescribed compounds for their activity against human CK2α and CK2α′ subunits. Both proteins were overexpressed in E. coli and purified to homogeneity by affinity chromatography using glutathione-sepharose (GE Healthcare). The inhibitory effect was examined by increasing concentrations of the compound. As in the former study we used two different substrates. In Table 1 all results using the synthetic peptide RRRADDSDDDDD or yeast P2B as a substrate are summarized. First of all we tested the flavonoid aglycones chrysoeriol (1), tricin (3), apigenin (4), luteolin (5), pedalitin (2), isokaempferide (6), kaempferol (7) and quercetin (8) for their influence on both CK2 catalytic subunits using yeast acidic ribosomal protein P2B (Fig. 3). The best inhibitory effect showed chrysoeriol (1), quercetin (8) and pedalitin (2) with IC50 values between 0.1 and 0.4 μM for CK2α′ as well as 0.5 and 7.0 μM for CK2α. The well known CK2 inhibitor apigenin possesses less effectiveness with IC50 values of 9.8 and 2.3 μM, for CK2α and CK2α′, respectively (Fig. 4). Luteolin (5) and kaempferol (7) decreased the phosphorylating activity weaker than chrysoeriol (1) but better than apigenin (IC50 = 1.0 μM). In all described cases the differences between the effect on both catalytic subunits are similar. CK2α′ is affected much stronger, up to 17-fold as in case of pedalitin (2). Within the tested compounds we also found examples that only CK2α′ was inhibited, like tricin (3, Fig. 4) and glycoside scutellarin (9). Otherwise aurone cernuoside (10) is the only inhibitor with a similar effect on both subunits. Fig. 3 represents the inhibitory activity of chrysoeriol (1), pedalitin (2), tricin (3), apigenin (4), scutellarin (9) and cernuoside (10). Furthermore, some glycosides of luteolin, apigenin, scutellarein, isokaempferide, kaempferol and quercetin were also involved in this study. Both luteolin derivatives, 7-O-glucoside (5a) and 6-hydroxyluteolin 7-O-glucoside (5b), are better inhibitors of CK2α′ than luteolin (Fig. 4). Examining the CK2α activity in the presence of these compounds the inhibitory effect is lost in case of a glycosylation at position C-7 (5a), whereas the compound with a hydroxyl group at position C-6 (5b) possesses similar influence. The apigenin derivative with the O-linked glucose moiety at position C-7 (4a) affects the CK2α activities in a weaker extent than apigenin. Glucuronic acid attached at the same position (4b) revealed a further decrease of the phosphorylating activity of CK2α′. This effect was not seen when a methylated glucuronic acid (4c) is attached, anyway, this compound still exhibited inhibitory potential against CK2α′. In general, compounds with an O-linked glucose moiety revealed mixed results. In case of the scutellarein 7-O-glucoside (9a) the effect was similar like for the 7-O-glucuronide - scutellarin (9). On the other hand, the substitution with glucose lowered the inhibitory potency as in case of derivatives of kaempferol (7a), isokaempferide (6a) and quercetin (8a). Similar observation like the increased inhibitory activity of apigenin 7-O-glucuronide (4b) was made in case of isokaempferide 7-O-glucuronide (6b) having a 4–5 fold stronger effect on CK2α′.