Scientific paper ^-Substituted Indole-2 and 3-Carboxamide Derivatives as Inhibitors of Human Protein Kinase CK2: In Vitro Assay and Molecular Modelling Study Süreyya Ölgen,1* Andreas Gratz,2 Zühal Kili^ Kurt1 and Joachim Jose2 1 Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry Tandogan, Ankara, 06100, Turkey 2 Institut für Pharmazeutische und Medizinische Chemie, Westfälische WilhelmsUniversität Münster, Hittorfstr. 58-62, 48149 Münster, Germany * Corresponding author: E-mail: olgen@ankara.edu.tr Tel: +90 (312) 203 30 73; Fax: +90 (312) 213 10 81 Received: 14-12-2012 Abstract Protein kinase CK2 (Casein Kinase 2) is involved in cell growth; proliferation and suppression of apoptosis. Hence, it strongly promotes cell survival and can be considered an important target for human cancers. In the present study, a series of N-substituted indole-2- and 3-carboxamide derivatives were tested for inhibitions of human recombinant protein kinase CK2 to evaluate their anticancer properties. The inhibition test revealed that the most active compound 4 (1-ben-zyl-N-(2,4-dichlorobenzyl)-1H-indole-2-carboxamide) showed an IC50 value of 14.6 |M towards human protein kinase CK2. A molecular docking study of the compounds with CK2 was performed and revealed the binding mode of the most active compound 4, underlying its inhibitory activity. Keywords: Casein Kinase-2, Indole derivatives, Inhibitors, Docking 1. Introduction The acidophilic Ser/Thr protein kinase CK2 (Casein Kinase 2) plays important roles in gene expression, DNA repair, RNA and protein synthesis, apoptosis, transformation and signal transduction from extracellular growth factors.1 In several mice models, it was shown that CK2 cooperates with other protooncogenes to generate lymphomas and promotes cytotoxic effects.2 Overexpression of protein kinase CK2 is an unfavorable prognostic marker in prostate, lung cancers and acute myeloid leukemia and suggests that CK2 may represents an important therapeutic target. Furthermore, many viral proteins are reported as CK2 substrates, indicating that CK2 kinase may play a role in viral infections as well. For this reason, CK2 inhibitors have become an attractive target as anticancer and anti-viral com-pounds.3 In a number of different cancers such as prostate,4 mammary gland,5 lung6 and others7 abnormally high levels of CK2 have been observed. In cancer cells, increased CK2 activity prevents apoptosis8 and promotes the transforming potential of oncogenes. It affects the chaperone machinery protecting the "onco-kinome", enhances the multidrug re- sistance (MRD) phenotype, counteracts the efficacy of antitumor drugs and supports angiogenesis.9 CK2 has emerged as an upcoming tumor target in human kinome and diverse strategies have been released to inhibit CK2 functions. The ATP binding site of CK2 is smaller than most of the other protein kinases, which allows for designing selective ATP-competitive inhibitors. Among the many known inhibitors of CK2 today, only CX-4945 (Fig. 1) has been reached in clinical trials for cancer as an orally bioavailable ATP-com-petitive inhibitor of protein kinase CK2.10 A number of different classes of small molecule inhibitors of CK2 have been described.11,12 Benzimidazole, benzotriazole and indole derivatives have been thoroughly investigated and led to the development of more potent compounds.13 Among them, several 4, 5, 6, 7-tetrabromo-1#-benzimidazole (TBBi), (IC50 = 0.40 pM), 4, 5, 6, 7-te-trabromo-1#-benzotriazole (TBBt) (IC50 = 0.30 pM), and 4,5,6,7-tetraiodobenzimidazole (TIBI) derivatives were found to be powerful inhibitors of CK214-17, (Fig. 1). Moreover, some polyhalogeno benzimidazole18,19 and 2-di-methylamino-4,5,6,7-tetrabromo-1H-benzimidazole compounds exhibited considerable inhibition against CK2.9,20 Fig. 1. Indole and benzimidazole derivatives known as inhibitors of CK2 From the group of indole based compounds, a po-dophyllotoxine indolo analog (W16) (Fig. 1), was identified as a potent inhibitor, which inhibited CK2 with IC50 values of 30 pM.21 In addition, several indazole