Acetoxymethyl Ester of Tetrabromobenzimidazole ... - ACS Publications

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Acetoxymethyl Ester of Tetrabromobenzimidazole−Peptoid Conjugate for Inhibition of Protein Kinase CK2 in Living Cells Kaido Viht,† Siiri Saaver,† Jürgen Vahter,† Erki Enkvist,† Darja Lavogina,† Hedi Sinijar̈ v,† Gerda Raidaru,† Barbara Guerra,‡ Olaf-Georg Issinger,‡,§ and Asko Uri*,† †

Institute of Chemistry, University of Tartu, Ravila 14A, 50411 Tartu, Estonia Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark § KinaseDetect Aps, Skovvej 22, 6340 Kruså, Denmark ‡

S Supporting Information *

ABSTRACT: CK2 is a ubiquitous serine/threonine protein kinase, which has the potential to catalyze the generation of a large proportion of the human phosphoproteome. Due to its role in numerous cellular functions and general anti-apoptotic activity, CK2 is an important target of research with therapeutic potential. This emphasizes the need for cell-permeable highly potent and selective inhibitors and photoluminescence probes of CK2 for investigating the protein phosphorylation networks in living cells. Previously, we had developed bisubstrate inhibitors for CK2 (CK2-targeted ARCs) that showed remarkable affinity (KD < 1 nM) and selectivity, but lacked proteolytic stability and plasma membrane permeability. In this report, the structures of CK2targeted ARCs were modified for the application in live cells. Based on structure−activity studies, proteolytically stable achiral oligoanionic peptoid conjugates of 4,5,6,7-tetrabromo-1H-benzimidazole (TBBz) were constructed. Affinity of the conjugates toward CK2 reached subnanomolar range. Acetoxymethyl (AM) prodrug strategy was applied for loading TBBz−peptoid conjugates into living cells. The uptake of inhibitors was visualized by live cell imaging and the reduction of the phosphorylation levels of two CK2-related phosphosites, Cdc37 pSer13 and NFκB pSer529, was demonstrated by Western blot analysis.

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INTRODUCTION Sequence analysis of the phosphoproteome suggests that the contribution of individual protein kinases (PKs) to protein phosphorylation is extremely variable.1 The formation of the majority of phosphosites is catalyzed by a small number of pleiotropic PKs. Among these, ubiquitous serine/threonine PK CK2 (the acronym being derived from the misnomer “casein kinase 2”) has the potential to generate a substantial proportion of the human phosphoproteome.2 Although a great body of knowledge has been accumulated about CK2 since its discovery in 1954, the physiological role of this PK has not been completely understood and its regulation mechanisms have remained obscure. In cells, CK2 is predominantly present in the form of the holoenzyme, a heterotetramer composed of two catalytic subunits (isoforms α and/or α′), joined by a dimer of noncatalytic regulatory (β) subunits,3 although independent roles of these subunits have also been suggested.4 CK2 has generally been considered a constitutively active enzyme with both the free catalytic subunit and the holoenzyme possessing catalytic activity that neither relies on the phosphorylation status of CK2 nor on the binding of second messengers.5 However, various mechanisms for the regulation/modulation of its activity have been proposed.6 For example, it has been established that CK2β affects substrate selectivity and stabilizes © XXXX American Chemical Society

the active form of CK2 and it has been proposed that aggregation of the holoenzyme controls the activity of CK2.7−9 The unbalanced expression of the subunits in tumor cells and the general pro-survival function makes CK2 a target for cancer therapy.10 Highly selective ATP-competitive inhibitors of CK2 have been developed based on the specific structure of the nucleotide-binding site of the catalytic subunit.11 However, many ATP-competitive inhibitors of CK2 tend to inhibit a number of other PKs with similar or better potency.12 For example, an orally administered ATP-competitive inhibitor of CK2, CX-4945 (Silmitasertib), that is in clinical trials in combination with other chemotherapeutics,13 has recently been identified as a potent inhibitor of CLKs (Cdc2-like kinases).14 Furthermore, proteomic studies with inhibitors of CK2 have revealed interactions with several nucleotide binding proteins outside the PK superfamily.15 Although absolute specificity is not always mandatory for successful drug development,16 highly selective cell-permeable inhibitors are invaluable tools for investigating the involvement of protein phosphorylation networks in the regulation of cellular processes.17 Received: July 10, 2015 Revised: October 30, 2015

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DOI: 10.1021/acs.bioconjchem.5b00383 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry In general, the intracellular efficiency of cell-permeable inhibitors of PKs may suffer from several factors, such as competition from the high intracellular concentration of ATP, fast extrusion from the cell, lack of specificity, and so forth. In addition, the phosphosites used as reporters of the activity of the particular PK respond differently to pharmacological inhibition, due to the variable phosphorylation stoichiometry and turnover rate of these sites.18 On one hand, these factors result in EC50-values that are orders of magnitude higher than the dissociation or inhibition constants (K D and K i , respectively) established with purified enzymes,19 which means that very potent inhibitors exert an effect at a relatively high concentration range where off-target activity is more likely to occur. On the other hand, inhibitors targeting the same PK may display unique biological properties, due to noncoinciding selectivity profiles. For example, the inhibitor-specific cellular activity of widely used inhibitors of CK2, 4,5,6,7-tetrabromo1H-benzimidazole (TBBz, also termed TBBi or TBI),20 4,5,6,7tetrabromobenzotriazole (TBB), and 2-dimethylamino-4,5,6,7tetrabromo-1H-benzimidazole (DMAT) was explained by the involvement of off-target effects.21 The variable responsiveness of phosphosites on the inhibition of CK2 can be illustrated by the faster response of Akt pSer129 following treatment with CX-4945, in comparison to another target site of CK2, pSer13 of cochaperon protein Cdc37 (cell division cycle 37),22 whose phosphorylation level was significantly reduced after longer incubation time and higher concentration of the same inhibitor.23 This was explained by the slower dephosphorylation rate of pSer13 of Cdc37.23,24 However, fast response of Cdc37 pSer13 on the incubation of cells with other potent inhibitors of CK2 has been reported.25 As expected from its anti-apoptotic role, pharmacological inhibition of CK2 negatively affects cell viability, but the magnitude and mechanism of this effect are variable among different inhibitors and cell lines.26−28 Following these considerations, inhibitors with variable physicochemical characteristics are needed. CK2 is an acidophilic PK, i.e., it preferably phosphorylates substrates with clusters of negatively charged amino acid residues flanking the phosphorylatable serine/threonine residue.29 Therefore, CK2 is inhibited in a protein substrate competitive manner by polyanions, such as heparin,30,31 negatively charged peptides,32−36 and nucleic acids.37−39 The inhibitory potency of these compounds is enhanced with increased number of negatively charged residues, reaching lownanomolar affinity with higher molecular weight homologues.31,39 Recently, we showed that the potency and selectivity of CK2 inhibition can be remarkably increased by the bisubstrate inhibitor approach (reviewed40), i.e., construction of conjugates that simultaneously occupy the binding sites of both the phosphodonor nucleotide and phosphoacceptor protein substrate of the PK. Specifically, the bisubstrate inhibitors designed by us (termed ARCs) targeted to CK2, were constructed by conjugation of ATP-competitive inhibitors, such as TBBz or benzoselenadiazole, with oligo-L-aspartate peptides that mimic the protein substrate recognition (consensus) sequence of CK2.41,42 These fragments were joined via a hydrophobic linker derived from octanoic acid (Oca). Labeling of these inhibitors with fluorescent dyes yielded probes (such as ARC-1504, structure A in Scheme 1) for the determination of CK2 and characterization of its inhibitors.41−43 More recently, bisubstrate inhibitors with similar structures comprising TBBz moiety and oligoanionic peptide were characterized by Cozza et al.44 As a common

