Letters pubs.acs.org/acschemicalbiology
A Note of Caution on the Role of Halogen Bonds for Protein Kinase/ Inhibitor Recognition Suggested by High- And Low-Salt CK2α Complex Structures Barbara Guerra,† Nils Bischoff,‡ Volodymyr G. Bdzhola,§ Sergiy M. Yarmoluk,§ Olaf-Georg Issinger,† Andriy G. Golub,§ and Karsten Niefind*,‡ †
University of Southern Denmark, Department of Biochemistry and Molecular Biology, Campusvej 55, DK-5230 Odense, Denmark University of Cologne, Institute of Biochemistry, Otto-Fischer-Str. 12-14, D-50674 Cologne, Germany § Department of Medicinal Chemistry, Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, 150 Zabolotnogo Str., Kyiv 03680, Ukraine ‡
S Supporting Information *
ABSTRACT: CK2 is a Ser/Thr kinase recruited by tumor cells to avoid cell death. 4′-Carboxy-6,8-dibromo-flavonol (FLC26) is a nanomolar CK2 inhibitor reducing the physiological phosphorylation of CK2 biomarkers and inducing cell death. Its binding mode to the ATP site was predicted to depend primarily on noncovalent interactions not comprising halogen bonds. We confirm this by two independent cocrystal structures which additionally show that FLC26 is selective for an open, protein kinase-untypical conformation of the hinge/helix αD region. The structures suggest how the bromo substituents, found previously in lead optimization studies, contribute to the inhibitory efficacy. In this context, one of the complex structures, obtained by crystallization with the kosmotropic salt NaCl, revealed an unconventional π-halogen bond between the 8-bromo substituent of FLC26 and an aromatic side chain which is absent under low-salt conditions. The kosmotropic salt sensitivity of π-halogen bonds is a novel feature which requires attention in structural comparisons and halogenbond-based explanations.
A
Consequently, more than 20 CK2α/inhibitor structures with halogen bonds exist in the PDB. Flavonols, i.e. flavonoid compounds with a 3-hydroxy group at the flavone matrix (Figure 1b) and with CK2 inhibition potential,11 form a recent example for the introduction of halogen substituents into a molecular framework in order to generate inhibitors with nanomolar affinity. On the basis of virtual screening and molecular docking, Golub et al.12 designed a set of ATP-competitive 4′-carboxy flavonol compounds and predicted a detailed binding mode for them in which exclusively the rings B and C form specific interactions with the hinge region and other parts of CK2α (Figure 1b). They then used ring A for optimization and managed to reduce the IC50 value from 1.3 μM (Ki = 410 nM) for the unsubstituted 4′-carboxy flavonol (R5/6/7/8 = H in Figure 1b) via 80 nM (Ki = 20 nM) for 6-bromo 4′-carboxy flavonol to finally 9 nM (Ki =2.5 nM) for “FLC26,” the best of this series of CK2 inhibitors in which both R6 and R8 (Figure 1b) are brominated. In spite of this success, it remained open (i) whether the predicted binding mode (Figure 1b)12 is correct, (ii) why derivatizations at the primarily uninvolved A-ring and in
s part of organic compounds, chlorine, bromine, and iodine are able to form halogen bonds, i.e. weak noncovalent attractions with electron rich atoms and moieties governed by the so-called “σ-hole” in line of the covalent Chalogen bond.1,2 Currently, there are more than 2300 ligands in the Protein Data Base (PDB) containing these elements. Accordingly frequent are halogen bonds. Their number will increase further since they are valuable for affinity and selectivity optimization in inhibitor design.3 This is particularly true for eukaryotic protein kinases (EPKs) where the backbone of the hinge region connecting the two canonical kinase domains (Figure 1a) contributes to ATP-binding and provides a number of carbonyl oxygen atoms that can be exploited as potential halogen bond acceptors.4,5 Further, apart from prospective inhibitor design, halogen bonds can be used for the retrospective rationalization of the properties of classical EPK inhibitors like that performed with 5,6-dichloro-1-β-Dribofuranosylbenzimidazole (DRB) for the selective inhibition of cyclin-dependent kinases (CDK).6 In the case of protein kinase CK2a CMGC subfamily EPK associated with cancer and other human diseases,7 the subject of intensive inhibitor design,8 and composed of two catalytic chains (CK2α and/or its isoform CK2α′; Figure 1a) attached to a dimer of regulatory subunits (CK2β)9halogen atoms have been used for inhibitor optimization since a long time.10 © XXXX American Chemical Society
Received: September 16, 2014 Accepted: May 11, 2015
A
DOI: 10.1021/acschembio.5b00235 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology Figure 1. continued
CK2α. A section of the low-salt CK2α/FLC26 complex is drawn with final electron density around FLC26 (cutoff 1σ). For comparison, an ADP analogue and the inhibitor DMAT are drawn after overlay of the protein matrices. (d) Conformational selectivity of FLC26. The open conformation of the hinge/αD region is exemplified by the CK2α/CX4945 complex (black C atoms) and the closed conformation by the CK2α/emodin complex29 (green C atoms). FLC26 was drawn after overlay of the protein matrices. The distances refer to the CK2α/CX4945 complex. (e) Hydrophobic embedding of the A ring of FLC26. The nearest-atom distances of the bromo substituents are indicated.
