Inhibition of the STAT3 Protein by a Dinuclear Macrocyclic Complex

Article pubs.acs.org/IC

Inhibition of the STAT3 Protein by a Dinuclear Macrocyclic Complex Lígia M. Mesquita,† Federico Herrera,† Catarina V. Esteves,† Pedro Lamosa,† Vânia André,‡ Pedro Mateus,*,† and Rita Delgado*,† †

Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal ‡ Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Avenida Rovisco Pais, 1049-001 Lisboa, Portugal S Supporting Information *

ABSTRACT: A new diethylenetriamine-derived macrocycle bearing 2-methylpyridyl arms and containing m-xylyl spacers, L, was prepared, and its dinuclear copper(II) and zinc(II) complexes were used as receptors for the recognition in aqueous solution of a phosphorylated peptide derived from a sequence of the STAT3 protein. A detailed study of the acid−base behavior of L and of its complexation properties as well as of the association of the phosphorylated peptide to the receptor was carried out by potentiometry in aqueous solution at 298.2 K and I = 0.10 M in KNO3. The data revealed that the receptor forms stable associations with several protonated forms of the substrate, with constant values ranging from 3.32 to 4.25 log units. The affinity of the receptor for the phosphorylated substrate studied is higher at a pH value where the receptor is mainly in the [Cu2L]4+ form and the pY residue of the substrate is in the dianionic form (pH 6.55). These results, also supported by 31P NMR studies, showed that the phosphopeptide is bound through the phosphoryl group in a bridging mode. Additionally, the receptor inhibited binding between active (phosphorylated) STAT3 and its target DNA sequence in a dose-dependent manner (IC50 63 ± 3.4 μM) in human nuclear extracts in vitro. Treatment of whole cells with the inhibitor revealed that it is bioactive in living cells and has oncostatic properties that could be interesting for the fight against cancer and other pathologies involving the STAT3 protein.

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INTRODUCTION STAT3 (Signal Transduction and Activator of Transcription 3) is a member of the STAT family of latent cytosolic transcription factors that transmits external signals from the surface membrane to target genes in the nucleus.1 STAT3 is constitutively activated in multiple human cancers including ovarian, breast, prostate, leukemia, and lymphoma.2 Increasing evidence of the potential clinical benefits of blocking constitutive STAT3 signaling validates the STAT3 protein as a target for drug intervention in cancer therapy.3 Phosphorylation of a tyrosine residue (Y705) by growth factor receptor tyrosine kinases, Janus kinases, and Src family kinases4 promotes the formation of transcriptionally active STAT3 dimers in which the phosphopeptide-binding module (SH2 domain) of one protein molecule binds to the phosphotyrosine (pY) residue of the other and vice versa. The dimer then migrates to the nucleus, binds to specific DNA sequences, and initiates transcription of several genes involved in cell progression, differentiation, and survival.5 Inhibition or disruption of STAT3 dimer formation provides an effective approach of targeting this protein for blocking its signaling activity and functional effects.6 There are two targets for STAT3 inhibitors: the pY residue and the SH2 domain. The majority of the effort has focused on the development of SH2 domain antagonists (peptidic, peptidomimetic, small molecules, © XXXX American Chemical Society

or platinum complexes) that compete for binding to the STAT3 SH2 domain.7 However, this strategy has been limited by the large planar interfacial areas involved in protein-binding interfaces8 and the poor cell permeability and metabolic susceptibility of the inhibitors.7 The converse approach of finding a suitable STAT3 SH2 domain-mimic capable of binding the pY residue has been pioneered by Gunning et al.9,10 and used bis(dipicolylamine) dinuclear copper(II) complexes to bind the phosphate moiety of pY, thus disrupting STAT3 dimerization in cancer cell extracts and obtaining promising antitumor activity.9 In the present work, with the knowledge that dinuclear complexes of ditopic hexaazamacrocycles have proven useful as receptors for the uptake of phosphorylated substrates,11 a member of this family of macrocycles was evaluated as a possible alternative to bis(dipicolylamine)-derived ligands. With hexaazamacrocyclic ligands, each metal center is invariably left with coordination sites occupied by one or two water molecules or weakly coordinated counterions, and the distance between the two metal centers is modulated by rigid spacers.12 Moreover, arms can be appended to provide supplementary Received: January 15, 2016

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DOI: 10.1021/acs.inorgchem.6b00116 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry Chart 1. Structures of the Ligand L and the Peptide Studied in This Work

