5,6-diiodo-1H-benzotriazole: new TBBt analogue that minutely affects mitochondrial activity

4,5,6,7-Tetrabromo-1H-benzotriazole is widely used as the reference ATP-competitive inhibitor of protein kinase CK2. Herein, we study its new analogs: 5,6-diiodo- and 5,6-diiodo-4,7-dibromo-1H-benzotriazole. We used biophysical (MST, ITC) and biochemical (enzymatic assay) methods to describe the interactions of halogenated benzotriazoles with the catalytic subunit of human protein kinase CK2 (hCK2α). To trace the biological activity, we measured their cytotoxicity against four reference cancer cell lines and the effect on the mitochondrial inner membrane potential. The results obtained lead to the conclusion that iodinated compounds are an attractive alternative to brominated ones. One of them retains the cytotoxicity against selected cancer cell lines of the reference TBBt with a smaller side effect on mitochondrial activity. Both iodinated compounds are candidate leaders in the further development of CK2 inhibitors.

4,5,6,7-Tetrabromo-1H-benzotriazole is widely used as the reference ATP-competitive inhibitor of protein kinase CK2. Herein, we study its new analogs: 5,6-diiodo-and 5,6-diiodo-4,7-dibromo-1Hbenzotriazole. We used biophysical (MST, ITC) and biochemical (enzymatic assay) methods to describe the interactions of halogenated benzotriazoles with the catalytic subunit of human protein kinase CK2 (hCK2α). To trace the biological activity, we measured their cytotoxicity against four reference cancer cell lines and the effect on the mitochondrial inner membrane potential. The results obtained lead to the conclusion that iodinated compounds are an attractive alternative to brominated ones. One of them retains the cytotoxicity against selected cancer cell lines of the reference TBBt with a smaller side effect on mitochondrial activity. Both iodinated compounds are candidate leaders in the further development of CK2 inhibitors.
Protein kinase CK2 is highly pleiotropic and constitutively active serine/threonine kinase, which catalyzes the phosphorylation of numerous protein substrates, which are often related to gene expression or protein synthesis. CK2 is essential to cell viability, apoptosis, proliferation and survival, angiogenesis, DNA-damage repair, the ER-stress response, the regulation of carbohydrate metabolism and development of nervous system. Many CK2 substrates are essential for a pathogen life cycle 1,2 , so its activity is a subject of long term studies [3][4][5] . Moreover, kinase CK2 is directly related to cancer research, since its protein substrates have been implicated in several human cancers, including breast, lung, colon, prostate, as well as hematologic malignancies 6 . Human CK2 is the holoenzyme consisting of two catalytic subunits (α and/or α') and two regulatory ones (β). The activity of this enzyme is associated with the catalytic subunit, while the regulatory ones are responsible for substrate recognition and stabilization of the holoenzyme complex 7 . 4,5,6,7-Tetrabtomo-1H-benzotriazole (TBBt or BBBB) is widely used as the reference ATP-competitive selective inhibitor of protein kinase CK2 8 . It was originally developed as the analogue of 5,6-dichloro-1-(β-Dribofuranosyl)benzimidazole (DBR), which was found to inhibit mRNA, but not rRNA or tRNA, synthesis in eukaryotic cells 9,10 . TBBt and its analogue 4,5,6,7-tetrabromo-1H-benzimidazole (TBBz) were also starting points for synthesis of potent CK2 inhibitors, including compounds further substituted in five-membered ring (e.g. 4,5,6,7-tetrabromo-2-(dimethylamino)benzimidazole 11 ) as well as inhibitor-peptide conjugates [12][13][14][15] .