particular the introduction of bromine were so efficient, and (iii), finally, why the bromination of the 8-position was successful although the Br8 atom was predicted to point away from the hinge backbone (Figure 1b) and thus away from the typical halogen bond acceptors normally exploited by halogenated EPK inhibitors.4 To address these questions, we determined CK2α/FLC26 complex structures supplemented by cell-culture data that emphasize the CK2 inhibitory potential of FLC26 within cells. An unexpected outcome of the structures was the kosmotropic-salt dependency of a particular halogen bond with an aromatic ring as a Lewis base, suggesting that environmental factors like the solvent composition deserve attention when halogen-bond patterns of enzyme/inhibitor complexes are compared. First, we tested the membrane permeability of FLC26 with six different human cell lines in comparison to the established CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT).13 In general, already a concentration of 10 μM FLC26 led to a CK2-inhibitory effect in extracts of treated cells equivalent to 25 μM DMAT (Figure 2a). Thus, FLC26 is able to pass the cell membrane efficiently. Due to the antiapoptotic function of CK2 in cells,7 CK2 inhibitors typically induce apoptosis as shown for DMAT,13 CX-4945 (Silmitasertib),14 E9,15 and other compounds. We analyzed two human cancer cell lines in this respect and detected increasing DNA fragmentation after incubation with FLC26 suggesting activation of apoptosis (Figure 2b). Cell death induction by FLC26 was particularly significant in PANC-1 cells fitting to the fact that these cells had the highest membrane permeability for FLC26 among the set of tested cell lines (Figure 2a). Endogenous CK2 substrate proteins can serve as biomarkers in order to test whether CK2 is the physiological target of an inhibitor. Recently, the HSP90 cochaperone CDC3716 and the transcription factor NFκB17 were used in this way. Following this approach, we performed phospho-specific Western blot analyses with two different cell lines and revealed that the phosphorylation state of CDC37 and NFκB is in fact reduced after FLC26 treatment (Figure 2c). In summary, these cellular data confirm that FLC26 is a candidate for drug development targeting CK2-related diseases.7 To determine the structural basis of the FLC26/CK2α interaction and to probe the predicted binding mode (Figure 1b), we used “hsCK2α1−335,” a C-terminal truncation construct of human CK2α.18 A preincubated mixture of hsCK2α1−335 and FLC26 was crystallized under two conditions differing strongly in the concentration of a kosmotropic salt, namely either with polyethylene glycol 4000 or with sodium chloride as the main precipitant (Table 1). The use of two different crystallization media with deviating NaCl concentrations was motivated by
Figure 1. FLC26 and its principle binding mode to the ATP-site of human CK2α. (a) Overview of the CK2α/FLC26 complex indicating the ligand binding site and the bilobal EPK architecture. Predominantly, the picture shows subunit A of the low-salt CK2α/FLC26 complex structure including the bound FLC26 inhibitor. FLC26 as coordinated by hsCK2α1−335 under high-salt conditions is drawn in black after 3D-fit of the protein chains. (b) Predicted interactions of 4′-carboxy flavonol compounds with CK2α. The figure was taken from Golub, A.G.; et al. Structure-based discovery of novel flavonol inhibitors of human protein kinase CK2. Mol. Cell. Biochem. 2011, 356, 107−115, with kind permission from Springer Science+Business Media and modified by red labeling. (c) Hydrogen bonds (gray dotted lines) and π−π interaction (blue dotted line) between FLC26 and B
DOI: 10.1021/acschembio.5b00235 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology
Figure 2. CK2 inhibitory impact of FLC26. (a) Membrane permeability of FLC26 probed by incubation of different tumor cell lines with increasing inhibitor concentrations followed by cell disruption and CK2 activity determination in soluble extracts. Twenty-five micromolar DMAT was used as a positive control. The assays were performed in triplicate; average values ± the standard deviation are shown. (b) Cell death induction by FLC26. PANC-1 and M059K cells were incubated with 10 μM or 5 μM FLC26, respectively. The fraction of dead cells was determined by flow cytometry after propodium iodide staining. Data from one representative experiment are shown. (c) Impact of FLC26 on CK2 biomarker phosphorylation in M059K and Mia PaCa-2 cells. The CK2-dependent phosphorylation of physiological substrate proteins (CDC37 and NFκB) and its depletion by FLC26 was detected by Western blot analysis. Lanes 1 and 4 refer to cells incubated with DMSO only. In all of the other cases, cells were incubated with 50 μM FLC26 for either 5 h (lanes 2 and 5) or 24 h (lanes 3 and 6), respectively. (d) Impact of FLC26 on the catalytic activity of hsCK2α1−335 as a function of the NaCl concentration. A radioactivity-based enzyme assay with saturation concentrations of ATP and of the peptide substrate RRRADDSDDDDD was performed with and without 250 nM FLC26 in the presence of eight different NaCl concentrations. The black and white histogram bars illustrate averages of two independent measurements, respectively. The inhibitory efficacy (gray bars) represents the percentage of depletion of CK2α activity by 250 nM FLC26.
and its 4′-carboxy substituent on the inhibitor side is similar to a substructure of the CK2α complex with the highly potent and selective CK2 inhibitor CX-494514 (Figure 1d). During FLC26 development, the flavonol-typical 3-hydroxy substituent of flavone ring C (Figure 1b) lowered the IC50 value by an order of magnitude,12 which was explained by the proximity of an H-bond acceptor (4-oxo) next to a donor (3hydroxy), thus mimicking the hydrogen bonding potential of ATP. Actually, the two predicted H-bonds to Glu114 and Val116 (Figure 1b) are present in all three CK2α/FLC26 couples of our two complex structures (Figure 1c). In summary, the flavone rings B and C determine the position and orientation of FLC26 within the ATP site of CK2α unambiguously and exactly as predicted (Figure 1b,c). Thus, the ring A is enforced to occupy an outer subsite known as “hydrophobic region II” in a popular EPK pharmacophore model21 (Figure 1c). As a consequence, its two Br-substituents point toward the surface, away from the typical halogen bond acceptors of the hinge backbone. 4 Thus, the strong optimization effects observed for the introduction of Br6 (decrease of Ki from 410 to 20 nM) and of Br8 (Ki = 2.5 nM)12 must be independent of hinge/halogen bonds in contrast to what is normally found for halogen-containing inhibitors of
the fact that kosmotropic salts support hydrophobic interactions.19 Recently, they were shown to significantly affect the conformation of the hinge/helix αD region (blue in Figure 1a) in human CK2α being critical for the binding of ATPcompetitive ligands.20 The low-salt crystals of the hsCK2α1−335/ FLC26 complex (referred to as “CK2α/FLC26” from here on) contain two protomers per asymmetric unit and the high-salt crystals only one (Table 1). The high-salt structure is significantly better resolved than the low-salt one, but all three protein chains are covered by electron density almost completely. In all three CK2α subunits, the ATP site is occupied by a well-defined FLC26 molecule (Figure 1c). The orientations and positions of the three FLC26 copies relative to the enzyme are similar (Figures 1a and 3a) irrespective of the salt concentration in the environment. In either case, the four noncovalent interactions illustrated in Figure 1b are found exactly as predicted and intended by Golub et al.:12 the 4′-carboxy group forms an ion pair with Lys68 (Figure 1c), a conserved lysine required to fix the triphospho moiety of the cosubstrate ATP, while the phenyl ring B is an aromatic stacking partner of the “gatekeeper” side chain Phe113 (Figure 1b,c). The whole ensemble of Lys68 and Phe113 on the enzyme side and ring B C
DOI: 10.1021/acschembio.5b00235 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology
a dynamic equilibrium, albeit with clearly distinct subpopulations.25 The alternative “closed” conformation would be incompatible with FLC26 binding due to unavoidable clashes of Asn117/Asn118 with the Br6 atom (Figure 1d). A comparative inspection of Phe121 (Figure 3b,c), the start residue of helix αD, emphasizes the power of this Br6-imposed conformational selectivity of FLC26. Phe121 rotamer transitions were computationally identified as the origin of the highest activation energy barrier (about 33.5 kJ/mol) between the two hinge/helix αD conformations.25 In closed-conformation CK2α structures (e.g., black reference structure 3BQC in Figure 3c), Phe121 is part of the catalytic spine (C-spine) that is one of two essential stacks of hydrophobic residues connecting the two kinase lobes in EPKs.26 The hydrophobic assembly is optimized in this state, and as kosmotropic salts generally support hydrophobic interactions,19 it was found so far in all CK2α structures crystallized under high-salt conditions, irrespective of any bound ligand.20 In contrast, in CK2α with open hinge/helix αD conformation Phe121 is turned away from its canonical C-spine position (black reference structure 3NSZ in Figure 3a). As expected, FLC26 binding supports this open state as visible in the low-salt CK2α/FLC26 structure (Figure 3b). Remarkably, this conformational impact of FLC26 is even stronger than an opposing kosmotropic salt effect: under high-salt conditions Phe121 approaches its C-spine position (Figure 3c), but unlike any other high-salt CK2α structure, it does not reach it and remains partly disordered because it is constrained by the hinge region around Asn118, which in turn is fixed in the open conformation by the Br6 atom of FLC26 (Figure 1d). Br8, the second bromo substituent of the A ring, is hydrophobically sandwiched as well (Figure 1e), but the fact that chloro and bromo substituents are particularly effective at this position12 indicates an additional halogen-specific effect. Consistently, the Br8 atom forms a halogen bond to the aromatic π system of Tyr50 (Figure 3a), albeit only in the highsalt CK2α/FLC26 structure and not in the low-salt one. Tyr50 is part of the glycine-rich loop (Figures 1a and 3a). This loop is known to be flexible, but for its genuine function of ATPbindingand in the low-salt CK2α/FLC26 structure as well it adopts an extended conformation. To approach the Br8-atom of FLC26, Tyr50 is displaced by about 14 Å, which requires an extreme local distortion involving a partial melting of the strands β1 and β2 (Figure 3a). In addition to the halogen bond to Br8, Tyr50 is anchored by a hydrogen bond and π−π stacking to His160 (Figure 3a), so that the glycine-rich loop despite its inherent disorder in many EPK structures is remarkably well-defined by electron density in this particular state. We assessed the significance of this unusual π-halogen bond in three ways: (i) We calculated the typical distance and orientation parameters of π-halogen bonds; the resulting values of 3.7 Å and 155.7° (Figure 3a) fit nicely to statistical data obtained from 53 comparable interactions from the PDB.3 (ii) To check the impact of the kosmotropic salt used for crystallization on the glycine-rich loop conformation, we overlaid the two CK2α/FLC26 complexes with all high-salt CK2α structures in the PDB. This 3D-comparison revealed a disposition of the loop to distort in a high-salt environment (Figure 3d), but simultaneously it documents that the collapse within the high-salt CK2α/FLC26 complex is unprecedented and requires the additional Br8/Tyr50 halogen bond. (iii) Finally, we used enzyme kinetics to probe whether a salt-
Table 1. X-ray Diffraction Data Collection and Refinement Statistics hsCK2α1−335/FLC26low salt main precipitant temperature (K) wavelength (Å) space group lattice constants a, b, c (Å) protomers per asymmetr. unit resolution (Å) (highest shell) Rsym (%) CC1/2 I/σI total reflections unique reflections completeness (%) multiplicity Wilson B-factor (Å2) no. of reflections used for Rfree Rwork/Rfree (%) mean coordinate error (Å) no. of non-hydrogen atoms protein ligand/ion water B factors (Å2) protein ligand/ion water RMS deviations bond lengths (Å) bond angles (deg) Ramachandran plot favored (%) allowed (%) outliers (%) a
hsCK2α1−335/FLC26high salt
crystallization 30% PEG4000 data collection 100 1.0000 P43212 126.00, 126.00, 124.14
100 0.8123 P43212 72.59, 72.59, 135.51
2
1
44.55−3.00 (3.18− 3.00)a 17.2 (89.1)a 0.994 (0.759)a 10.6 (2.0)a 133 197 (18055)a 20 425 (2978)a 98.5 (91.3)a 6.5 (6.1)a 32.25 refinement 1018
19.69−2.10 (2.16− 2.10)a 7.1 (55.5)a 0.998 (0.788)a 17.8 (3.6)a 180 836 (12663)a 21 710 (1703)a 98.9 (97.5)a 8.3 (7.4)a 35.19
18.7/23.8 0.350
17.7/21.9 0.250
5630 52 36
2816 26 179
57.0 36.9 28.4
53.3 49.1 47.9
0.002 0.660
0.003 0.890
96.0 0.0 0.3
96.0 0.0 0.3
4.4 M NaCl
1077
The numbers in parentheses refer to the highest resolution shell.