Scheme 1. Synthetic Procedure for the Preparation of L

of the phosphonate group of phosphotyrosine (pY), and the respective value is on the order of that determined for the protonation of phenylphosphate (PhP2−). pY remains in the dianionic form over a wide pH range, with the corresponding H2pST3 species reaching its maximum percentage at pH 7.90, and its monoanionic form predominates only below pH 5.55 (see the speciation diagram shown in Figure 1). Because of

coordinating functions or additional intermolecular interactions. Herein a new diethylenetriamine-derived macrocycle bearing 2-methylpyridyl arms and containing m-xylyl spacers (L; Chart 1) is described. The dinuclear copper(II) and zinc(II) complexes of L were first evaluated as receptors for a phosphorylated peptide, H2pST3 (Chart 1), derived from a sequence of the STAT3 protein in aqueous solution to validate their usefulness. In order to evaluate whether the dinuclear complexes of L could inhibit/disrupt phosphorylation-mediated STAT3 interactions and thus function as potential chemotherapeutic agents, studies were performed in nuclear extracts in whole cancer cells.

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RESULTS AND DISCUSSION Synthesis of the Ligand L. The ligand L was prepared by a “one-pot” procedure consisting of a [2 + 2] cyclization reaction between isophthaldehyde and 4-(2-pyridylmethyl)-1,4,7-triazaheptane, followed by reduction with sodium borohydride (see Scheme 1), as described for related compounds containing hydroxyethyl13 and methylimidazole14 pendant arms. Acid−Base Behavior of the Peptidic Substrates and Their Copper(II) Coordination. Knowledge of the acid−base behavior and coordination properties of the model peptide of the phosphorylated sequence of the STAT3 protein targeted is fundamental for subsequent binding studies. Therefore, the protonation constants of the peptidic substrate as well as the stability constants of its copper(II) complexes were determined by potentiometric titrations in aqueous solution at 298.2 ± 0.1 K and an ionic strength of 0.10 ± 0.01 M in KNO3. The results are presented in Tables S1 and S2 in the Supporting Information. Three protonation constants were determined for the H2pST3 peptide. The two first protonation constants are assigned to the two amine groups of the lysine residues, while the third protonation constant, KH3 , is ascribed to protonation

Figure 1. Species distribution diagram for the protonation of H2pST3. Cpeptide = 1.0 × 10−3 M.

their high basicity, the amide centers stay protonated in water solution at the pH range possible to use by potentiometry, and so it was impossible to determine their protonation constants. The peptidic substrate exhibits a low affinity for copper(II) (Table S2 in the Supporting Information), as expected of peptides with blocked N-termini containing only weakly coordinating side chains.15 This means that the side chains of the peptide, pY in particular, do not participate in the coordination. B


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Inorganic Chemistry

Table 1. Stepwise Equilibrium Constants of the Protonation of L (KH i ) and Its Copper(II) and Zinc(II) Complexation (KMmHhLl) a in Aqueous Solution log KMmHhLlb equilibrium reaction L + H+ ⇌ HL+ HL+ + H+ ⇌ H2L2+ H2L2+ + H+ ⇌ H3L3+ H3L3+ + H+ ⇌ H4L4+ H4L4+ + H+ ⇌ H5L5+ H5L5+ + H+ ⇌ H6L6+

log

KHi b

9.87(1) 8.92(1) 7.75(1) 7.04(1) 3.70(1) 3.05(1)

equilibrium reaction

copper(II)

[MH3L]5+ + H+ ⇌ [MH4L]6+ [MH2L]4+ + H+ ⇌ [MH3L]5+ [MHL]3+ + H+ ⇌ [MH2L]4+ [ML]2+ + H+ ⇌ [MHL]3+ M2+ + L ⇌ [ML]2+ [ML(OH)]+ + H+ ⇌ [ML]2+ [ML(OH)2] + H+ ⇌ [ML(OH)]+ [M2HL]5+ + H+ ⇌ [M2H2L]6+ [M2L]4+ + H+ ⇌ [M2HL]5+ [ML]2+ + M2+ ⇌ [M2L]4+ [M2L(OH)]3+ + H+ ⇌ [M2L]4+ [M2L(OH)2]2+ + H+ ⇌ [M2L(OH)]3+ [M2L(OH)3] + + H+ ⇌ [M2L(OH)2]2+

2.55(7) 3.57(3) 7.21(4) 8.54(9) 16.32(8) 9.3(1) 3.63(3) 3.76(9) 13.18(8) 7.35(2) 8.11(3)

zinc(II)

7.31(2) 8.86(4) 10.52(3) 9.74(6) 11.18(8)

7.86(3) 8.08(1) 8.94(1) 11.16(2)

T = 298.2 ± 0.1 K; I = 0.10 ± 0.01 M in KNO3. The values of all of the overall constants are presented in Tables S3 and S4 in the Supporting Information. bValues in parentheses are standard deviations in the last significant figure. a

Figure 2. Species distribution diagrams calculated for the complexes of copper(II) with L in a 2:1 ratio (a) and Zn2+ with L in a 2:1 ratio (b). CCu = CZn = 2CL = 2.0 × 10−3 M.