Scheme 1. Design Strategy of Cell-Permeable Acetoxymethyl (AM) Esters of TBBz−oligocarboxylic Acid Conjugates, Starting from Previously Reported41 Inhibitors ARC-1502 (part A, R = H) and ARC-1504 (part A, R = PromoFluor647)a

a

The peptide chain was substituted with more stable peptoid counterpart resulting in oligocarboxylic TBBz−peptoid conjugates (B) that were converted into per-AM-esters (C) for delivery into cell. The general structure of the active scaffold of the inhibitor consists of TBBz (yellow), targeted to the ATP-binding pocket of CK2, and oligoanionic tail (red), mimicking the consensus sequence of CK2. These parts are connected via a linker (blue). Amino functional structure (green) could be introduced to the C-terminus of the conjugates for labeling (R).

feature of this type of compound, the attachment of negatively charged peptide to ATP-competitive inhibitor TBBz remarkably (several orders of magnitude) increased the potency as well as selectivity of inhibition, as CK2 was the most inhibited PK in a panel of 140 PKs.41,44 As another feature, the introduction of negatively charged peptide to the structure rendered these inhibitors cell membrane impermeable that had an advantage when targeting ecto-CK2.44 However, the intracellular application of inhibitors comprising peptide chain built from L-amino acids is compromised by their limited metabolic stability and poor plasma membrane permeability. In the present work, a technology is proposed for the construction of cell membrane permeable bisubstrate inhibitors of CK2. The technology is based on the modification of the oligocarboxylic acid part of the inhibitor for increasing its stability in biofluids and for enabling its loading into cell in the form of acetoxymethyl (AM) ester prodrug (Scheme 1). The latter strategy (“ester loading technique”) had previously been used for increasing the bioavailability of various anionic compounds, such as metal ion sensors and second messengers.45−47 Once exposed to the intracellular esterase activity, AM esters are hydrolyzed and the negatively charged compound is trapped inside the cell. Here, a structure−activity relationship is presented for TBBz−oligocarboxylic acid B


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Bioconjugate Chemistry conjugates. As a proof of principle, one active scaffold with optimized structure was selected and its improved uptake in the form of per-AM ester was demonstrated by live cell imaging and by the intracellular inhibition of CK2-mediated phosphorylation of Cdc37 and nuclear factor kappa B (NFκB). In addition, the effect of the novel prodrug of bisubstrate inhibitor of CK2 on cell viability was tested in human cervical cancer (HeLa) and human pancreatic carcinoma (MIA PaCa-2) cell lines.

Chart 1. Structures of Inhibitors Introduced in This Study

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RESULTS AND DISCUSSION Development of per-AM Ester of TBBz-Oligocarboxylic Acid Conjugate for Live Cell Studies. The general outline of the design of cell-permeable per-AM ester of TBBzoligocarboxylic acid conjugate is presented in Scheme 1. The development was started from the previously described compound ARC-1502 (structure A in Scheme 1, KD = 0.5 nM of its complex with CK2α) as the lead structure. ARC-1502 comprised the peptide sequence D-Asp-[L-Asp]5 that incorporated altogether 7 negatively charged carboxylate groups at physiological pH. According to our previous experience with the construction of oligo-arginine conjugates of adenosine analogues (bisubstrate inhibitors of basophilic PKs48), the inhibitory potency increased with the number of arginine residues in the peptide moiety, and peptides composed of Darginine residues were superior over their all-L-counterparts for achieving high-affinity binding with the PKs. It was now important to establish whether similar trends for structure− affinity relationships held for the acidophilic CK2. Specifically, we aimed to find out whether the oligoaspartate sequence could be shortened to reduce the total negative charge and structural complexity of the conjugate, and to establish whether D-aspartic acid residues could be substituted for L-aspartic acid residues to increase resistance of the conjugate to proteolytic degradation. TBBz was kept in the role of the fragment targeting the ATPbinding pocket of CK2 to enable comparison with the previously reported inhibitors.41 Additionally, the presence of a common TBBz chromophore in the structures made the concentrations of the stock solutions directly comparable by the absorbance maximum in the spectrum of TBBz at 272 nm. Four bromine atoms of TBBz led to a characteristic MS peak fingerprint that facilitated the interpretation of HPLC-MS data. TBBz, and its derivatives incorporating from one to six carboxylate groups, including a well characterized inhibitor (4,5,6,7-tetrabromo-1H-benzimidazol-1-yl)acetic acid (K68),49 8-(4,5,6,7-tetrabromo-1H-benzimidazol-1-yl)octanoic acid (TBBz-Oca),41 and conjugates of the latter compound with peptides comprising either 1−5 L- or 1−5 D-aspartic acid residues (Chart 1A), were synthesized and characterized by their binding ability to CK2α in the previously reported binding/displacement assay with fluorescence anisotropy (FA) readout.41 Surprisingly, the affinities of enantiomeric conjugates were practically identical, indicating that CK2α did not reveal a preference for chirality of amino acid residues forming the peptide part of this type of compound. In line with earlier studies with polyanionic inhibitors of CK2, the affinity of the conjugates gradually increased with the number of incorporated anionic residues (Table 1). An exception in this series was K68 that, in accordance with previous studies,49 showed a higher KD value (1500 nM) than TBBz (640 nM). The attachment of a single carboxylate group via a longer hydrophobic linker may afford more favorable positioning of the inhibitor in complex with CK2α, as shown by 6-fold higher affinity of TBBz-Oca

Table 1. Dissociation Constants of the Complexes of the Compounds and CK2α, as Determined in the Binding/ Displacement Assay with FA Readouta compound TBBz20 TBBz-[CH2]n-COOH TBBz-Oca-[L-Asp]n

TBBz-Oca-[D-Asp]n

TBBz-Oca-[Nasp]n

TBBz-Oca-[Nasp]n-NH2

TBBz-Oca-[Nasp]n-Nlys

n

code or abbreviation

1 7 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 3 6 2 3 4 5 6

K6849 TBBz-Oca41 ARC-1506 ARC-1507 ARC-1508 ARC-1518 ARC-1519 ARC-1846 ARC-1847 ARC-1848 ARC-1818 ARC-1819 ARC-1840 ARC-1841 ARC-1842 ARC-1843 ARC-1844 ARC-1814 ARC-1816 ARC-1817 ARC-1831 ARC-1832 ARC-1833 ARC-1834 ARC-1835 ARC-1836

TBBz-Oca-[Nasp]3Nlys(BODIPY FL) TBBz-Oca-[Nasp]3-Nlys(ATTO 647N)