CK2exemplified by DMAT22 (Figure 1c) or DRB23or of other EPKs.4−6 In fact, the Br6 atom of FLC26 does not form any halogen bond to the enzyme (Figure 1c,d). Rather, together with the A ring, it is sandwiched by hydrophobic side chains (Figure 1e). This coincides with the observation that hydrophobic substituents at the 6 position (Figure 1b) increase the affinity of FLC26 precursors from methyl via methoxy, chloro, ethyl, to finally bromo.12 Apart from hydrophobicity, this succession reveals a spatial effect with larger substituents at position 6 being more effective than small ones. The structural background is that the Br6 atom forms favorable contacts to atoms of the C-terminal part of the hinge region (Val116 to Asn118; Figure 1d), provided that the latter adopts the so-called “open” hinge/αD conformation.24 This is one of two principle structural states of the interdomain hinge and the subsequent helix αD existing in human CK2α in D
DOI: 10.1021/acschembio.5b00235 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology
Figure 3. Confirmed and potential salt-effects on local conformations and halogen bonds. (a) Overlay of the glycine-rich loop regions of the high-salt CK2α/FLC26 complex (green; electron density with cutoff 1σ covering FLC26, Tyr50, and His160), the low-salt CK2α/FLC26 complex (yellow; electron density with cutoff 1σ covering Tyr50), andas a referencea high-resolution structure of human CK2α (black). An extreme distortion of the loop accompanied by additional π-halogen bond between FLC26 and Tyr50 exists exclusively in the high-salt structure. (b) C-terminal part of the hinge/αD region in the low-salt CK2α/FLC26 complex covered by electron density (cutoff 1σ). The critical side chain Phe12123−25 is turned away from the C-spine (green). As a reference, the open conformation is drawn from the near-atomic resolution structure 3NSZ with black C atoms. (c) C-terminal part of the hinge/αD region in the high-salt CK2α/FLC26 complex accompanied by electron density (cutoff 1σ). For comparison, the closed hinge/αD conformation is drawn (black C atoms) from the CK2α/emodin complex29 (3BQC) with Phe121 harbored by its canonical hydrophobic cavity at the C-spine cavity (green). (d) High-salt and π-halogen bond impact on the glycine-rich loop conformation of human CK2α. The high- and low-salt CK2α/FLC26 complexes were superimposed with one protomer of all other human CK2α structures in the PDB obtained from crystallization with a kosmotropic salt precipitant. The main precipitants used for cystallization are indicated together with the PDB codes. (e) DRB binding to CDK9 (magenta-colored C atoms) and for comparison to CDK2 (yellow C atoms). Halogen bonds and a chlorine-mediated hydrogen bond are drawn as found by Baumli et al.6 For the CDK2/DRB complex, an additional potentially salt-dependent π-halogen interaction with Phe80 is indicated in black.