5.5. The stepwise stability constant for [Cu2L]4+ (log K = 13.18) is much higher than that for the parent macrocycle (log K = 10.86)11e and for the corresponding tren-derived cryptand with m-xylyl spacers (log K = 9.40),17 indicating the effect of the additional coordination of the pyridine group of L and the lesser strain created by the free group compared with the one in a constrained cryptand. At pH >5.5, the successive hydrolysis of two water molecules directly coordinated to each copper center takes place. The low pH value at which the [Cu2L(OH)]3+ species is formed (1.95 log units lower than that of [CuL(OH)]+) indicates a strong binding to the metal centers and suggests that the hydroxide anion bridges the two copper(II) centers, as was also proposed for the related compounds.11e,17 As expected, the zinc(II) complexes of L exhibit lower stability constants than the copper(II) ones, and, consequently, the corresponding complexes occur at higher pH values. The [Zn2L]4+ species is stable over a relatively wide pH range and reaches its maximum at pH 6.90. Three hydroxo complexes were found at higher pH, which suggests that the zinc(II) centers may have coordination numbers higher than 5, in contrast with what was observed for copper(II).

Acid−Base Behavior of L and Copper(II) and Zinc(II) Complexation Studies. The protonation constants of L and its stability constants with copper(II) and zinc(II) were determined by potentiometry in the conditions mentioned in the previous section. The results are collected in Table 1 (see also Tables S3 and S4 in the Supporting Information), and the corresponding species distribution diagrams are represented in Figure S1 in the Supporting Information and Figure 2. The acid−base behavior of L is similar to that of other related diethylenetriamine-derived macrocycles.16 Mono- and dinuclear complexes were found for both metal ions, although at 2:1 M:L ratio, the dinuclear species are clearly predominant (see Figure 2), but even at 1:1 M:L ratio, the dinuclear complexes are formed in a significant percentage (Figure S2 in the Supporting Information), confirming the high tendency of L to form dinuclear complexes. For copper(II), the dinuclear species exist even at very low pH in the form of the [Cu2H2L]6+ species. As the pH is raised, the last two amine donors sequentially deprotonate and coordinate to the metal centers, giving rise to the [Cu2L]4+ complex cation. This species is stable over a wide pH range and reaches its maximum at pH C


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Inorganic Chemistry Single-Crystal X-ray Diffraction Studies. The molecular structure of [Cu2L(Cl)2⊃Cl]Cl·2H2O is shown in Figure 3

form an angle of 31.78(8)°, which means that the six equilateral nitrogen atoms are almost coplanar. In fact, the two pyridyl nitrogen atoms, N37 and N43, are located at only 0.776(4) and 0.980(4) Å above the plane defined by the four secondary amine nitrogen atoms, N4, N13, N19, and N28. The macrocycle adopts a conformation in which the m-xylyl spacers and the methylene groups of the diethylenetriamine subunit are turned toward the same side of the macrocycle, resembling the cone conformation observed in calix[4]arenes (Figure S3a−c in the Supporting Information). A chloride anion was found encapsulated within this cone, placed at 1.000(2) Å above the centroid defined by the four secondary amine nitrogen atoms and held by four N−H···Cl hydrogen bonds (Figure 3; see also Figure S3d and Table S5 in the Supporting Information). The present structure differs from that determined for the copper(II) complex of the parent macrocycle, [Cu2L2(SO4)2] (L2 is the same macrocycle of L but without arms).11e In that case, the macrocycle adopts a chairlike conformation, with one of the diethylenetriamine subunits turned toward one side of the macrocyclic plane while the other is turned toward the opposite side. The Cu···Cu distance is 5.31 Å, which is shorter than the one found in [Cu2L(Cl)2⊃Cl]+. Also different in [Cu2L2(SO4)2], the counteranions bridge two copper centers of neighboring molecules, favoring the formation of an extended polymeric structure instead of anion encapsulation. In [Cu2L(Cl)2⊃Cl]+, as well as in two other related complexes found in the literature, the appended arms have the advantage of leaving the copper centers with only one vacant binding site, which disfavors the formation of polymeric structures. Indeed, related compounds containing hydroxyethyl13b and methylimidazole14 pendant arms were shown to bind a substrate in a bridging manner, leaving the two copper centers at 5.81 and 5.89 Å, respectively. Together, the structures of these four dicopper(II) complexes show that this macrocyclic architecture is rather flexible and can adopt different conformations, including the ones that allow binding of an anion in a bridging mode. Cascade Species Formed by the Copper(II) Complexes of L. Given the higher stability of copper(II) complexes of L relative to those of zinc(II), the former were selected as the receptor for uptake of the phosphorylated substrates and subsequent studies. The association constants of copper(II) complexes of L with the substrates were determined by potentiometry at the experimental conditions used before. The results are collected in Table 3 (see also Table S6 in the Supporting Information), together with the results for phenylphosphate (PhP 2− ) also determined in this work for comparison.

