Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • Since protein kinase CK has over known

    2019-10-09

    Since protein kinase CK2 has over 300 known substrates we examined if the inhibitory effects described above could also be detected using another substrate. Therefore, we employed the synthetic peptide RRRADDSDDDDD which is often used in CK2 inhibitory studies. Quite contrary to the phosphorylation of P2B we obtained mixed results with the small peptide resembling the consensus sequence. In most cases CK2α′ was better inhibited than CK2α but this was not the rule as in case of substrate P2B. Chrysoeriol (1), apigenin (4), luteolin (5), pedalitin (2), kaempferol (7) possess higher inhibitory potency against both catalytic subunits compared to the IC50 obtained using P2B. One substance, scutellarin (9), has similar effect on the phosphorylating activities independently of which substrate was used. Tricin (3), quercetin (8) and cernuoside (10) were more active against CK2α′ subunit than CK2α. Glycosides of apigenin (4a-c) had similar or weaker effect towards CK2α′ than towards CK2α. Isokaempferide (6) inhibited the CK2α′ activity similarly independent from the used substrate. CK2α was moderately inhibited using the peptide substrate. The glucuronic derivative of isokaempferide (6b) similar like in case of P2B showed better inhibitory potential towards CK2α′ than the parent compound. Both derivatives of luteolin (5a and 5b) possess the same inhibitory effect towards CK2α′ comparing phosphorylation of both substrates. CK2α was much better inhibited by the glucosylated derivative (5b) using P2B. Experiments with scutellarin (9) and scutellarein 7-O-glucoside (9a) gave opposite results than when tested with P2B. The derivative with a glucose moiety at position C-7 (9a) possess quite good inhibitory effect towards CK2α and slightly weaker effect on CK2α′. Further tested compounds linarin (11), pectolinarin (11a), erigeroside (13), 6″-caffeoylerigeroside (13a) and chlorogenic Primidone mass (14) had very weak effect (IC50 values over 40 μM) on CK2 activity independently from the used substrate and examined subunit. To assess the mechanism by which the flavonoid compounds inhibit the CK2 activity we used different ATP concentrations in the reaction mixture. All compounds decreasing the catalytic activities showed a purely ATP-competitive mode of inhibition. The Ki values ranged between 0.1 and 0.5 μM using P2B, and lower values we estimated using the peptide substrate. Chrysoeriol the best inhibitor in this study has an inhibition constant of 17 and 100 nM for CK2α′ and CK2α, respectively, using the synthetic peptide as phosphate acceptor (Fig. 5). Comparing the active sites of different protein kinases we can observe a relatively small one in case of CK2 due to bulky side chains reducing the space for co-factors or potential inhibitors. The most important amino acids involved in the interaction (binding) with inhibitors are: Leu85, Val95, Leu111, Phe113 and Ile174 (hydrophobic region I), Val45 and Tyr115 (hydrophobic region II), and Val53, Ile66, Val116 and Met163 (adenine region). It has been shown that apigenin and luteolin possess a planar structure which is necessary to fit into the narrow ATP-binding site of the CK2α molecule (Lolli et al., 2012). Due to the lack of crystal structures of CK2α subunits and these flavonoid inhibitors, it is not possible to explain the different inhibition potential of one compound towards both subunits. A co-crystallization of both catalytic subunits and 3-(4,5,6,7-tetrabromo-1H-benzotriazol-1-yl)propan-1-ol (MB002) revealed a different binding mode (Bischoff et al., 2011). This might be also the reason for the different inhibitory potential of the tested flavonoid compounds. CK2 possesses a wide range of substrates. Up to date there are over 350 proteins identified which are phosphorylated by this kinase (Bian et al., 2013). In a former study we examined the effect of CK2 inhibitors towards five CK2 isoforms from S. cerevisiae using different protein substrates (Janeczko et al., 2011). However, the tested inhibitors are purely ATP-competitive the presented results show that the substrate/enzyme complex has influence on the affinity for inhibitors. It is imaginable that the ATP-binding pocket undergoes conformational changes and, therefore, might be less or better available for inhibitors.