ARC-1849

KD (SD) [nM] 640 1500 250 45 25 7.2 1.9 1.2 63 21 5.6 1.8 0.7 92 30 8.7 4.2 1.7 160 20 1.5 110 34 10 3.4 1.5 3.0

(120) (300) (100) (5) (7) (2.5) (0.6) (0.1) (5) (4) (0.1) (0.5) (0.4) (1) (4) (0.7) (0.9) (0.3) (50) (3) (0.1) (10) (2) (3) (0.7) (0.5) (0.7)

41 (4)

The reported values represent the means ± SD of at least two parallel experiments. a

C


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Bioconjugate Chemistry

in acidic conditions, a complication experienced during RPHPLC purification of TBBz−peptide conjugates. The affinity of TBBz−peptoid conjugates toward CK2α followed the same trend as their peptide counterparts, with smaller KD-values obtained for homologues with longer oligocarboxylic acid chain (Table 1). If compared to the peptide counterparts with the same number of carboxyl groups, a slight (less than two times in average) decrease of affinity of TBBz−peptoid conjugates was observed, showing that the peptoid could be efficiently used as the protein substrate mimicking fragment in the design of bisubstrate inhibitors for CK2. Amidation of one carboxyl group at the C-terminal residue of the conjugates decreased the affinity almost by the same extent as shortening the peptoid chain by one Nasp residue; a similar effect was observed when a positively charged ammonium group was introduced to the C-terminus of the conjugate. The affinity of the monoanionic peptoid conjugate ARC-1814 was close to the affinity of TBBz-Oca. Taken together, with all TBBz derivatives introduced in this report, the affinity gain was in correlation with the charge of the oligocarboxylic acid moiety. Given that at physiological pH, TBBz (pKa ≈ 9) is predominantly in the neutral form,26 carboxyl groups are all deprotonated,57 and the Nlys residue bears an ammonium ion, the relationship between log KD and the number of carboxyl groups in the structure was linear (r2 > 0.94) between 0 to −6 net charge of the compounds (Figure 1). In this range, all carboxyl groups, irrespective of their chiral

(KD = 250 nM) if compared with K68; however, decreased aqueous solubility induced by a longer hydrophobic linker should be taken into account. The affinity of TBBz− oligoaspartate conjugates reached a one-digit nanomolar KD value already upon conjugation with the triaspartate peptide, and the tight-binding affinity range was reached with compounds that incorporated 4 and more aspartate residues. The conjugates comprising longer oligomers of aspartic acid residues showed affinities close to that of ARC-1502, leading to nearly 3 orders of magnitude smaller KD values than observed for the parent compound TBBz. These results revealed that inhibitors with high affinity could also be achieved by the attachment of a shorter oligoaspartate moiety to TBBz than that in the structure of ARC-1502, and that biologically more stable all-D-peptide could be substituted for the oligo-Laspartate moiety. Although it possesses increased proteolytic stability in biofluids, TBBz-oligo-D-aspartate conjugate was not yet a suitable scaffold for the AM ester-based prodrug internalization technology, due to the instability of the esterified form of aspartate-comprising peptides. Namely, as a well-known sidereaction in peptide synthesis, aspartic acid residues can rearrange when treated with acids and bases, especially in the esterified form.50 This reaction involves an attack of the amide nitrogen of the adjacent residue on the carbonyl group of the side chain of the aspartic acid, resulting in the formation of a cyclic aspartimide intermediate that upon hydrolysis yields a mixture of α- and β-peptides, accompanied by partial racemization. In peptide synthesis, this rearrangement is suppressed by optimized synthesis protocols, utilization of sterically hindered side-chain protecting groups, or blocking of specific amide nitrogens.50,51 Acetoxymethylation of aspartic acid residues using well-optimized procedures has been reported;52 however, in the present study an attempt to alkylate TBBz−triaspartate conjugate with bromomethyl acetate in the presence of an amine base was unsuccessful as only the corresponding aspartimide was formed (data not shown). Therefore, considering the established structure− activity relationships for TBBz−oligoaspartate conjugates, it was hypothesized that the inhibitor could tolerate structurally more drastic substitution of the peptide part with isomeric oligomers of N-substituted glycine (peptoids) (reviewed53). Although with altered steric constraints of the backbone, TBBz−peptoid conjugates would lack chiral centers and amide NH-bonds, thus simultaneously eliminating the risk of aspartimide formation and racemization that could otherwise compromise the structural integrity of the compounds. Also, the peptoid backbone should well meet the biological stability criterion.54 To test this hypothesis, a series of conjugates was assembled where the peptide fragment was replaced with the corresponding peptoid mimicking the oligoaspartate (Chart 1B). The peptoid backbone of the conjugates was built up according to the solid-phase submonomer strategy.55 The Cterminal peptoid carboxylic acids were prepared on 2chlorotrityl chloride (2CTC) resin.56 A selection of TBBz− peptoid conjugates was also synthesized in the C-terminally amidated form (Chart 1B) to see whether the structurally equivalent carboxylate groups connected to the C-terminal peptoid monomer have an equal contribution to the affinity of the conjugates. In addition, conjugates comprising a C-terminal peptoid analogue of lysine (Nlys) were made for the subsequent labeling of the conjugate (Chart 1C). The peptoid conjugates had lower propensity for crystallization and gelation

Figure 1. Effect of the number of carboxyl groups (N) on the dissociation constants of the complexes between CK2α and the following compounds: TBBz (blue ●), K68 (green +), TBBz-Oca (brown ○), TBBz-Oca-[L-Asp]n (red ■), TBBz-Oca-[D-Asp]n (green ▲),TBBz-Oca-[Nasp]n (purple ▼), TBBz-Oca-[Nasp]n-NH2 (orange ◊), and TBBz-Oca-[Nasp]n-Nlys (black ×). The reported values represent the means ± SD of at least two parallel experiments.

positioning in the case of the peptide chain, possessed an equal contribution to the binding energy of −2.5 ± 0.2 kJ/mol, as calculated from about 3-fold different KD values between the consecutive homologues of the conjugates. Possibly, the compounds are flexible enough to adopt conformations that allow the carboxylate groups to find interaction partners among the numerous positively charged amino acid residues on the protein substrate-binding site of the catalytic subunit of CK2α, forming an electrostatic “zipper” between the peptide part of the conjugate and PK. The flexibility of the peptide chain of TBBz−oligoaspartate conjugate is also exemplified by previously reported cocrystal structure of CK2α with bisubstrate inhibitor ARC-1154, where no electron density could be resolved for the peptide moiety.41 Decrease of affinity upon introduction of the C-terminal ammonium group can be explained by the formation of an intramolecular salt bridge that D


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Bioconjugate Chemistry competes with PK for interaction with the carboxylate group, and it is in agreement with the widely accepted knowledge that cationic residues are negative determinants of the substrate recognition by CK2.29 Indeed, the reduction of affinity by Cterminal lysine residue in the peptide part of CK2-directed ARC-inhibitor had previously been reported.42 Altogether, as a practical consequence, the variation of the length of the oligocarboxylic acid moiety offers the means to tune the affinity of the bisubstrate inhibitors of CK2. In addition to the established structure−affinity relationship, it was important to ensure that the novel oligocarboxylic TBBz−peptoid conjugates indeed inhibit CK2-catalyzed phosphorylation as the binding affinity of inhibitors of PKs is not always strongly correlated to their inhibitory potency.58 An in vitro kinetic assay was conducted that was based on ratiometric quantification of the phosphorylated fluorescently labeled synthetic peptide substrate (analogue of CK2tide) by TLC analysis.41 An excellent correlation was found between the observed inhibition IC50 and dissociation KD values (r2 = 0.94, log scale) for the peptoid series of the conjugates and for the lower molecular weight derivatives of TBBz used in this study (Figure 2).