Figure 3. Crystal structure of the [Cu2L(Cl)2⊃Cl]+ complex cation determined from single-crystal X-ray diffraction data.

along with the relevant atomic notation adopted. Selected bond lengths and angles are given in Table 2. Each copper center is Table 2. Selected Bond Distances (Å) and Angles (deg) in the Coordination Spheres of the [Cu2L(Cl)2⊃Cl]Cl·2H2O Complex Bond Lengths/Å Cu1−Cl1 Cu1−N1 Cu1−N4 Cu1−N28 Cu1−N37 N1−Cu1−Cl1 N4−Cu1−N28 N4−Cu1−N37 N28−Cu1−N37 N1−Cu1−N4 N1−Cu1−N28 N1−Cu1−N37 Cl1−Cu1−N4 Cl1−Cu1−N28 Cl1−Cu1−N37

2.249(1) Cu2−Cl2 2.032(3) Cu2−N13 2.068(3) Cu2−N16 2.168(3) Cu2−N19 2.047(3) Cu2−N43 Bond Angles/deg

2.235(1) 2.076(3) 2.029(3) 2.145(3) 2.050(3)

177.43(9) 107.4(1) 132.8(1) 115.2(1) 84.3(1) 83.0(1) 81.6(1) 97.46(9) 94.68(8) 98.48(9)

178.21(9) 109.0(1) 135.8(1) 110.7(1) 84.8(1) 83.4(1) 81.2(1) 96.92(9) 95.64(8) 97.69(9)

N16−Cu2−Cl2 N13−Cu2−N19 N13−Cu2−N43 N19−Cu2−N43 N13−Cu2−N16 N16−Cu2−N19 N16−Cu2−N43 Cl2−Cu2−N13 Cl2−Cu2−N19 Cl2−Cu2−N43

pentacoordinate and bound by three nitrogen atoms of a diethylenetriamine subunit of L to a pyridyl nitrogen atom and to a chloride anion. The trigonal distortion calculated using the index structural parameter τ (τ = 0 for a perfect squarepyramidal geometry and τ = 1 for an ideal trigonal-bipyramidal geometry)18 assumes values of 0.74 and 0.71 for Cu1 and Cu2, respectively, which is consistent with distorted trigonalbipyramidal stereochemistries. The distortion from a regular trigonal bipyramid is evident because the Neq−Cu−Neq angles deviate from 120°: 132.8(1), 115.2(1), and 107.4(1)° in Cu1 and 135.8(1), 110.7(1), and 109.0(1)° in Cu2. In addition, the copper(II) centers are located slightly above the trigonal plane [0.257(2) and 0.249(2) Å for Cu1 and Cu2, respectively] and pulled toward the apical chloride anions. The macrocyclic ligand is folded in such way that the two copper centers are located at 7.3622(9) Å from one another and the two apical positions occupied by the chloride anions are turned toward the outside of the cavity. Consequently, the two trigonal planes of the coordination spheres of the two copper centers instead of being parallel (or almost parallel)

Table 3. Stepwise Association Constants (KCumHhLlSs) for the Indicated Equilibria in Aqueous Solutiona equilibrium reaction

log KCumHhLlSsb

[Cu2L] + PhP ⇌ [Cu2L(PhP)] [Cu2L]4+ + H3ST32+ ⇌ [Cu2L(H3ST3)]6+ [Cu2L]4+ + H3pST3+ ⇌ [Cu2L(H3pST3)]5+ [Cu2L]4+ + H2pST3 ⇌ [Cu2L(H2pST3)]4+ [Cu2L(OH)]3+ + HpST3 ⇌ [Cu2L(OH)(HpST3)]3+

4.48(1) c 3.32(3) 4.25(2) 3.32(7)

4+

2−

2+

T = (298.2 ± 0.1) K; I = (0.10 ± 0.01) M in KNO3. bValues in parentheses are standard deviations in the last significant figures. cNo association constant could be determined with the nonphosphorylated peptide.

a

D


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Inorganic Chemistry

Figure 4. (a) Species distribution diagram calculated for a solution of copper(II), L, and H2pST3. (b) Diagram of the overall amounts of supramolecular species Σ[Cu2(HhpST3)L](2+h)+ formed between Σ[Cu2HhL](4+h)+ and Σ(HhpST3(2−h)−). CL = CCu/2 = CpST3/5 = 1.0 × 10−3 M.