derivatives were found to inhibit CK2 to a considerable ex-tent.22,23 A benzopyridoindole derivative, 19E (11-chloro-8-methyl-7H-benzo[e]pyrido[4,3-b]-3-ol, Fig. 1), showed a good pharmacologic profile, causing a marked inhibition (IC50 = 0.67 pM) of CK2 activity associated with cell cycle arrest and apoptosis in human cancer cells.24 More recently, substituted indeno[1,2-b]indole derivatives, 4b, 4d, 4g (Fig. 1), were found to be potent inhibitors of CK2 with IC50 of 0.11 pM, 0.82 pM and 1.44 pM, respectively.25 Three dimensional quantitative structure-activity relationship (QSAR) and molecular docking are useful approaches for drug design. Combining these techniques could offer more insight to understanding the structural features of active site of protein, and thus could more effectively design new potent inhibitors. Recently novel CK2 inhibitors have been identified by these computational methods.18,26 7-substituted indoloquinazoline (IQA, 5- oxo-5,6-dihydroindolo-(1,2-a)-quinazoline-7-yl-acetic acid, Fig. 1) was obtained as a potent CK2 inhibitor (IC50 = 0.40 pM) out of a 400.000-compound library by using a virtual screening approach.27,28 In addition, N-aryl indole-3-acetic acid derivative (compound I, Fig. 1) and 2,6-di-substituted pyrazine derivative (compound II, Fig. 1), were discovered as new molecules, which exhibited high CK2 inhibitory activity in both enzymatic (IC50 = 9.3 nM) and cell-based (IC50 = 0.9 nM) assays.29 As part of our interest in the area of protein kinase CK2 inhibitors, we previously reported promising 3-(sub-stituted-benzylidene)-1,3-dihydro-indolin-2-one, and thione derivatives as inhibitors of human CK2.30 To investigate more indole derivatives as inhibitors of human CK2, we have tested some ^-substituted indole-2- (1-10) and 3-carboxamide (11-20) derivatives against CK2 enzyme to continue our effort to finding new inhibitors. Moreover, docking analyses were performed to evaluate binding properties of the compounds compared with the potent CK2 inhibitor 19E and relationships between the biological activities and the binding affinities of the compounds were established. 2. Results and Discussion 2. 1. Experimental A recently developed assay for the determination of protein kinase CK2 activity based on the quantification of phosphorylated substrate by cappillary electrophoresis was used to test the inhibitory activities of the compounds.31'32 The inhibition values of all new derivatives which were tested for their antioxidant properties33'34 toward protein kinase CK2 are presented in Table 1. Inhibition was tested during the linear range (Fig. 2). Enzymatic reaction was set up in a volume of 2 mL but without the addition of an inhibitor. 200 pL aliquots were taken from the reaction tube in intervals indicated in the diagram. The CE/UV assay was used to quantify the amount of phosp-hopeptide formation represented by the area of the pro- duct peak in each of the 10 aliquots. Inhibition of CK2 ho-loenzyme was measured after incubation with compound 4 in concentrations ranging from 0.01 to 100 pM using the CE/UV assay. The dose-response curve of compound 4 was obtained from three independent CK2 kinase assays. Reduction of CK2 activity was plotted in a semi-logarithmic dose-response curve. The IC50 value was obtained by extrapolating the compound concentration at a residual CK2 activity of 50% from a four-parameter logarithmic curve fit (Fig. 3). Although compound 4, which has 2,4-dichloro benzamide group at second position was found the most active with an IC50 value of 14.6 pM, the corresponding compound 14 at third position did not show any inhibition. Reference compound 19E inhibits CK2 with an IC50 value of 0.67 pM. Table 1. Inhibition of human protein kinase CK2 by A-substituted indole-2- and 3-carboxamide derivatives Comp. No X Ri R2 % CK2 Inhibition3 LogPb 1 H H H 17 5.28 (± 0.43) 2 H H Cl 5 5.83 (± 0.50) 3 H H F 3 5.32 (± 0.46) 4 H Cl Cl 30 (IC50 = 14.60 |M) 6.38 (± 0.54) 5 H F F -14 5.55 (± 0.44) 6 F H H -1 5.32 (± 0.46) 7 F H Cl -1 6.02 (± 0.46) 8 F H F -1 5.49 (± 0.46) 9 F Cl