Table 2. Inhibition of Recombinant CK2α and CK2α′ and the Corresponding Reconstituted Holoenzymes by ARC1842a kinase CK2α CK2α′ CK2α2β2 CK2α′2β2

IC50 (SD) [nM] 47 67 46 40

(28) (34) (13) (8)

a Measured at 100 μM ATP and 30 μM 5-TAMRA-RADDSDDDDD. The reported values represent the means ± SD of at least two parallel experiments.

synthase kinase 3 beta) that showed about 8-fold higher residual activity. If compared to TBBz, ARC-1842 exhibited much narrower selectivity, with stronger preference for acidophilic PKs. Thirteen PKs were inhibited over 50% by 10 μM ARC-1842, whereas five of those had residual activity below 10% that roughly corresponds to submicromolar IC50 value. In comparison, more than half of the set of PKs were inhibited over 50% in the presence of the same concentration of TBBz.59 The selectivity data for ARC-1842 was similar to that of ARC-1502,41 with a few rearrangements in the rank of inhibition potency; e.g., ARC-1842 showed higher preference for TAK1 than ARC-1502. Altogether, the data confirmed that substitution of the peptide part of the bisubstrate inhibitor of CK2 with peptoid chain does not drastically alter the selectivity of inhibition. For live cell imaging, two fluorescence-labeled derivatives based on the scaffold of ARC-1842 were constructed: ARC1836, labeled with green dye BODIPY FL, and ARC-1849, labeled with red dye ATTO 647N (Chart 1C). The dyes were attached to the amino group in the structure of ARC-1832 which differed from ARC-1842 by an additional Nlys residue in the C-terminus. The structures of the chosen fluorescent dyes withstand the conditions of acetoxymethylation and possess no negative charges (e.g., sulfonates) that could compromise with the cell penetration characteristics of the conjugate. For cellular uptake, the conjugates were converted into tetra-AM esters, ARC-1859 (Chart 1B), ARC-1837, and ARC-1850 (Chart 1C). The attachment of the BODIPY FL dye to Nlys residue introduced to the C-terminus of the conjugate was well tolerated as even higher affinity for the labeled ARC-1836 was observed if compared to ARC-1842 (Table 1). This could be explained by additional hydrophobic interactions favoring the complexation of the conjugate with CK2. Labeling with BODIPY FL also eliminates the positive charge of the Cterminal Nlys residue of ARC-1832 that explains the 10-fold affinity difference in favor of ARC-1836. Possibly due to steric bulk and positive charge of the dye, labeling with ATTO 647N was less tolerated as shown by increased KD value for ARC1849 (Table 1). Saturating acetoxymethylation of carboxyl groups completely abolished the positive effect of the peptoid chain on the affinity of the conjugate as the inhibitory potency of tetra-AM ester ARC-1859 was practically equal to that of TBBz (Supporting Information Table S1). These measurements were performed in the presence of Pluronic F-127, a commonly used nonionic surfactant for solubilizing hydrophobic AM esters of calcium indicators,60 which slightly decreased the inhibitory potency of TBBz. On the other hand, equal affinities of TBBz and ARC-1859 point to the nitrogen of the imidazole ring of TBBz as a suitable position for structural modification such as introduction of labels or

Figure 2. Correlation between dissociation constants KD and inhibition IC50 values of derivatives of TBBz, determined by FAbased displacement assay and phosphorylation assay, respectively. The reported values represent the means ± SD from at least two parallel experiments. The KD values were taken from Table 1 and the IC50 values are given in Supporting Information Table S1.

A middle conjugate of the series ARC-1842 as a compromise between affinity (KD < 10 nM) and structural complexity (four carboxyl groups, Mr < 1 kDa) was chosen for further characterization and for subsequent demonstration of the applicability of the AM prodrug strategy for loading oligocarboxylic TBBz−peptoid conjugate into cells. The competitiveness with respect to ATP was tested by kinetic analysis that demonstrated competitive inhibition mode for ARC-1842 (Supporting Information Figure S1). A Ki value of 10 ± 3 nM was derived from the data of the double reciprocal plot, which matched the KD value of ARC-1842 (Table 1). The selectivity of ARC-1842 was assessed with different forms of CK2 and in a panel of 50 PKs across the kinome. ARC-1842 did not significantly discriminate between the isoforms (α and α′) of the catalytic subunit of CK2 or between the free catalytic subunits and the corresponding holoenzymes (α2β2 and α′2β2) (Table 2). The selectivity profiling against 50 PKs (Supporting Information Table S2) revealed the same tendencies as observed with other TBBzbased bisubstrate inhibitors of CK2 published to date. In the presence of 10 μM ARC-1842, CK2 was the most inhibited PK (0.6% residual activity), followed by TAK1 (transforming growth factor β activated kinase-1) and GSK3β (glycogen E


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Figure 3. Uptake of ARC-1836 and ARC-1837 by HeLa cells, incubated for 1 and 2 h with 1 μM and 10 μM compounds, as indicated. Due to weaker fluorescence signal, the intensity of the light source for ARC-1836 was 2.5 times higher. The images are pseuducolored.

Figure 4. Western blot analysis of whole lysates from MIA PaCa-2 cells. Control experiment refers to cells treated with vehicle (0.1% DMSO v/v). βActin detection was used as a control for equal loading. Values beneath protein bands refer to the densitometric analysis of band signals expressed in percentage. (A) Cells were incubated with ARC-1859 (5 μM or 10 μM, 1 h) and harvested after 5 or 24 h. (B) Cells were incubated with ARC-1842, ARC-1859, and CX-4945, all at the final concentration of 10 μM, for 5 h.

groups) had poorly entered the cells in the same time scale and possessed a strong endosomal staining pattern (Figure 3). The ATTO 647N-labeled compounds demonstrated the same tendencies (Supporting Information Figure S3). The corresponding AM ester ARC-1850 entered cells much more efficiently than its nonesterified counterpart ARC-1849, whereas compounds at 10 μM concentration caused much more intensive staining than at 1 μM concentration. Staining of the cytoplasm upon treatment with ARC-1850 was detected, although nuclear exclusion was less pronounced than with ARC-1837 and more aggregation of the compound was evident. On the other hand, less staining of the plasma membrane was visible with the ATTO 647N-labeled compound (ARC-1850) than with the corresponding BODIPY FL-labeled compound (ARC-1837). Overall, it was confirmed that the carboxylate-rich TBBz−peptoid conjugates could be loaded into cells using the ester prodrug technique, and the uptake of the conjugates in the form of AM esters changed profoundly their distribution profile in cells if compared to the nonesterified compounds. Hydrolytic Stability of an AM Ester of TBBz−Peptoid Conjugate. The hydrolytic stability of the BODIPY FL-labeled AM ester ARC-1837 in different conditions was tested using RP-HPLC analysis that separated the conjugates according to the number of attached AM groups (Supporting Information Figure S4). Stock solution (2 mM) of ARC-1837 in DMSO at −20 °C was stable, as only 6% of partially hydrolyzed compound lacking one AM group was detected after one month storage. In aqueous buffer (pH = 7.5) at 40 °C, AM ester was slowly hydrolyzed. After 3 h incubation, about 20% of ARC-1837 had lost one AM group, and after 24 h incubation,