The receptor forms stable associations with several protonated forms of the phosphorylated peptide, with constant values ranging from 3.32 to 4.25 log units, and only species of 1:1 receptor/substrate stoichiometry were found. The association constants increase as the overall charge of the substrate decreases, as would be expected based on electrostatic effects. Several protonated forms of the phosphorylated substrate bind to the receptor over a very wide pH range (2.5−10), and the [Cu2L(H2pST3)]4+ associated species reaches the maximum percentage at pH 6.55 (Figure 4). At this pH value, the receptor is mainly in the [Cu2L]4+ form (Figure 2a), while the phosphorylated peptide exists mainly in the form of the H2pST3 species (Figure 1), in which the respective pY residue is dianionic and the two lysine residues are still protonated. Accordingly, it was found that the association constant for the [Cu2L]4+ + H2pST3 ⇌ [Cu2L(H2pST3)]4+ equilibrium is of the same magnitude as that obtained for [Cu2L]4+ + PhP2− ⇌ [Cu2L(PhP)]2+, which strongly suggests that the peptide is bound to the receptor through the pY residue. The fact that the nonphosphorylated version of the peptide19 is not bound to the receptor also supports this binding mode. In addition, the association constant of the entity involving the H2pST3 peptide is much larger than that found for the coordination of PhP2− to Cu2+ (2.77 log units; see Table S2 in the Supporting Information), which also points to coordination of the peptide in a bridging mode. In order to get other proof of the phosphopeptide-binding event, NMR studies were performed using the dinuclear zinc(II) complex of L as the receptor. In spite of the broad resonances presented by the dinuclear zinc(II) complex 1H NMR spectrum (Figure S4 in the Supporting Information), probably due to exchange between different conformations taking place at an intermediate rate relative to the NMR time scale, the 1H NMR peaks of the free and receptor-bound phosphopeptide were easily assigned using the 1H−1H TOCSY NMR experiment (see Figures S5 and S6 in the Supporting Information). Assignments and chemical shift variations Δδ (δbound − δfree) of proton signals are gathered in Table S7 in the Supporting Information. In Figure 5, a histogram containing all of the obtained chemical shift changes (Δδ) for protons of the H2pST3 peptide in the absence and presence of 1 equiv of a dinuclear zinc(II) complex receptor is shown. It can be observed in this histogram

Figure 5. Histogram of the observed 1H chemical shift changes (Δδ) in ppm for the H2pST3 phosphopeptide upon binding by the dinuclear zinc complex receptor at pH 7.2. The bars exhibit the colors of the corresponding parts in the structure of the H2pST3 peptide also included in the diagram.

that the protons most affected, by the addition of the receptor, are those of the acetyl group and pY, thus indicating that the peptide is bound to the receptor through the pY residue. Besides being the most shifted, these resonances are also quite broadened because of intermediate receptor/substrate exchange on the NMR time scale. The resonances of the protons of the remaining residues are not broadened and undergo much less shift. To further confirm the binding mode of the phosphopeptide through the pY residue, a 31P NMR study was performed using the dinuclear zinc(II) complex of L as the receptor. As can be observed in Figure 6, the chemical shift due to the phosphorus atom of H2pST3 changed from −0.43 to −1.39 ppm upon the addition of 1 equiv of [Zn2L]4+. In addition, the signal of the bound phosphorylated substrate is remarkably broadened to the point of almost disappearing into the baseline, because of intermediate receptor/substrate exchange on the NMR time scale, indicative of phosphoryl complexation. Upfield shifts of similar magnitudes were observed in the binding of PhP2− and phosphorylated peptides to a dinuclear zinc(II) complex of E


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Inorganic Chemistry

well with those of the dinuclear complexes studied by Gunning et al. (IC50 = 43−74 μM).9a Furthermore, when several proliferative cell lines were exposed to the inhibitor at different concentrations (0.1−150 μM), they stopped proliferating (Figure 8). The cell number was significantly decreased after only 24 h of exposure, and this was not due to cell death because there was no release of the cell contents into the extracellular medium (Figure 8b). Cells looked healthy, but they suffered a striking change in morphology, becoming flatter and tightly attached to the surface of the plates (data not shown). This is consistent with inhibition of the STAT3 activity because STAT3 activation is involved in the proliferation and tumorigenesis of glioma cancer cells, for example.21 These results indicate that the dinuclear copper(II) complex of L is bioactive in living cells, inhibiting their proliferation and enhancing their attachment to the substrate. These oncostatic properties could be interesting for the fight against cancer and other pathologies involving the STAT3 protein. In order to confirm if the dicopper(II) complex was able to inhibit the STAT3 activity in living cells, U251 cells were incubated with leukemia inhibitory factor (LIF; 100 ng/mL) for 0, 15, 30, and 60 min in the presence or absence of the inhibitor (150 μM), nuclear proteins were extracted, and the STAT3 translocation to the nucleus was analyzed (Figure 9). As expected, LIF activated STAT3 and induced its translocation to the nucleus in a time-dependent manner, with a peak at 60 min. The inhibitor was able to completely block LIF-induced STAT3 translocation, confirming that it is active in living cells.