Cl -2 6.59 (± 0.51) 10 F F F -12 5.70 (± 0.50) 11 H H H -2 5.18 (± 0.43) 12 H H Cl n.a 5.66 (± 0.45) 13 H H F -2 5.17 (± 0.42) 14 H Cl Cl -1 6.30 (± 0.50) 15 H F F 2 5.41 (± 0.44) 16 F H H 5 5.23 (± 0.42) 17 F H Cl 22 5.89 (± 0.44) 18 F H F 6 5.36 (± 0.46) 19 F Cl Cl 7 6.45 (± 0.49) 20 F F F 3 5.50 (± 0.51) 19E IC50 = 0.67 |Mc Emodin IC50 = 0.27 |M TBB IC5„ = 1.33 |M a CK2 activity of a sample including the test compound was calculated as a fraction of the CK2 activity at 10 pM concentration, measured without test compound but with DMSO (positive control). b LogP values calculated from website: www.vcclab.org IC50 was taken from PDB data bank. 4000- M ] Fig. 3. IC5Q determination of compound 4 as a CK2 inhibitor in a dose-dependent manner designed as CK2 inhibitors. Despite the small number of indole compounds studied and the efficacy of CK2 inhibition could not be clearly explained by structure-activity relationships, the obtained results prompted us to evaluate binding conformation and the degree of fit into the CK2 binding sites of compounds 1-20 by using Autodock 4.2.1 and Vina 1.0.2. The crystallographic structure of human 19E-CK2 complex (entry 3OWL of the Brookhaven protein data bank) was chosen as the starting point to perform molecular docking. The ligand 19E (Fig. 1) is a potent CK2 inhibitor that docked onto CK2 to validate the docking process. The reliability of the docking protocol was first tested by simulations of the binding mode of CK2 inhibitor 19E and comparison of the modeled complexes with the available three-dimensional structures derived by X-ray crystallography (3OWL). A root mean square deviation (rmsd) value of 1.641 A, calculated on the whole structure, was found with no significant conformational rearrangement of the hinge region. The program successfully reproduced the X-ray coordinates of the inhibitor binding conformations (rmsd values ranging from 1.034-2.078 A between docked and crystallographic con-formers). During our work, we also tried the dockings for compound 12 and 13 which have interactions with the protein, but the rmsd values were higher than 2.08 which is outside the error range for interaction between ligand and target. The docking result shows that 19E occupies in the ATP binding site (Fig. 4) and overlapped with cystlal-lographic 19E. One hydrogen bond was identified between OH of crystallographic 19E and NH2 proton of VAL116. Other hydrogen bond was observed between OH hydrogen of crystallographic 19E and CO of GLU114. Other gatekeeper amino acids of CK2 binding site are LYS 68,VAL 53, ILE 174 and VAL 66 (Fig. 4). The docking results of compounds 1-20 suggested that all of the compounds occupied in the ATP binding site of CK2 but with a different orientation than 19E. Each docked compound was assigned a score according to its Fig. 4. Comparison of experimentally determined (orange) and docked (cyan) positions of 19E in the 3OWL active site. binding mode onto the binding site of enzymes. Compounds showed similar interactions and overlapped in the active site, except compounds 1, 3, 4, 11, 12, 13, 15 and 16 (Fig. 5). Compound 1 did not show any hydrogen bond between active site amino acids and occupy in the ATP binding site. Compound 17 interacted with ATP binding site and a hydrogen bond was observed between the oxygen of amide carbonyl and NH2 hydrogen of ASN 118. The results suggest that ligands with lowest binding affinities have a better chance of interaction with enzymes and have also a good probability of acting as inhibitors. Among the active compounds 1, 4 and 17, one hydrogen bond was identified between the amide carbonyl oxygen of compound 4, and NH2 hydrogen of LYS 68 (Fig. 6). Fig. 5. Overlay of the compounds inside the active site of the CK2. Fig. 6. Interaction of compound 4 with CK2 binding site. 3. Conclusion In this study, a combination of docking studies and biological evaluation allowed us to get into the relationships between biological activity and binding properties of compounds under investigation. Nevertheless, there were no certain relationships