immobilization of the inhibitor to carriers. In addition, ARC1836 was displaced from complex with CK2 by ATP and phosphoacceptor-competitive inhibitor heparin (Supporting Information Figure S2). Collectively, these data further highlight the importance of concurrent association of the structural fragments of oligocarboxylic TBBz−peptoid conjugates with the active site of CK2 that is in agreement with the bisubstrate character of the inhibitors. Recently, similar conclusions were drawn about TBBz−peptide conjugates, whose dual mode of binding to CK2 was verified by mutational mapping.44 Cellular Uptake of AM Esters of Fluorescence-Labeled TBBz−Peptoid Conjugates. Upon incubation of HeLa cells with BODIPY FL-labeled ARC-1837, diffuse distribution of the compound in cytoplasm was visible while the cell nuclei were less stained (Figure 3). This observation was dramatically different from the previous studies with arginine-rich ARCs which stained intense speckles in the cell nuclei.61 However, it should be noted that for CK2α, both cytoplasmic and nuclear localization has been reported.62 The uptake of ARC-1837 was more intensive at 10 μM than at 1 μM concentration of the applied ligand in the incubation solution, whereas no remarkable difference between 1 and 2 h incubation was evident. In regions with increased cell confluence, intense staining of the cell plasma membrane upon treatment with ARC-1837 was observed; however, this might have been caused by nonspecific binding of the BODIPY FL moiety to the membrane regions with increased thickness. In contrast, ARC1836 (the compound that lacked AM protection of carboxyl F


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Bioconjugate Chemistry only 6% of starting per-esterified compound ARC-1837 was left and four products were formed, including the target compound ARC-1836 and various amounts of partially hydrolyzed derivatives with one, two, and three AM groups. The hydrolysis of AM esters was much faster in cellular milieu, as the completely unprotected conjugate ARC-1836 was detected as the dominating product after 1 h incubation in HeLa cells at 37 °C. These experiments demonstrated that the per-esterified derivative of the TBBz−peptoid conjugate was stable for storage and that it was efficiently hydrolyzed inside the cell releasing the active compound. Inhibition of Intracellular CK2 by TBBz−Peptoid Conjugates and the Effect on Cell Viability. The inhibition of CK2 in live cells was investigated in MIA PaCa-2 cell line. Western blot analysis of the whole cell lysate was performed for determination of the phosphorylation status of two CK2regulated phosphosites, Cdc37 S13 and NFκB S529.63 Incubation of cells with ARC-1859 reduced the phosphorylation levels of both selected biomarkers in a concentrationdependent manner (Figure 4A). After 24 h, a slight recovery of both NFκB and Cdc37 phosphorylation was observed in cells treated with 5 μM inhibitor (Figure 4A) that was possibly caused by slow extrusion of the inhibitor from cells. On the contrary, the phosphorylation levels of both substrates rather decreased within 24 h after treatment with 10 μM ARC-1859 (Figure 4A). In comparison with other inhibitors at the same concentration (10 μM, Figure 4B), ARC-1859 treatment decreased the phosphorylation level of the substrates by the same extent as CX-4945, while ARC-1842 did not have significant influence as expected from the poor uptake of unesterified conjugate. In conclusion, the results confirm that oligoanionic TBBz−peptoid conjugates can be used for targeting intracellular CK2 when loaded into cell in the form of AM ester prodrug. Finally, the effect of ARC-1859 on the viability of HeLa and MIA PaCa-2 cells was compared with that of unesterified ARC1842 and previously known inhibitors CX-4945 and TBBz. No effect with ARC-1842 was observed during the time frame of the experiments (Figure 5 and Supporting Information Figure S5). This is in line with earlier studies showing that the inhibition of ecto-CK2 did not affect cell viability.44 On the contrary, ARC-1859 showed concentration-dependent reduction of viability in both cell lines. The extent of reduction increased with incubation time, reaching about 80% viability loss after 48 h treatment with 20 μM ARC-1859. However, more than 80% viable cells were detected after treatment with 5 μM ARC-1859 for the maximal incubation time used in the experiments with either cell line. The effect of ARC-1859 on cell viability was similar to that of CX-4945 in HeLa cells (Figure 5); surprisingly, no reduction of viability by CX-4945 at the same concentration range was detected in MIA PaCa-2 cells (Supporting Information Figure S5). Although the explanation of this discrepancy may be complex, the inhibition of other kinases (e.g., TAK1 that is poorly inhibited by CX-4945) likely contributes to the cytotoxicity of ARC-1842. TBBz was used as the reference compound in HeLa cell line that at 5 μM concentration already caused about 50% loss of viability after 48 h incubation (Figure 5). This indicates that the increased affinity and selectivity achieved by conjugation of TBBz with oligocarboxylic peptoid are paralleled with reduced cytotoxicity. Together with the inhibition data presented above, these results suggest that, as with other inhibitors of CK2,24 conditions such as incubation time and concentration of the inhibitor can be

Figure 5. Cytotoxic effect of ARC-1842 (blue ●), ARC-1859 (red ■), CX-4945 (green ▼), or TBBz (purple □) in HeLa cells (trypan blue test). Control experiment (c = 0) refers to cells treated with vehicle (DMSO and Pluronic F-127). HeLa cells were treated for 24 h (A) or 48 h (B) with increasing concentrations of compounds, as indicated. The reported values represent the means ± SD from three parallel experiments.

defined where the inhibition of CK2-catalyzed phosphorylation is observed without revealing drastic cytotoxicity. Overall, this supports the application of such inhibitors for cellular studies. Conclusions and Perspectives. In summary, oligoanionic TBBz−peptoid conjugates are potent and selective inhibitors of CK2 that can be loaded into cell in the form of acetoxymethylated prodrug, supplementing the repertoire of compounds that inhibit the phosphorylation of physiological substrates of CK2 in living cells. To the best of our knowledge, this is the first example of the use of AM prodrug strategy for increasing the bioavailability of compounds with oligoanionic peptoid backbone. Moreover, the conjugation of ATPcompetitive inhibitors with the negatively charged peptoids in combination with AM ester loading technique represents a general approach for the construction of cell-permeable prodrugs of highly potent and selective inhibitors of CK2. Other structural fragments targeted to the nucleotide binding pocket of CK2 could be used instead of TBBz and the length of the peptoid moiety could be varied, offering a distinct control over the affinity of the inhibitor. Given that the conjugates can be labeled with fluorescent dyes, this technology has the potential for the construction of high-affinity fluorescent probes targeting intracellular CK2. From the synthesis point of view, the peptoid chain of the conjugates can be assembled using simple protocols and building blocks, without the requirement for expensive orthogonal protecting group strategies, which makes the synthesis more atomically economical if compared to traditional peptide synthesis. The described conjugates of Nsubstituted glycine oligomers and TBBz are achiral, which avoids stereochemical concerns. Interestingly, the affinity of the interaction of the conjugate with the CK2α does not depend on the chirality of the moiety bearing negative charges, and the relationship between the charge and the binding energy of the inhibitors is linear. Apparently, the affinity of the conjugates G