Figure 6. 31P NMR spectra of solutions containing the H2pST3 substrate and increasing amounts of [Zn2L]4+ (0, 0.05, 0.10, 0.25, and 1.00 equiv, from red to dark green) in H2O/D2O (90:10) at pH 7.2. The signal centered at 0.0 ppm corresponds to external H3PO4 used as the reference.

dipicolylamine used as an artificial receptor, in which X-ray analysis revealed that the phosphoryl moiety bridges the two metal centers.20 Biological Assays. The ability of the dinuclear copper(II) complex of L to inhibit STAT3 activity in vitro, in nuclear extracts from HEK293 human kidney cells, was determined by electromobility shift assays (EMSAs). A mixture of human nuclear extracts from cells containing activated STAT3 (HEK293) and biotin-labeled DNA was treated with a wide range of concentrations of the inhibitor (10−150 μM) for 30 min, and the STAT3 dimer/DNA binding in vitro was measured. It was found that the binding of the STAT3 dimer to DNA is inhibited in a dose-dependent manner (see Figure 7). The IC50 for STAT3 inhibition was 63 ± 3.4 μM, which is quite remarkable considering that the range of IC50 values found in the literature for small-molecule STAT3 inhibitors is between 0.1 and 106 μM.7 In addition, the IC50 value compares

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CONCLUSIONS A new diethylenetriamine-derived macrocycle bearing 2methylpyridyl arms and containing m-xylyl spacers was prepared in good yield by a “one-pot” procedure, and its dinuclear copper(II) and zinc(II) complexes were studied as receptors for the recognition of a phosphorylated peptide derived from a sequence of the STAT3 protein in aqueous solution. A detailed determination of the protonation constants of the ligand and peptide as well as of their copper(II) complexes in aqueous solution allowed the complete study of the association constants of the dinuclear receptor with the phosphorylated peptide in the same conditions. It was found that the receptor forms stable associations with several protonated forms of the phosphorylated peptide, with constant values ranging from 3.32 to 4.25 log units. The affinity of the receptor for the phosphorylated substrate studied is higher at a pH value where the receptor is mainly in the [Cu2L]4+ form and the pY residue of the substrate is in the dianionic form (pH 6.55). These results, combined with the 31P NMR data, showed that the phosphopeptide is bound through the phosphoryl group in a bridging mode. Biological assays have shown that the dicopper(II) complex of L inhibits the STAT3 activity in vitro in nuclear extracts, as determined by EMSA. It was found that binding of the STAT3 protein to DNA is inhibited in a dose-dependent manner, with a IC50 value for STAT3 inhibition of 63 ± 3.4 μM. Treatment of whole cells with the inhibitor, followed by extraction of nuclear proteins, showed complete suppression of LIF-induced STAT3 translocation to the nucleus, confirming that the dicopper(II) complex of L is active in living cells. Furthermore, when several proliferative cell lines were exposed to the inhibitor at different concentrations (0.1−150 μM), prolifer-

Figure 7. Association between STAT3 and DNA disrupted by the inhibitor in a dose-dependent manner. Top image: EMSAs performed using nuclear extracts (HEK293) containing STAT3 and biotinlabeled DNA in the presence of increasing amounts of the dicopper(II) complex. In EMSAs, the degree of STAT3/DNA association is directly proportional to the intensity of the bands. Bottom graph: Band intensity normalized versus the control group. The IC50 value corresponds to a relative band intensity of 0.5. F