We have already identified 5,6-dibormo-1H-benzotriazole (HBBH) as a ligand strongly binding to hCK2α 16,17 . Its affinity to hCK2α estimated by Differential Scanning Fluorimetry was very close to that displayed by TBBt 16 . The binding thermodynamics studied with MST and ITC proved that HBBH binds to hCK2α minutely weaker than TBBt 17 . The difference in the dissociation constant for the strongly binding site is even much smaller than DSF data suggested. We have also shown that HBBH is much less hydrophobic and less acidic than TBBt 18 , which may make it less toxic. Herein, we study the properties of two iodinated compounds: 5,6-diiodo-1H-benzotriazole (HIIH) and 4,7-dibromo-5,6-diiodo-1H-benzotriazole (BIIB). Both can be obtained from the commercially available 4,5-diiodobenzene-1,2-diamine 19 under a single-step protocol, avoiding direct site-specific bromination of benzotriazole. Their binding affinities to hCK2α are comparable to that of TBBt, but HIIH displayed better physicochemical properties, being less hydrophobic, less acidic and much more soluble in aqueous medium 20 . Herein, we have used a wider set of physicochemical methods (Microscale Thermophoresis, MST, and Isothermal Titration Calorimetry, ITC) for the analysis of the thermodynamics of protein-ligand interactions. We have additionally determined the IC 50 values against hCK2α for HIIH and BIIB and two non-iodinated reference ligands (HBBH, TBBt). Since TBBt decreases the viability 21 and is often used as the reference kinase CK2 inhibitor 27 22 as the toxic compound that affects ATP/ADP ratio in cells 23 and impairs neuronal excitability by increasing action potential threshold and lowering firing frequency 24 . Since its cytotoxicity can be explained not only by the suppression of activation of apoptotic proteins, but also by the decrease of mitochondrial membrane potential 25 , we additionally monitored how our compounds affect the mitochondrial inner membrane potential, which may be further related to their toxicity, especially in context of cardiotoxicity 26 .
We have determined interactions between these four compounds and the catalytic subunit of human protein kinase CK2 using two different biophysical techniques: MST and ITC. MST, which enables direct estimation of binding affinity, K d , in a pseudo-titration mode has already been applied in detailed thermodynamic studies of the interactions between kinase hCK2α and its ligands 12,17 . The determined binding affinities are summarized in Table 1, and the pseudo-titration data are shown in Supplementary Figures S5-S8. For all ligands, contrary to the standard dose-response curve expected for a single binding site, a distinct "hill-like" shape was observed, indicating the presence of at least two different types of binding sites 17 . The first transition (small "up" effect reflecting binding in a solvent-protected pocket) corresponds to binding at the ATP-binding site, while the second (large  values, in response to tested compounds. a Literature data 18,20 . b Two independent binding sites were identified, the second one should be assigned to the solvent exposed region located at the interface between alpha and beta subunits of CK2 32 . c This value was constrained to the MST-derived K d1 . www.nature.com/scientificreports/ "down" effect of binding on the protein surface) should be assigned to the solvent exposed region located at the interface between alpha and beta subunits of CK2. Both sites have recently been identified in crystal structures of variously brominated benzotriazoles bound by hCK2α 32 . Tetrahalogeno derivatives bind to hCK2α stronger than their dihalogeno analogues. For diiodo derivatives these differences, according to Student's t-test, are statistically significant (K d1 = 15 ± 7 vs. 3 ± 6 nM, t(6,6) = 3.19, p = 0.01; K d2 = 374 ± 83 vs. 43 ± 22 nM, t(6,6) = 9.44, p < 0.001), while for bromo derivatives only affinity to weakly binding site differ significantly (K d1 = 29 ± 13 vs. 16 ± 18 nM, t(4,4) = 1.17, p = 0.29; K d2 = 327 ± 95 vs. 130 ± 88 nM, t(4,4) = 3.04, p = 0.02). Both diiodo derivatives bind to hCK2α stronger than their bromo counter partners (Fig. 2), however for the strongly binding site (affinity of which is close to the detection limit of our Monolith NT.115 hardware) these differences are not significant (t(4,6) = 2.24, p = 0.06 and t(4,6) = 1.68, p = 0.13 for di-and tetrahalogeno derivatives, respectively).
It is worth pointing that K d2 values determined for TBBt and HBBH moderately differ from those determined previously for the same system 17 . These discrepancies must be attributed to the different method of protein labelling with the fluorescent dye, of which that targeting lysine side-chains may compete with the ligand for a surfaceexposed hydrophobic weak binding site. Thus, the application of amine-reactive RED-NHS dye (MO-L011) that targets surface-exposed lysine residues may cause modification of the residue(s) in the vicinity of K44, K71 (Fig. S18), and the bulky dye may compete with the ligand for this site. K78, which is crucial for ligand binding at the ATP-binding site, is more shielded from the modification. No such perturbations are expected for the currently used His-Tag Labeling Kit RED-tris-NTA 2nd Generation, which modifies solely the peripheral region of hCK2α.