Article


acids56 and Fmoc Rink Amide MBHA (0.45 mmol/g) resin for obtaining the corresponding amides. 2CTC resin was loaded with bromoacetic acid in DCM solution according to the protocol described above. The acylation was performed with a solution of bromoacetic acid (5 equiv) and DIC (5 equiv) in DMF (1 h, 40 °C). The N-alkylation of Gly(OtBu) and N-Boc1,4-butanediamine with bromoacylated resin was conducted with the amine (20 equiv) and DIPEA (25 equiv) in DMF (2− 4 h, 40 °C). The reactions were monitored with the pnitrobenzylpyridine test.70 TBBz-Oca was coupled to the resin-bound peptide or peptoid chain upon HBTU/HOBt (1.5−2 equiv of TBBz-Oca, 1.47−1.9 equiv HBTU/HOBt and 6 equiv NMM) or COMUmediated (2 equiv TBBz-Oca, 2 equiv COMU, 4 equiv DIPEA) activation in DMF (2 h to overnight, rt). The final resin-bound product was washed several times with isopropanol and DCE and dried. The fully deprotected conjugates were released from the resin by treatment with TFA:H2O:TIPS (90:5:5 by volume; 2− 3 h, rt). The crude product was precipitated with ether or the solvents were removed in vacuum and the residue was triturated with ether. The obtained solid was purified by RPHPLC. The fluorescent labeling was done in a solution of the BODIPY FL SE-ester (Invitrogen) or ATTO 647N SE ester (ATTO-TEC GmbH) (1−1.5 equiv) with the aid of TEA (10− 50 equiv) in DMF (3−6 h, rt). The solvent was removed in vacuum and the residue was purified by RP-HPLC. AM esters were prepared by the 4 h treatment of the conjugates with bromomethyl acetate (2 equiv per carboxyl group) and DIPEA (10 equiv) in ACN at rt. The AM ester was isolated by RP-HPLC. Preparative yield of 70% for ARC-1859 was determined (around 1 μmol synthesis scale). The molecular masses of the final compounds were verified by ESI HRMS (HPLC and MS data are presented in Supporting Information). Binding/Displacement Assay. The assay was carried out in buffer (50 mM HEPES pH = 7.5, 150 mM NaCl, 0.005% Tween 20, 5 mM DTT) in a final volume of 20 μL on black 384-well polystyrene microplates with nonbinding surface (Corning #3676). Experiments were run in 2−3 parallels. The concentration of CK2α was determined by titration of a fixed concentration (20 nM) of PromoFluor-647-labeled fluorescent probe ARC-1504 (available from Kinasera OÜ , Estonia) with the solution of the enzymes (2-fold dilutions). The binding constants for fluorescent compounds were determined in solutions containing either ARC-1836 or ARC1849 (both 1 nM) and CK2α (2-fold dilutions). Displacement experiments with unlabeled compounds were performed by the addition of the competing ligand (3-fold dilutions) to either ARC-1504 (2 nM) in complex with CK2α (3 nM) or ARC1836 (2 nM) in complex with CK2α (10 nM). The microplates were incubated for 10−20 min at 30 °C. The FA changes relative to the signal of the uncomplexed fluorescent probe were registered on a PHERAstar platereader (BMG Labtech) with FA optical modules suitable for detecting PromoFluor-647 and ATTO 647N [ex 590 (50) nm, em 675 (50) nm] and BODIPY FL [ex 485 (10) nm, em 520 (10) nm]. The obtained data were analyzed as previously described71 to yield the concentration of the catalytically active CK2 and dissociation constant values for the complexes between the investigated compounds and CK2.

further increases upon elongation of the negatively charged peptoid chain that will be established in future studies.

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EXPERIMENTAL PROCEDURES Materials and Instrumentation. All chemicals were used without further purification. The chirality of Fmoc-L-Asp(tBu) and Fmoc-D-Asp(tBu) was confirmed by polarimetry (MCP300 polarimeter, Anton Paar). TBBz,64 K68, TBBz-Oca, RAD2SD5, and its TAMRA-labeled derivative41 were synthesized by previously reported methods. Human recombinant CK2α1−335, CK2β, and CK2α′ were prepared as described previously.65−67 The concentrations of the ligands were determined by Nanodrop 2000c (Thermo Scientific) spectrophotometer using the extinction coefficients reported in manufacturer’s datasheets or in specified literature sources: ε272 nm(unlabeled TBBzconjugates) = 10 000 M−1 cm−1,41 ε505 nm(BODIPY FL-label) = 80 000 M−1 cm−1, ε653 nm(PromoFluor-647-label) = 250 000 M−1 cm−1, ε644 nm(ATTO 647N-label) = 150 000 M−1 cm−1. HPLC-MS was performed with Schimadzu LC Solution (Prominence) system connected to LCMS-2020 ESI-MS spectrometer. Purification of the compounds was performed with RP-HPLC using a C18 column at 40 °C. Linear ACN/ water + 0.1% aqueous TFA gradient at a flow rate of 1 mL/min was used. HRMS of compounds were measured with Thermo Electron LTQ Orbitrap and combined Varian 910-FT-ICR and Varian J320 3Q spectrometers in positive ion mode. Eppendorf Research (Eppendorf) pipettes and an 8-channel 125 μL Voyager pipet (Integra Biosciences AG) were used for volumetric measurements. Liquid handling accuracy was increased by the application of low binding centrifuge tubes (Protein LoBind, Eppendorf AG) and low retention pipet tips (Repel Polymer Technology, Starlab GmbH) when possible. Data were analyzed with Graphpad Prism software (v 6.04, GraphPad Software, La Jolla California USA). Analysis of cell and Western blot images was performed with ImageJ (v 1.47) software. Chemical Synthesis. The peptide chain of the conjugates was built up according to the Fmoc solid-phase peptide synthesis methods on 2CTC resin (1.1 mmol/g) and preloaded Fmoc- L-Asp(OtBu)-Wang and Fmoc- D-Asp(OtBu)-Wang (both 0.6 mmol/g) resins (Iris Biotech GmbH). The first Fmoc-L-Asp(tBu) or Fmoc-D-Asp(tBu) was loaded to 2CTC resin with a solution of the amino acid (2 equiv) and DIPEA (4 equiv) in DCM:DMF (2:1 by volume; 2 h, rt), followed by quenching of unreacted functional groups with a mixture of DCM:MeOH:DIPEA (17:1:1 by volume; 3 × 3 min, rt). The Fmoc-group was removed by double treatment (5 + 15 min, rt) with piperidine:DMF (1:4 by volume) with an addition of HOBt (0.1 M) for suppression of aspartimide formation.68 The extension of the peptide chain was performed with solutions containing the protected amino acids (3 equiv) activated with either HBTU/HOBt (2.85−2.95 eq each) and NMM (9−10 equiv) or COMU (3 equiv)69 and 2,4,6-collidine (3.1 equiv) in DMF (1−2 h, rt). The mixture was allowed to preactivate for a few minutes before applying the solution to resin. The completeness of the coupling steps was verified by Kaiser test; capping (50 equiv Ac2O in pyridine; 30 min, rt) was performed when necessary. The peptoid chain of the conjugates was assembled by submonomer method of peptoid synthesis55 on 2CTC (1.1 mmol/g) resin for receiving C-terminal peptoid carboxylic H