Article

Inorganic Chemistry

Figure 8. Bioactive dinuclear copper(II) complex of L inhibiting the proliferation of various cancer cell lines. Brain cancer cells from different species (U251, C6, and HT22 cell lines) were incubated with a series of concentrations of the inhibitor for 24 h. (a) The inhibitor decreased the number of viable cells in a dose-dependent manner. (b) The inhibitor did not induce the release of intracellular contents to the medium, indicating that the decrease in the cell number is due to inhibition of cell proliferation and not toxicity. Syntheses of L and Its Copper(II) Complex. Synthesis of L. A solution of isophthalaldehyde (592 mg, 4.41 mmol) in methanol (44 mL) was added dropwise to a magnetically stirred solution of 4-(2pyridylmethyl)-1,4,7-triazaheptane (857 mg, 4.41 mmol) in methanol (44 mL). The mixture was left under stirring at room temperature for 12 h. Solid NaBH4 (3.34 g, 88 mmol) was added in small portions to avoid excessive foaming. Upon this addition, the mixture was left under stirring at room temperature until the bubbling ceased and under reflux for 3 h. The solution was filtered off and evaporated under vacuum almost to dryness, then water was added (20 mL), and the remaining methanol was evaporated. Afterward, the solution was made strongly basic with 6 M KOH and extracted with dichloromethane (3 × 50 mL). The organic layer was dried with anhydrous Na2SO4 and evaporated to give a yellow oil, which was dissolved in the minimum amount possible of methanol, and then concentrated HClO4 (1.6 mL) was added dropwise, under magnetic stirring. The precipitate formed was filtered off and washed with cold methanol. Yield: 1.58 g (60%). 1 H NMR (400 MHz, D2O): δ 2.99 (8 H, t, J = 6.53 Hz, CH2NCH2py), 3.26 (8 H, t, J = 6.43 Hz, CH2NCH2-bz), 4.12 (4 H, s, NCH2-py), 4.25 (8 H, s, NCH2-bz), 7.49−7.58 (8 H, m, H-bz), 7.82 (2 H, t, J = 6.74 Hz, H4-py), 7.82 (2 H, d, J = 7.92 Hz, H3-py), 8.37 (2 H, t, J = 7.92 Hz, H5-py), 8.55 (2 H, d, J = 5.56 Hz, H6-py). Anal. Calcd for C36H48N8·6HClO4·CH3OH: C, 36.20; H, 4.76; N, 9.13. Found: C, 36.30; H, 4.98; N, 9.41. The deprotonated compound was obtained by dissolving the precipitate in water and raising the pH to 13 with a NaOH solution and then extracted with dichloromethane (3 × 50 mL). The organic layer was dried with anhydrous Na2SO4 and evaporated to give a yellow oil. Overall yield: 0.767 g (59%). 1H NMR (400 MHz, CDCl3): δ 1.87 (4 H, s, NH), 2.66 (16 H, m, NCH2CH2N), 3.63 (8 H, s, NCH2-bz), 3.72 (4 H, s, NCH2-py), 7.10−7.12 (6 H, m, H2,H4-bz + H5-py), 7.15−7.19 (2 H, m, H3-bz), 7.22 (2 H, s, H6-bz), 7.33 (2 H, d, J = 7.7 Hz, H3-py), 7.60 (2 H, t, J = 7.7 Hz, H4-py), 8.47 (2 H, d, J = 7.7 Hz, H6-py). 13C NMR (400 MHz, CDCl3): δ 47.13 (NCH2CH2N), 53.85 (NCH2-bz), 54.26 (NCH2CH2N), 61.29 (NCH2-py), 122.08 (C5-py), 123.02 (C3-py), 126.70 (C2-bz), 127.63 (C6-bz), 128.32 (C3-bz), 136.52 (C4-py), 140.72 (C1,5-bz), 149.23 (C6-py), 160.00 (C2-py). ESI-MS (H2O/ CH3OH): m/z 593.3 ([M + H]+). See Figures S12−S19 in the Supporting Information. Synthesis of [Cu2L(Cl)2⊃Cl]Cl·2H2O. An aqueous solution of 0.05 M CuCl2 (0.16 mL, 0.008 mmol) was added to L·6HCl (3.25 mg, 0.004 mmol) in aqueous solution. The pH was adjusted to 5.5 with diluted aqueous KOH and the solution (2 mL) stirred at room temperature for 2 h. Then the solution was evaporated to dryness, the complex was dissolved in methanol, and the insoluble matter was filtered off. This procedure was repeated until no more precipitation was observed. The green solution was left slowly evaporating at room temperature. Blue single crystals suitable for X-ray crystallographic determination were

Figure 9. Dicopper(II) complex inhibiting STAT3 translocation in living cells. U251 human glioma cells were treated with LIF (100 ng/ mL) for 0, 15, 30, and 60 min in the presence or absence of the inhibitor, and nuclear proteins were extracted and submitted to electrophoresis. LIF induced a time-dependent increase in the STAT3 levels in the nucleus, as expected, peaking at 60 min. The inhibitor completely blocked the STAT3 translocation. Lamin B is a nuclear protein that was used as a loading control.

ation was arrested. These results suggest that the dicopper(II) complex of L is bioactive in living cells and has oncostatic properties that may be of interest for the fight against cancer and other pathologies involving the STAT3 protein.