systems by disturbing ordered water clusters otherwise shielding the aromatic groups. Cases of halogen-bond pattern flexibility of EPK inhibitors are documented,6,22 but to our knowledge this is the first report that kosmotropic salt effects are relevant in that context. If this is a general feature of π-halogen bonds beyond the CK2α/ FLC26 case, it becomes more difficult to rationalize protein/ ligand interactions as soon as interactions of this type are involved. In particular, iflike shown in this study (Figure 3c)an extreme salt-supported structural distortion in the protein partner is enabled by a π-halogen bond, it likewise seems to be possible that π-halogen bonds induce orientational
supported halogen bond is reflected in the inhibitory power of FLC26. This approach is restricted by the long-known fact that the activity of CK2α is strongly reduced with growing salt concentrations.27 Within this limitation, we observed the expected increase of the inhibitory efficacy of FLC26 parallel to the NaCl concentration (Figure 2d). In summary, these results are consistent with a dynamic conformational equilibrium of the glycine-rich loop which is affected by FLC26 via a surprising π-halogen bond supported by the kosmotropic salt in the environment. Such salts affect the water structure in the first hydration shell of a protein;19 therefore, they may favor halogen bonds with aromatic π E
DOI: 10.1021/acschembio.5b00235 ACS Chem. Biol. XXXX, XXX, XXX−XXX
ACS Chemical Biology
■
and conformational adaptions on the ligand side in a saltdependent manner. This could, for example, be the case for the above-mentioned CDK6 and CK223 inhibitor DRB, which adopts partially different orientations in its complex structures with CDK9 and CDK2 (Figure 3e), the former crystallized with 20% polyethylene glycol 1000 (low salt) but the latter with 1.1 M ammonium sulfate (high salt).6 DRB forms two halogen bonds with the hinge region of CDK9 (Figure 3e). In complex with CDK2, two alternate DRB orientations occur, the minor one (12% occupancy6) being equivalent to the CDK9-bound state, while in the major onedepicted in Figure 3eonly one halogen bond to the hinge is formed. This difference of the number of hinge-halogen bonds is consistent with a higher inhibitory efficacy of DRB with CDK9.6 However, in the highsalt CDK2/DRB structure, the not-hinge-bound Cl2-atom seems to compensate this loss partially and is directed toward Phe80 (Figure 3e). With a σ-hole angle of 155.4° and a Cl2/ centroid distance of 4.0 Å, the geometry data of this Cl2/Phe80 interaction are in the range of the average values (161.5°/3.85 Å) over 124 C−Cl···π halogen bonds in the PDB;3 further, its centroid/centroid distance is 7.0 Å (Figure 3e) which is close to the minimum of an energy profile calculated for these types of interactions.28 Hence, kosmotropic salt impacts on the halogenbond patterns of DRB may interfere with genuine differences between CDK9 and CDK2, so that the halogen-bond based rationalization of the DRB selectivity6 would benefit from complex structures obtained under comparable crystallization conditions. In summary, salt and solvent effects deserve careful attention in the study of protein complexes with halogenated ligands. The analysis of the CK2α/FLC26 complexes shows that without the high-salt structure, we would have missed the Br8/ Tyr50 halogen-bond, while without the low-salt structure we would have failed to notice its salt-dependent character. Hence, for experimental investigations, complementary structures obtained from high- and from low-salt crystallization conditions are advisible in order to capture kosmotropic salt-sensitive πhalogen bonds, and in theoretical studies on π-halogen bonds the inclusion of such subtle environmental effects should be considered.
■
METHODS
■
ASSOCIATED CONTENT
Letters
AUTHOR INFORMATION
Corresponding Author
*E-mail: Karsten.Niefi
[email protected]. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We thank the staff of the EMBL outstation in Hamburg (Germany) and of the Swiss Light Source in Villigen (Switzerland) for assistance with X-ray data collection. We are grateful to G. Schwarz and U. Baumann (both Cologne, Germany) for access to protein crystallography equipment. The work was funded by the National Academy of Sciences of Ukraine (grant 0107U003345), the Danish Council for Independent Research (grant 1323-00212A) and the Deutsche Forschungsgemeinschaft (grant NI 643/4-1).