Article

Bioconjugate Chemistry Phosphorylation Assay. The phosphorylation assay was carried out as previously described.41 All experiments were run in duplicate at 30 °C. The inhibitory potency was measured at the following concentrations of the components: inhibitors (3fold dilutions), CK2α or CK2α′ (0.8 nM) in the presence or absence of CK2β (10 nM), ATP (100 μM), peptide substrate 5-TAMRA-RAD2SD5 (30 μM), Mg(OAc)2 (10 mM), Pluronic F-127 (Molecular Probes; 0.02% or not included) and other buffer components as used in binding/displacement assay. The phosphorylation reaction was monitored for 30 min after initiation by the addition of peptide substrate. The Lineweaver−Burk analysis was performed at the following concentrations: ARC-1842 (0, 20, or 60 nM), CK2α (1 nM), ATP (10, 20, 40, or 80 μM), 5-TAMRA-RAD2SD5 (20 μM), and Mg(OAc)2 (10 mM). The phosphorylation was initiated by the addition of enzyme and the reaction was monitored for 90 min. Hydrolysis of the AM Ester ARC-1837 in Buffer. A solution of ARC-1837 (10 μM) in buffer [50 mM HEPES pH = 7.5, 150 mM NaCl, 0.005% Tween 20, 0.1% Pluronic F-127] diluted from stock solution in DMSO (2 mM) was incubated at 37 °C and the hydrolysis reaction was monitored at fixed time points (1.5, 3, and 24 h) by RP-HPLC (gradient 30−90% ACN/30 min). Since the presence of detergents interfered with MS analysis, the peaks were identified by their Rt values when compared to the data from the control experiment where the hydrolysis mixture of the same compound (50% DMSO in the buffer without Pluronic F-127, 70 °C, 4 h) was analyzed by RPHPLC-MS, at the same gradient conditions. Hydrolysis of the AM Ester ARC-1837 in Cells. HeLa cells (a kind gift from Beatson Institute for Cancer Research, Glasgow) were grown on 6-well culture plates at an initial density of 300 000 cells/well in the medium (DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/ mL streptomycin) at 37 °C in a humidified 5% CO 2 environment. After 2 days, the medium was removed and the cells were washed twice with PBS. The cells were incubated with a solution of ARC-1837 (10 μM solution in serum- and antibiotic-free DMEM medium containing 1% DMSO and 0.1% Pluronic F-127, 500 μL/well) for 1 h at 37 °C in humidified 5% CO2 environment. The cells were washed twice with PBS and suspended by the aid of 0.25% trypsin-EDTA solution in PBS (400 μL/well, 3 min). The suspension was centrifuged for 5 min at 100 g and the pellet was washed with PBS (1 mL). The cells were resuspended in 200 μL of lysis buffer with 1% Triton-X. Cells were lysed on ice for 30 min with mixing after every 10 min. The proteins were precipitated with ACN (400 μL) and the suspensions were centrifuged at 20 000 g for 30 min. The supernatants were collected and dried in vacuum. The obtained samples were analyzed by RP-HPLC (gradient 30−90% ACN/30 min) as described above. Live Cell Imaging. 20 000−25 000 cells were seeded on an 8-well microscopy chamber (Ibidi) and grown as described above. After 1−2 days, the medium was removed and the cells were incubated with solutions of ARC-compounds (1 μM or 10 μM, 1 or 2 h, in a volume of 200 μL) in serum- and antibioticfree DMEM medium containing 1% DMSO and 0.1% Pluronic F-127 at 37 °C in humidified 5% CO2 environment. The solutions were then removed and the cells were washed twice briefly with PBS, followed by a prolonged wash with indicatorfree RPMI buffer (1 h at 37 °C in humidified 5% CO2 environment). During the microscopic analysis, cells were kept in HBSS buffer (200 μL/well) at rt. Cells were imaged

with a microscope (TILL Photonics) equipped with xenon high stability lamp (150 W), 20× oil objective, and filters for BODIPY FL label [ex 475 (35) nm, em 525 (45) nm] and ATTO 647N label [ex 628 (40) nm, em 690 (40) nm]. The experiments were performed at 50 or 100 ms exposure time. Inhibition of CK2 in MIA PaCa-2 Cell Culture. MIA PaCa-2 cells (American Type Culture Collection, ATCC) were grown in medium (DMEM supplemented with 10% fetal bovine serum, 2.5% horse serum, and 1 mM L-glutamine) at 37 °C under a 5% CO2 atmosphere. Cells were loaded with ARC1859 (5 or 10 μM) in PBS containing DMSO (0.1%) and Pluronic F-127 (0.02%) for 1 h. Then, the loading buffer was removed and cells were incubated for additional 5 or 24 h in culturing medium. In a separate experiment, cells were loaded with CX-4945 (Selleckchem), ARC-1842, or ARC-1859 (all at 10 μM) for 5 h. Prior to harvesting, cells were washed with cold PBS, collected by centrifugation, and resuspended in lysis buffer (50 mM Tris/HCl pH = 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM DTT, 1 mM Na3VO4, 30 mM βglycerophosphate, 10 mM NaF, 100 nM ocadaic acid) containing a protease inhibitor cocktail (Roche). After sonication, cells were centrifuged at 10 000 g for 30 min at 4 °C. Whole cells extracts (30 μg) were subjected to Western blot analysis as previously described.72 Proteins were detected by probing Western blot membranes with the following antibodies: rabbit polyclonal antiphospho-NFκB p65 (Ser529, Abcam), rabbit monoclonal anti-NFκB p65, (Cell Signaling Technology), mouse monoclonal anti-Cdc37 (Santa Cruz Biotechnology), mouse monoclonal anti-β-actin (Sigma), and affinity-purified antiphospho-Cdc37 (Ser13) antibody (a kind gift from Dr. I. Miyata, University of Kyoto, Japan). Cell Viability Assay. HeLa cells were grown as described above in a 24-well plate to ∼50% confluency. Thereafter, the cells were washed with 2 mL of DPBS and incubated with ARC-1842, CX-4945 (Synkinase), TBBz, or ARC-1859 (all at 0, 1, 5, 10, or 20 μM) in serum-free DMEM high glucose medium containing 1% DMSO and 0.1% Pluronic F-127 (300 μL total volume) for 24 and 48 h in triplicate. Cells incubated in culture medium without DMSO and Pluronic F-127 were used as control. Thereafter each well was washed twice with 1 mL of DPBS and the solutions were transferred into a screwcapped tube. The cells were incubated with 0.25% trypsinEDTA (100 μL) for 2 min, suspended in DPBS (1 mL), and added to the screw-capped tube. After centrifugation at 100 g for 5 min, the supernatant was removed and the cells were resuspended in the indicator-free RPMI medium (200 μL). For quantitative analysis, 30 μL of the cell suspension and 30 μL of 0.4% trypan blue solution were mixed thoroughly. The suspension was analyzed twice in 10 μL volumes by cell counter (Bio-Rad, TC-10). MIA PaCa-2 cells were grown as described above and were incubated with ARC-1842, CX-4945 (Selleckchem), or ARC1859 (all at 0, 5, 10, or 20 μM) for 24, 48, and 72 h. The proportion of viable cells was determined by WST-1 assay (Roche). Cells incubated in culture medium with DMSO were used as control.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.5b00383. I