The binding of halogenated compounds was additionally followed with Isothermal Titration Calorimetry that enables determination of the stoichiometry, free energy and heat of ligand binding. We have successfully used this approach to study interactions of other ligands with hCK2α 12,17 . In line with MST data, two binding sites were identified for two the most hydrophobic tetrahalogenated compounds (TBBt and BIIB), which in MST experiments displayed the lowest K d2 values (i.e. the strongest binding). The model assuming a single binding site was found sufficient for the two remaining less hydrophobic derivatives, HBBH and HIIH. Inspection of the ITC-derived thermodynamic data, summarised in Table 1, shows for all but one ligand binging event the strong entropy-enthalpy compensation (slope 0.94 ± 0.04, Fig. 3). This common trend reaffirms our interpretation that the binding of halogenated benzotriazoles to the catalytic subunit of protein kinase CK2 is predominately driven by hydrophobic effect 16,18,20,30,31 . In this view, the excess heat of binding of BIIB at the ATP binding site (− 8 kJ/mol) may be assigned to the contribution of halogen bond(s), which are putatively formed between two iodine atoms of BIIB and two backbone carbonyl groups of residues located in the hinge region, resembling a virtual interaction that has been recently identified in the crystal structure of TBBt at hCK2α ATP binding site 32 .
We have additionally measured in vitro inhibition of hCK2α activity, IC 50 , by four studied compounds ( Table 1, Suplementary Figures S9-S12). According to Student's t-test no statistically significant differences between 5,6-diiodo-and 5,6,dibromo-derivatives were observed (t(5,5) = 1.52, p = 0.08 and t(4,5) = 1.59, p = 0.08 for di-and tertahalogeno benzotriazols, respectively), while 5,6-dihalogenated compounds differ significantly from their tetrahalogeno analogues (t(5,4) = 2.29, p = 0.03 and t(5,5) = 3.77, p = 0.003 for 5,6-dibromo-and 5,6-diiodo-benzotriazols, respectively). Overall, their inhibitory activity is moderately correlated with K d1 values, which results from the large bias of the determined K d1 values. However, IC 50 is sufficiently correlated with the literature ΔT m values 18,20 determined with Thermal Shift Assay (nanoDSF) (Fig. 4A), which states the efficient method for screening of protein-ligand interactions. Moreover, IC 50 values also correlate with calculated in silico LogP (Fig. 4B), pointing again that hydrophobic interactions drive binding of halogenated benzotriazoles. www.nature.com/scientificreports/ Since the biophysical methods confirmed that all tested ligands target hCK2α, we have further determined in vitro cytotoxic activity against four cancer cell lines: epidermoid carcinoma (A-431), hepatocellular carcinoma (HEPG2), and colorectal carcinoma (HCT116 and HCT116p53 -/-). Additionally, the reference anticancer drug, cispaltin, was used as the positive control. Despite the fact that the obtained results were significantly biased due to a very limited aqueous solubility of the tested compounds, we were able to analyse the low-concentration part of the dose-response curves. Effect of aggregation of halogenated benzotriazole derivatives, which biased thermodynamic measurements at micromolar concentration of the ligand has been already described 17 . Aggregation of the studied compounds after dissolution in aqueous medium was monitored with DLS (Supplementary Figure S17). The shape of the autocorrelation curve is indicative for the existence of nano-aggregates. In all cases the shoulder at 100-600 µs is indicative of particles of the radius of 50-100 nm, however the similar ACF distribution was observed at different solute concentration: the highest for HBBH (160 µM), and the lowest for the most hydrophobic BIIB (40 µM). It should be pointed that aggregation makes ligand unavailable for cells, so further addition did not affected the viability, since the concentration of dissolved monomeric compound remains saturated. This effect was the most significant in case of the less soluble BIIB, however for any of tested compounds a complete dose-response effect undistorted in the high-concentration region could not be recorded. To overcome this limitation we have used the simplified approach, in which IC 50 values were fitted globally for the restricted set of low-concentration data obtained in three independent experiments, while up and down asymptotes were constrained to OD values measured in each experiment for untreated cell culture and for pure   Table 1.