■
REFERENCES
(1) Politzer, P., and Murray, J. S. (2013) Halogen bonding: an interim discussion. ChemPhysChem 14, 278−294. (2) Wilcken, R., Zimmermann, M. O., Lange, A., Joerger, A. C., and Boeckler, F. M. (2013) Principles and applications of halogen bonding in medicinal chemistry and chemical biology. J. Med. Chem. 56, 1363− 1388. (3) Lu, Y., Wang, Y., and Zhu, W. (2010) Nonbonding interactions of organic halogens in biological systems: implications for drug discovery and biomolecular design. Phys. Chem. Chem. Phys. 12, 4543− 4551. (4) Grant, S. K., and Lunney, E. A. (2011) Kinase inhibition that hinges on halogen bonds. Chem. Biol. 18, 3−4. (5) Fedorov, O., Huber, K., Eisenreich, A., Filippakopoulos, P., King, O., Bullock, A. N., Szklarczyk, D., Jensen, L. J., Fabbro, D., Trappe, J., Rauch, U., Bracher, F., and Knapp, S. (2011) Specific CLK inhibitors from a novel chemotype for regulation of alternative splicing. Chem. Biol. 18, 67−76. (6) Baumli, S., Endicott, J. A., and Johnson, L. N. (2010) Halogen bonds form the basis for selective P-TEFb inhibition by DRB. Chem. Biol. 17, 931−936. (7) Guerra, B., and Issinger, O.-G. (2008) Protein kinase CK2 in human diseases. Curr. Med. Chem. 15, 1870−1886. (8) Prudent, R., and Cochet, C. (2009) New protein kinase CK2 inhibitors: jumping out of the catalytic box. Chem. Biol. 16, 112−120. (9) Niefind, K., Guerra, B., Ermakowa, I., and Issinger, O.-G. (2001) Crystal structure of human protein kinase CK2: insights into basic properties of the CK2 holoenzyme. EMBO J. 20, 5320−5331. (10) Meggio, F., Shugar, D., and Pinna, L. A. (1990) Ribofuranosylbenzimidazole derivatives as inhibitors of casein kinase-2 and casein kinase-1. Eur. J. Biochem. 187, 89−94. (11) Lolli, G., Cozza, G., Mazzorana, M., Tibaldi, E., Cesaro, L., Donella-Deana, A., Meggio, F., Venerando, A., Franchin, C., Sarno, S., Battistutta, R., and Pinna, L. A. (2012) Inhibition of protein kinase CK2 by flavonoids and tyrphostins. A structural insight. Biochemistry 51, 6097−6107. (12) Golub, A. G., Bdzhola, V. G., Kyshenia, Y. V., Sapelkin, V. M., Prykhod’ko, A. O., Kukharenko, O. P., Ostrynska, O. V., and Yarmoluk, S. M. (2011) Structure-based discovery of novel flavonol inhibitors of human protein kinase CK2. Mol. Cell. Biochem. 356, 107− 115. (13) Pagano, M. A., Meggio, F., Ruzzene, M., Andrzejewska, M., Kazimierczuk, Z., and Pinna, L. A. (2004) 2-Dimethylamino-4,5,6,7tetrabromo-1H-benzimidazole: a novel powerful and selective inhibitor of protein kinase CK2. Biochem. Biophys. Res. Commun. 321, 1040− 1044. (14) Siddiqui-Jain, A., Drygin, D., Streiner, N., Chua, P., Pierre, F., O’Brien, S. E., Bliesath, J., Omori, M., Huser, N., Ho, C., Proffitt, C., Schwaebe, M. K., Ryckman, D. M., Rice, W. G., and Anderes, K. (2010) CX-4945, an orally bioavailable selective inhibitor of protein
FLC26 was synthesized according to Golub et al.11 and used from a 10 mM FLC26 stock solution in dimethyl sulfoxide (DMSO) for subsequent cell culture and cocrystallization studies. The enzyme construct hsCK2α1−33518 was prepared as described previously.29 Complex crystals were obtained by vapor diffusion either with 4.4 M NaCl and 0.1 M sodium citrate, pH 5.2 (high-salt conditions), or with 30% PEG4000, 15% (v/v) glycerol, 0.2 M ammonium acetate, and 0.1 M sodium citrate, pH 5.6 (low-salt conditions). X-ray diffraction data and refinement statistics are reported in Table 1. Full details of cell membrane permeability determination of FLC26, cell death and biomarker analysis, NaCl-dependent inhibition kinetics, and cocrystal structure-determination are described in the Supporting Information. The final atomic coordinates and structure factors were deposited in the PDB with accession codes 4UB7 (high-salt structure) and 4UBA (low-salt structure).