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dependent and CK2 independent roles reveal a secret identity for CK2beta. Int. J. Biol. Sci. 1 (2), 67−79. (5) Litchfield, D. W. (2003) Protein kinase CK2: structure, regulation and role in cellular decisions of life and death. Biochem. J. 369 (1), 1−15. (6) Montenarh, M. (2010) Cellular regulators of protein kinase CK2. Cell Tissue Res. 342 (2), 139−146. (7) 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, Proteins Proteomics 1804 (3), 484−492. (8) Lolli, G., Pinna, L. A., and Battistutta, R. (2012) Structural determinants of protein kinase CK2 regulation by autoinhibitory polymerization. ACS Chem. Biol. 7 (7), 1158−1163. (9) Hübner, G. M., Larsen, J. N., Guerra, B., Niefind, K., Vrecl, M., and Issinger, O.-G. (2014) Evidence for aggregation of protein kinase CK2 in the cell: a novel strategy for studying CK2 holoenzyme interaction by BRET(2). Mol. Cell. Biochem. 397 (1−2), 285−293. (10) Guerra, B., and Issinger, O.-G. (2008) Protein kinase CK2 in human diseases. Curr. Med. Chem. 15 (19), 1870−1886. (11) Cozza, G., Pinna, L. A., and Moro, S. (2013) Kinase CK2 inhibition: an update. Curr. Med. Chem. 20 (5), 671−693. (12) Sarno, S., Papinutto, E., Franchin, C., Bain, J., Elliott, M., Meggio, F., Kazimierczuk, Z., Orzeszko, A., Zanotti, G., Battistutta, R., et al. (2011) ATP site-directed inhibitors of protein kinase CK2: an update. Curr. Top. Med. Chem. 11 (11), 1340−1351. (13) https://clinicaltrials.gov/, identifier NCT02128282 (accessed in October, 2015). (14) Kim, H., Choi, K., Kang, H., Lee, S.-Y., Chi, S.-W., Lee, M.-S., Song, J., Im, D., Choi, Y., and Cho, S. (2014) Identification of a novel function of CX-4945 as a splicing regulator. PLoS One 9 (4), e94978. (15) Gyenis, L., Kuś, A., Bretner, M., and Litchfield, D. W. (2013) Functional proteomics strategy for validation of protein kinase inhibitors reveals new targets for a TBB-derived inhibitor of protein kinase CK2. J. Proteomics 81, 70−79. (16) Karaman, M. W., Herrgard, S., Treiber, D. K., Gallant, P., Atteridge, C. E., Campbell, B. T., Chan, K. W., Ciceri, P., Davis, M. I., Edeen, P. T., et al. (2008) A quantitative analysis of kinase inhibitor selectivity. Nat. Biotechnol. 26 (1), 127−132. (17) Cohen, P. (1999) The development and therapeutic potential of protein kinase inhibitors. Curr. Opin. Chem. Biol. 3 (4), 459−465. (18) Tsai, C. F., Wang, Y. T., Yen, H. Y., Tsou, C. C., Ku, W. C., Lin, P. Y., Chen, H. Y., Nesvizhskii, A. I., Ishihama, Y., and Chen, Y. J. (2015) Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics. Nat. Commun. 6, Article number 6622 10.1038/ ncomms7622. (19) Knight, Z. A., and Shokat, K. M. (2005) Features of selective kinase inhibitors. Chem. Biol. 12 (6), 621−637. (20) Andrzejewska, M., Pagano, M. A., Meggio, F., Brunati, A. M., and Kazimierczuk, Z. (2003) Polyhalogenobenzimidazoles: synthesis and their inhibitory activity against casein kinases. Bioorg. Med. Chem. 11 (18), 3997−4002. (21) Duncan, J. S., Gyenis, L., Lenehan, J., Bretner, M., Graves, L. M., Haystead, T. A., and Litchfield, D. W. (2008) An unbiased evaluation of CK2 inhibitors by chemoproteomics: characterization of inhibitor effects on CK2 and identification of novel inhibitor targets. Mol. Cell. Proteomics 7 (6), 1077−1088. (22) Miyata, Y., and Nishida, E. (2004) CK2 controls multiple protein kinases by phosphorylating a kinase-targeting molecular chaperone, Cdc37. Mol. Cell. Biol. 24 (9), 4065−4074. (23) Zanin, S., Borgo, C., Girardi, C., O’Brien, S. E., Miyata, Y., Pinna, L. A., Donella-Deana, A., and Ruzzene, M. (2012) Effects of the CK2 inhibitors CX-4945 and CX-5011 on drug-resistant cells. PLoS One 7 (11), e49193. (24) Franchin, C., Cesaro, L., Salvi, M., Millioni, R., Iori, E., Cifani, P., James, P., Arrigoni, G., and Pinna, L. A. (2015) Quantitative analysis of a phosphoproteome readily altered by the protein kinase CK2

Inhibitory potencies of TBBz−peptoid conjugates, data from competitive inhibition and binding experiments, HPLC and HRMS data, viability studies with MIA PaCa2 cell line, selectivity data for ARC-1842, and live cell imaging data for ATTO 647N-labeled compounds (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +372 737 5275. Author Contributions

K.V., S.S., and J.V. carried out the chemical synthesis of the compounds. K.V. and S.S. performed inhibition and binding assays. S.S. and D.L. performed experiments with HeLa and B.G. with MIA PaCa-2 cells. S.S., G.R., and H.S. carried out HPLC analysis and purification. O.-G.I. produced and characterized the recombinant CK2 subunits. H.S. performed viability studies with HeLa cells. The work was designed by K.V., E.E., D.L., S.S., and A.U. All authors contributed to the writing of the manuscript. The authors declare no conflict of interest. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Estonian Research Council Grants IUT20-17 and PUT0007, and Danish Council for Independent Research-Natural Sciences (Grant 1323-00212A to B. Guerra). HRMS measurements by Varian 910-FT-ICR-MS system were supported by the Estonian national R & D infrastructure development program Measure 2.3 “Promotion of development activities and innovation” (Regulation No. 34) funded by the Enterprise Estonia Foundation. We are grateful to Tõiv Haljasorg (Institute of Chemistry, University of Tartu, Estonia) for performing HRMS measurements, and to Dr Lauri Vares (Institute of Technology, University of Tartu, Estonia) for providing access to the polarimeter.

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ABBREVIATIONS 2CTC, 2-chlorotrityl chloride; AM, acetoxymethyl; Cdc37, cell division cycle 37; CK2, casein kinase 2; CK2α, the catalytic subunit of CK2; CK2β, the regulatory subunit of CK2; DMAT, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole; FA, fluorescence anisotropy; GSK3β, glycogen synthase kinase 3 beta; K68, (4,5,6,7-tetrabromo-1H-benzimidazol-1-yl)acetic acid; TAK1, transforming growth factor β activated kinase-1; NFκB, nuclear factor kappa B; Oca, octanoic acid; PK, protein kinase; TBB, 4,5,6,7-tetrabromobenzotriazole; TBBz, 4,5,6,7tetrabromo-1H-benzimidazole

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REFERENCES

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