While the brominated compounds (HBBH and TBBt) similarly reduce cell viability, the iodinated ones display higher cytotoxicity. This effect is correlated with their higher activity in the enzymatic assay, however for all tested cell lines HIIH reduces the viability more efficiently than TBBt, and BIIB reduces viability of all cell lines even more, with IC 50 values estimated close to the values obtained for cisplatin, the reference anticancer drug. It is worth to mention, that the extremely low solubility of this compound preclude its application as a potential anticancer drug. Deletion of p53 in HCT116 did not significantly affected cell viability, so mechanism of inhibition of these compounds is probably not correlated with this protein.
To evaluate a contribution to the toxicity of the tested compounds not directly connected with kinase inhibition, we have monitored how they decrease the mitochondrial inner membrane (IM) potential (ΔΨ). This was monitored by fluorescence quenching of Rhodamine 123 in mitochondria isolated from the yeast Saccharomyces cerevisiae. In response to variations in ΔΨ this dye accumulates proportionally inside the mitochondrial matrix, where its fluorescence is quenched, thus fluorescence changes reflect the changes in the IM potential 33,34 . The traces shown in the Fig. 7 demonstrate how the fluorescence of Rhodamine 123 varied upon the following   To quantify the effect of ligand addition we have compared the ligand concentrations required to recover 50% of the initial fluorescence drop (dotted line), i.e. the average of value after addition of EtOH and fully decoupled ΔΨ. In the case of tetrahalohenated benzotriazoles it is less than 2 µM, while for the dihalogenated ones is much higher (> 5 µM). So, the predominately neutral HBBH and HIIH are at least twice less effective in decoupling ΔΨ than the anionic BIIB and TBBt.

Conclusions
Obtained results clearly indicate that HIIH can be successfully used as the leading compound for CK2 inhibitors, stating an attractive alternative for TBBt. Its interaction with hCK2α is similar to that of TBBt. Only slight differences in protein-ligand interaction may be caused by different physicochemical properties of HIIH. This includes the lower hydrophobicity, which is additional advantage of this compound. Moreover, synthesis of HIIH is much simpler than synthesis of TBBt based on bromination of benzotriazole. We showed that HIIH much more decreases viability of cancer cell lines than TBBt (p < 0.012). Moreover, its higher activity is not correlated with decoupling of mitochondrial inner membrane potential, since it decouples ΔΨ much less than TBBt does. So, its activity should be assigned directly to protein inhibition. Moreover, weaker impact on mitochondria makes HBBH less toxic than TBBt, the toxicity of which was associated with decrease in cellular ATP 25 . Even though we have identified BIIB (4,7-dibromo-5,6-diiodo-1H-benzotriazole) as much stronger CK2 inhibitor, which also very efficiently decreases cancer cells viability, and decouples ΔΨ not more than TBBt does. However, its synthesis is more demanding, basing on bromination of HIIH. BIIB is also more hydrophobic and less soluble than HIIH and TBBt, what makes it less useful as a leading compound for CK2 inhibitors. In general, the replacement of bromine atom with iodine (at least in the halogenated benzotriazoles) increases cytotoxic activity much more than it could be expected from the inhibitory activity determined in the enzymatic assay. This points the synergistic effect of the iodine atom which improves both the inhibitory activity of the compound and its transfer through the cell membrane.

Methods
Chemistry. All starting materials and solvents for reactions were purchased from Sigma Aldrich, Fluorochem, ABCR or Chempur. All compounds were synthesized according to protocols previously described by our team 20,30 . Structure of compounds was confirmed using 1 H NMR spectroscopy and Mass spectrometry. 1 H NMR spectra were recorded with Varian 500 MHz spectrometer. Either TMS or the residual solvent signal were used as the internal standard. High resolution mass spectra (HR:MS) were recorded on an TQ OrbitrapVelos instrument (Thermo Scientific). Purity of compounds was confirmed by HPLC, see Supporting Information for details.