between binding properties, structures and activity of compounds. The only conclusion that can be made is that the presence of hydrogen bond acceptor group (C=O) of the side chain at the indole ring is important for binding to the enzyme active site. Compound 4 showed different binding properties than the reference compound 19E. Compound 4 has a hydrogen bond between the oxygen of amide carbonyl and NH2 hydrogen of LYS 68, which is a different interaction than 19E. This difference is found also with the other indole compounds. In conclusion, it may be necessary to design some compounds with exactly the same binding properties with 19E to obtain better activity results. 4. Experimental ATP, Tris and EDTA were bought from Roth (Karlsruhe, Germany), MgCl2 from VWR (Darmstadt, Germany), NaCl and HCl from J. T. Baker (Deventer, The Netherlands), DTT from AppliChem (Darmstadt, Germany), and DMSO from Sigma-Aldrich (Steinheim, Germany) were purchased. CK2 substrate peptide had the amino acid sequence RRRDDDSDDD and was synthesized with its C-terminus conjugated to 5-[(2-ami-noethyl)amino]naphthalene-1-sulfonic acid (EDANS). It was synthesized by the Merrifield solid phase procedu-re.36 Fmoc was used as the N-terminal protecting group,37 and a final purification step was done by RP-HPLC. If not stated otherwise, all solutions were prepared with fresh ultrapure water and have been stored at 4 °C, for short time periods, or at -20 °C, if storage lasted longer than 14 days. 4. 1. Preparation of Recombinant Human CK2 Enzyme A purification protocol was set up according to the method of Granowski with modifications.38 For this purpose a-subunit (CSNK2A1) and P-subunit (CSNK2B) of protein kinase CK2 were expressed separately in a bacterial expression system (pT7-7/BL21(DE3).31 Freshly transformed bacterial cells were grown overnight at 37 °C until they reached the stationary phase. With the separate overnight culture for each subunit, CSNK2Al and CSNK2B, 6 L of fresh medium was inoculated and cultivated until an OD500 of 0.6 was reached. Protein expression was induced by adding IPTG to a final concentration of 1 mM and was run at 30 °C for 5-6 h for CSNK2Al and at 37 °C for 3 h for CSNK2B. Bacterial cells were harvested by centrifugation (6.000 x g at 4 °C for 10 min) and disrupted by sonification (3 x 30 s on ice). Cell debris was removed by centrifugation and the bacterial extracts by this strategy for both subunits were combined and subjected to a three-column purification procedures as described previously.31 The fractions containing active CK2 holoenzyme were determined by activity measurement. Fractions exhibiting CK2 enzymatic activity were combined and analyzed by SDS PAGE and western blot. They were stored in aliquots at - 80 °C until used for testing. 4. 2. CE-determination of CK2 Inhibition 80 pL of the CK2-supplemented kinase buffer (kinase buffer + 1pg CK2) was either preincubated with 2 pL test compound, previously dissolved in pure DMSO, or incubated with pure DMSO as a control, for 10 min at 37°C before it was mixed with 120 pL CE assay buffer (25 mM Tris/HCl, pH 8.5, supplemented with 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 pM ATP and 190 pM substrate peptide (RRRDDDSDDD-[EDANS]). After incubation for 15 min at 37°C, 2 pmol EDTA were added to stop the enzyme reaction. The samples were stored at 4°C until about 20 nL were injected into a bare fused silica capillary with 50 cm effective and 60 cm total length (50 pM ID and 375 pM OD, Beckman Coulter GmbH, Krefeld, Germany). Separations were conducted on a ProteomeLab PA800 System operated by 32 karat 7 software (Beckman Coulter GmbH, Krefeld, Germany) with 2 mol/L aqueous acetic acid as background electrolyte (voltage: 30 kV). Absorption detection was recorded at a wavelength of 214 nm. Before using the capillary for the first time, it was conditioned with 0.1 mol/L NaOH, then rinsed with deionized water and equilibrated with 2 mol/L acetic acid before the sample was injected. This procedure was also performed prior to every measurement. Peak