S Supporting Information *
Supplementary methods. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.5b00235. F
DOI: 10.1021/acschembio.5b00235 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology kinase CK2, inhibits prosurvival and angiogenic signaling and exhibits antitumor efficacy. Cancer Res. 70, 10288−10298. (15) Guerra, B., Rasmussen, T. D., Schnitzler, A., Jensen, H. H., Boldyreff, B. S., Miyata, Y., Marcussen, N., Niefind, K., and Issinger, O.-G. (2015) Protein kinase CK2 inhibition is associated with the destabilization of HIF-1α in human cancer cells. Cancer Lett. 356, 751−761. (16) Moucadel, V., Prudent, R., Sautel, C. F., Teillet, F., Barette, C., Lafanechere, L., Receveur-Brechot, V., and Cochet, C. (2011) Antitumoral activity of allosteric inhibitors of protein kinase CK2. Oncotarget 2, 997−1010. (17) Manni, S., Brancalion, A., Mandato, E., Tubi, L. Q., Colpo, A., Pizzi, M., Cappellesso, R., Zaffino, F., Di Maggio, S. A., Cabrelle, A., Marino, F., Zambello, R., Trentin, L., Adami, F., Gurrieri, C., Semenzato, G., and Piazza, F. (2013) Protein kinase CK2 inhibition down modulates the NF-κB and STAT3 survival pathways, enhances the cellular proteotoxic stress and synergistically boosts the cytotoxic effect of bortezomib on multiple myeloma and mantle cell lymphoma cells. PLoS One 8, e75280. (18) Ermakova, I., Boldyreff, B., Issinger, O.-G., and Niefind, K. (2003) Crystal structure of a C-terminal deletion mutant of human protein kinase CK2 catalytic subunit. J. Mol. Biol. 330, 925−934. (19) Zhang, Y., and Cremer, P. S. (2006) Interactions between macromolecules and ions: The Hofmeister series. Curr. Opin. Chem. Biol. 10, 658−663. (20) Klopffleisch, K., Issinger, O.-G., and Niefind, K. (2012) Lowdensity crystal packing of human protein kinase CK2 catalytic subunit in complex with resorufin or other ligands: a tool to study the unique hinge-region plasticity of the enzyme without packing bias. Acta Crystallogr. D68, 883−892. (21) Traxler, P., and Furet, P. (1999) Strategies toward the design of novel and selective protein tyrosine kinase inhibitors. Pharmacol. Ther. 82, 195−206. (22) Battistutta, R., Mazzorana, M., Sarno, S., Kazimierczuk, Z., Zanotti, G., and Pinna, L. A. (2005) Inspecting the structure-activity relationship of protein kinase CK2 inhibitors derived from tetrabromobenzimidazole. Chem. Biol. 12, 1211−1219. (23) Raaf, J., Brunstein, E., Issinger, O.-G., and Niefind, K. (2008) The CK2α/CK2β interface of human protein kinase CK2 harbors a binding pocket for small molecules. Chem. Biol. 15, 111−117. (24) Niefind, K., and Issinger, O.-G. (2010) Conformational plasticity of the catalytic subunit of protein kinase CK2 and its consequences for regulation and drug design. Biochim. Biophys. Acta 1804, 484−924. (25) Gouron, A., Milet, A., and Jamet, H. (2014) Conformational flexibility of human casein kinase catalytic subunit explored by metadynamics. Biophys. J. 106, 1134−1141. (26) Taylor, S. S., and Kornev, A. P. (2011) Protein kinases: evolution of dynamic regulatory proteins. Trends Biochem. Sci. 36, 65− 77. (27) Grankowski, N., Boldyreff, B., and Issinger, O.-G. (1991) Isolation and characterization of recombinant human casein kinase II subunits α and β from bacteria. Eur. J. Biochem. 198, 25−30. (28) Matter, H., Nazaré, M., Güssregen, S., Will, D. W., Schreuder, H., Bauer, A., Urmann, M., Ritter, K., Wagner, M., and Wehner, V. (2009) Evidence for C-Cl/C-Br···π interactions as an important contribution to protein-ligand binding affinity. Angew. Chem., Int. Ed. Engl. 48, 2911−2916. (29) Raaf, J., Klopffleisch, K., Issinger, O.-G., and Niefind, K. (2008) The catalytic subunit of human protein kinase CK2 structurally deviates from its maize homologue in complex with the nucleotide competitive inhibitor emodin. J. Mol. Biol. 377, 1−8.
G
DOI: 10.1021/acschembio.5b00235 ACS Chem. Biol. XXXX, XXX, XXX−XXX