Protein sample preparation. The catalytic subunit of human protein kinase His6-tagged at the C-termini CK2 was expressed and purified according to the method described previously 16 . Plasmid pET28 containing DNA encoding human CK2α was transformed into BL21(DE3) Escherichia coli cells. A single colony was inoculated in 30 ml LB broth supplemented with kanamycin (50 μg/ml) and incubated overnight at 37 °C. The culture (5 ml) was used to inoculate 1000 ml of Luria Broth with kanamycin (50 μg/ml) and cells were cultured at 37 °C until OD 600 reached 0.5, when hCK2α expression was induced by 0.5 mM isopropylthio-β-galactoside. Cells were then incubated at 37 °C for 4 h, harvested by centrifugation at 6000 rpm for 10 min at 4 °C, and stored at -20 °C. Thawed cells were suspended in 30 ml of buffer A (25 mM Tris-HCl pH 8, 500 mM NaCl, 10 mM imidazole, 5 mM β-mercaptoethanol) with 0.2 mM PMSF (phenylmethylsulfonyl fluoride) and protease inhibitor cocktail (Sigma-Aldrich P8849), and sonicated by five 1-min bursts. The cell lysate was centrifuged at 20,000 rpm for 40 min, and the supernatant fraction processed by Ni 2+ -NTA agarose chromatography under non-denaturing conditions. The protein was then purified by affinity chromatography with a HiTrap Heparin HP column (GE HealthCare), and eluted with a linear 0.4 to 1 M NaCl gradient buffered with 25 mM Tris-HCl, pH 8.5. Purified (His)6-tagged hCK2α was concentrated by ultrafiltration, rebuffered in 500 mM NaCl, 50% glycerol, 1 mM DTT, 25 mM Tris-HCl, pH 8.5, and stored at − 80 °C. The final yield of purified protein was 20 mg/l. Concentration of the protein was estimated by UV absorbance at 280 nm, assuming molar extinction coefficient ε = 61,895 M −1 cm −1 .

Microscale thermophoresis (MST).
Samples of protein (50 nM in 25 mM Hepes buffer pH 8 with 0.5 M NaCl and 1%DMSO content v/v) labeled with Monolith His-Tag Labeling Kit RED-tris-NTA kit (NanoTemper Technologies) were prepared with varying ligand concentration according to previously described protocol 16 . The measurements were performed using Monolith NT.115 (NanoTemper Technologies) apparatus in standard capillaries (Nanotemper Technologies). For each ligand six independent pseudo-titration experiments, each with 16 ligand dilutions, were performed. The thermophoretic effect was then analyzed globally assuming the common values of binding affinities for two independent sites (K d1 , K d2 ), while the asymptotic values for the apo form of the protein and 1:1 and 1:2 complexes with the ligand were estimated individually for each experiment. The model of two independent binding sites, implemented in origin, has been already described and tested 17 . It is worth noting that the used method of data treatment goes beyond the standard MST software capabilities. Cell viability assay. The viability of cells was assayed by measuring the conversion of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to the formazan (the rate of this reaction is proportional to the number of surviving cells). Cells were seeded in a 96-well plate at a density of 2500-3500 cells per well, 24 h before treatment. Treatment with measured compounds and cisplatin (2.5-160 µM) was performed for 72 h. MTT stock solution (Sigma-Aldrich) was added at the final concentration 0.5 mg/mL. After 4 h of incubation at 37 °C, water-insoluble formazan was dissolved in a lysis buffer containing 20% SDS, 50% DMF, 2.5% hydrochloric acid and 2.5% acetic acid. Optical densities were measured at 570 nm using a scanning multi-well spectrophotometer (PARADIGM Detection Platform; Beckman Coulter, Brea, CA). For each ligand the standard doseresponse curve was fitted to three independent experiments, however the concentration range was restricted to overcome the effect of uncontrolled aggregation. HBBH and HIIH were analysed in the concentration range of 1-10 µM, while TBBt and BIIB in the range of 1-5 and 1-2.5 µM, respectively. We have applied the simplified approach to estimate inhibitory activity: top and bottom asymptotes were constrained to the individual OD values measured for untreated cells and pure buffer, respectively, while the common IC 50 value was for each compound optimized globally for three independent experiments.