integrations usually had an RSD < 1 %. For determination of IC50 values a concentration-response analysis of the compound in eleven concentrations ranging from 0.5 nM to 50 pM was performed. One hundred percent CK2 activity was determined in the absence of the compound but in the presence of DMSO. A control value for no enzyme activity was obtained by adding all components but without the co-substrate ATP. A nonlinear regression analysis of the relative inhibition at different compound concentrations was conducted. Therefore a four parameter logistic fit of the data points was calculated by Graphpad Prism using the analysis option "sigmoid curve with variable slope" (GraphPad, La Jol-la, CA, USA). The IC50 value represents the concentration at the midpoint (50 %) of CK2 inhibition on a semi logarithmic dose-response plot. The CE assay was finally validated by determining the IC50 values of the known inhibitors of human CK2 Emodin and TBB, which turned out to be 0.27 pM and 1.33 pM, respectively, and which were in agreement with the IC50 values that have been determined before with radiometric as- 25 says.25 4. 3. Docking Calculations For this study, X-ray crystal structure of CK2-19E complex (PDB entry 3OWL) was downloaded from Protein Data Bank (PDB). Solvents of molecules were deleted and hydrogen atoms were added to the protein to obtain the docking grid the active site was defined using Au-togrid. The grid size was set to 40 x 40 x 40 points with grid spacing of 0.375A. The grid box was centered on the center of the ligand from the corresponding crystal structure complexes. 2D structures of inhibitors were established by using ChemBioDrawUltra 11.0 and minimized with Hyperchem 8.0.7 (Hypercube Inc, FL, USA) using the semi-emperical the Austin Model-1 (AM-1) method. To be exported to Autodock, the minimized structures were converted into the mol2 file format. The structures of proteins and ligands were combined using the Autodock 4.2.1 and Vina 1.0.2 software package (The Scripps Research Institute, La Jolla CA, USA). The rigid root and ro-tatable bonds were defined using ADT. The results were ranked by the scoring functions of ligand-fit, and analyzed for relatively lower binding energy, and potential in forming hydrogen bonds with CK2. The ligand was fully optimized inside the binding site during the docking simulations The conformation with the lowest predicted binding free energy of the most occurring binding modes in CK2 active pockets were evaluated to binding properties and relationships between biological activity-binding affinities of compounds. 5. Acknowledgements This work was partially supported by a grant from the Turkish Scientific and Technical Research Institute (HD-141, 106S127). 6. References 1. D. I. Perez, C. Gil, A. Martinez, Med. Res. Rev. 2011, 30, 924-954. 2. M. Mazzorana, L. P. Pinna, R. Battistutta, Mol. Cell Bio-chem. 2008, 316, 57-62. 3. R. Prudent, C. Cochet, New Chem. and Biol. 2009, 16, 112120. 4. S. Yenice, A. T. Davis, S. A. Goueli, A. Akdas, C. Limas, K. Ahmed, Prostate 1994, 24, 11-16. 5. E. Landesman-Bollag, R. Romieu-Mourez, D. H. Song, G. E. Sonenshein, R. D. Cardiff, D. C. Seldin, Oncogene 2001, 20, 3247-3257. 6. M. Daya-Makin, J. S. Sanghera, T. L. Mogentale, M. Lipp, J. 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Povzetek Protein kinaza CK2 (kazein kinaza 2) je vključena v procese celične rasti, proliferacije in zaviranja apoptoze. Tako močno spodbuja preživetje celic in jo lahko štejemo kot pomembno tarčo za zdravljene raka pri človeku. V tej študiji smo proučevali skupino N-substituiranih derivatov indol-2-karboksamida. Da bi preverili njihove učinke proti raku, smo spojine smo testirali kot inhibitorje rekombinantne protein kinase CK2. Testi inhibicije so pokazali, da je najučinkovitejša spojina 4 (1-benzil-N-(2,4-diklorobenzil)-1_ff-indol-2-karboksamid), z IC50 14.6 |M proti človeški protein kinazi CK2. Naredili smo tudi študije molekularnega sidranja spojin s CK2 in opredelili vezavni model, ki je pokazal inhibi-torno aktivnost najučinkovitejše spojine 4.