Protein kinase CK2 is a ubiquitous, highly pleiotropic and constitutively active Ser/Thr kinase that has more than 400 physiological targets and involved in many biological processes such as transcription, translation, cell cycle control, proliferation, cell survival and apoptosis. This enzyme takes part in the development of various diseases, including neurodegenerative and cardiovascular disease, inflammation, viral and parasitic infections and many cancer types etc. Thus, inhibitors of protein kinase CK2 can be an effective tool for studying its role in signaling pathways and used to develop of therapeutic agents. The aim of our research is to develop new low-molecular inhibitors of human protein kinase CK2 and to develop their binding models with enzyme ATP-acceptor site. Initially, the receptor-based virtual screening has been performed and a number of classes of potential CK2 inhibitors have been identified. One of them was 3,4,5,6-tetrahydrothiopirano[2,3-d]thiazole derivatives. Seven inhibitors from this class were tested in vitro and have IC50 values in the range of 0,2 to 24,5 microМ. The analysis of the binding modes of these compounds with CK2 ATP-acceptor site showed that the main influence on complex stability have hydrophobic contacts and hydrogen bond with Lys68. Another identified inhibitors of CK2 were flavonoids - flavone and flavonole derivatives. Biochemical tests in vitro revealed that 19 flavones out of 49 selected by virtual screening inhibit activity of CK2 (IC50<30 microM). The most active compound is FNH28 with IC50=0,1 microM. After studying the structure-activity relationship and inspecting flavones binding mode with ATP-acceptor site of CK2 the 4'-hydroxyflavone core was chosen as a template for further structural optimization. New synthesized derivatives show an incredibly high hit rate - 26 out of 28 compounds inhibit enzyme activity of CK2 in submicromolar range. The analysis of binding modes of newly synthesized flavones has shown that their strong binding with ATP-acceptor site is caused by interactions with its hydrophobic residues and formation of intermolecular hydrogen bonding network with key amino acids Val116, Lys68 and/or Asp175. It should be added that IC50 value of the 4'-hydroxyflavone FNH79 is nearly 100 times higher than IC50 of fisetin, the most active natural flavone known to inhibit CK2. Other identified flavonoids were carboxyl-containing flavones. In vitro tests of 13 compounds from this class showed that five derivatives have inhibitory potency at the range from 0.6 to 8 microM. The studying of their structure-activity relationship and docking complexes indicated that the most effect on the compounds activity has the presence of carboxyl group in position 4' of phenol ring and hydroxyl group in position 3 of the heterocycle. The chemical optimization step allows us to obtain 13 new 4'-carboxyflavonoles. All synthesized compounds have submicromolar activity (IC50=0.009-0.72 microM). The values of Ki for two more potent inhibitors FLC21 and FLC26 were 13 and 2.5 nM, correspondingly. The binding mode of these compounds with ATP-acceptor site showed an importance of hydrophobic substituents in structure of ligands for complexes stabilization. The next studied series of compounds were thieno[2,3-d]pyrymidine derivatives. Initially, we determined inhibitory activity of 28 substituted (thieno[2,3-d]pyrimidin-4-ylthio)carboxylic acids. Ten compounds from this class have negligible activity toward CK2, ten compounds have IC50 in the range 0.1-7 microM. It was estimated that the activity of 4-mercaptothieno[2,3-d]pyrimidines is a result of their competition with ATP molecule for the binding site (Ki values of the most active compound 6a is 40 nM). Key contacts that contribute significantly to the ligand binding are Van der Waals interactions of thieno[2,3-d]pyrimidine heterocycle with a number of hydrophobic residues (Leu45, Val53, Val66, Val116 and Ile174). In addition, active compounds form intermolecular hydrogen bonds in two areas (hinge region and phosphate-binding region) of active site, that is possible then R4 = propionic acid. 4-aminothieno[2,3-d]pyrimidines were synthesized as potentially active analogs of previously described compounds. But, biochemical in vitro tests revealed that only 2 out of 18 new compounds were active. The analysis of the complexes of these derivatives with ATP-binding site obtained with docking didn't explain why inhibitors activity disappeared. It has been suggested that compounds form intramolecular hydrogen bond. This hypothesis was confirmed during chemical optimization step which allowed us to design new more active compounds with chemical structure that prevents formation of this intramolecular hydrogen bond. The most active obtained inhibitors (NHTP23, NHTP25 and NHTP33) are ATP-competitive with Ki values 4.5 nM, 12.7 nM and 4 nM, respectively. Finally we studied effect of the most active from developed protein kinase CK2 inhibitors (with IC50 from 0.004 microM to 0.7 microM) on the activity of СК2alpha and СК2alpha' recombinant proteins. Biochemical tests showed that isozymes have different sensitivity toward same compounds. The most isoform-selective inhibitor is 4'-hydroxyflavone derivative (FLC26) with IC50 value of 0.02 microM (СК2alpha) and 0.003 microM (СК2alpha').