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EXPERIMENTAL SECTION

General Considerations. All solvents and reagents used were purchased with reagent grade quality and used as supplied without further purification, except 4-(2-pyridylmethyl)-1,4,7-triazaheptane, which was prepared according to literature methods.22 The H2pST3 peptide was obtained commercially through CASLO ApS (potentiometric titration, NMR, high-performance liquid chromatography, and mass spectrometry can be found in Figures S7−S11 in the Supporting Information). NMR spectra used for characterization of the products were recorded on a Bruker Avance III 400 instrument operating at a central proton frequency of 400.13 MHz and equipped with a twochannel 5 mm direct-detection broad-band probe with Z gradients. Spectra used in the 1H and 31P NMR studies were recorded on a Bruker Avance III 800 instrument operating at a central proton frequency of 800.33 MHz and equipped with a three-channel 5 mm inverse-detection probe with Z gradients. The reference used for the 1 H NMR measurements in CDCl3 was tetramethylsilane, and that in D2O was 3-(trimethylsilyl)-propanoic acid-d4 sodium salt. Peak assignments are based on peak integration and multiplicity for 1D 1 H NMR spectra and on COSY, NOESY, and HMQC experiments. Microanalyses were carried out by the ITQB Analytical Service. Caution! Although no problems were found during this work with the perchlorate salts, these compounds should be considered potentially explosive. G


Article

Inorganic Chemistry obtained within a few days. ESI-MS (H2O): m/z 825.0 ([Cu2L(Cl)3]+) (see Figure S20 in the Supporting Information). Single-Crystal X-ray Diffraction. Crystals of [Cu2L(Cl)2⊃Cl]Cl· 2H2O suitable for an X-ray diffraction study were mounted with Fomblin in a cryoloop. Data were collected on a Bruker AXS-KAPPA APEX II diffractometer with graphite-monochromated radiation (Mo Kα, λ = 0.71073 Å) at 150 K. The X-ray generator was operated at 50 kV and 30 mA, and the X-ray data collection was monitored by the APEX223 program. All data were corrected for Lorentzian, polarization, and absorption effects using the SAINT24 and SADABS24 programs. SIR9724 and SHELXS-9725 were used for structure solution, and SHELXL-97 was used for full-matrix least-squares refinement on F2. These three programs are included in the package of programs WINGX, version 2014.1.26 Non-hydrogen atoms were refined anisotropically. A full-matrix least-squares refinement was used for the non-hydrogen atoms with anisotropic thermal parameters. All of the hydrogen atoms connected to carbon atoms were inserted in idealized positions and allowed to refine in the parent carbon atom. The hydrogen atoms connected to the nitrogen atom and in the water molecules were located from the electron density map. Molecular diagrams presented are drawn with PyMOL.27 PLATON28 was used to calculate bond distances and angles as well as hydrogen-bonding interactions. Table 4 summarizes data collection and refinement details.

prepared from a Merck ampule diluted to 1000 mL of water (freshly boiled for about 2 h and allowed to cool under nitrogen). These solutions were discarded every time when the carbonate concentration was about 0.5% of the total amount of base. The titrant solutions were standardized by Gran’s method.30 Equipment and Working Conditions. The equipment used was described before.31 A Metrohm 6.0123.100 glass electrode and a Metrohm 6.0733.100 Ag/AgCl reference electrode were used for the measurements. The ionic strength of the experimental solutions was kept at 0.10 ± 0.01 M with KNO3, and the temperature was maintained at 298.2 ± 0.1 K. Atmospheric CO2 was excluded from the titration cell during experiments by passing purified nitrogen across the top of the experimental solution. Measurements. [H+] of the solutions was determined by measurement of the electromotive force of the cell, E = E′° + Q log [H+] + Ej. The term pH is defined as −log [H+]. E′°, Q, Ej, and K′w were determined by titration of a solution of known hydrogen-ion concentration at the same ionic strength, using the acid pH range of the titration. The liquid-junction potential, Ej, was found to be negligible under the experimental conditions used. The value of K′w was determined from data obtained in the alkaline range of the titration, considering E′° and Q valid for the entire pH range and found to be equal to 10−13.78 in our experimental conditions. Before and after each set of titrations, the glass electrode was calibrated as a [H+] probe by titration of a 1.000 × 10−3 M standard HNO3 solution with a standard KOH solution. Every measurement was carried out with 0.040 mmol of the ligand L in a total volume of 40.00 mL, except for the measurements regarding the peptide, where 0.010 mmol of the peptide in a total volume of 15.00 mL was used. The exact concentration of L·6HNO3 and of the peptidic substrate was obtained by titration with a standard KOH solution. Experiments with complexes of L and metal cations were performed in the presence of Cu(NO3)2 or Zn(NO3)2 in 1:1 and 2:1 CM2+/CL ratios. The ternary system measurements were carried out in the simultaneous presence of L, Cu(NO3)2, and substrates at 2:1:1 M:L:S ratios (L = ligand, M = metal, and S = substrate). In each titration, 85−120 points were collected, and a minimum of two titrations were performed. The substrates were independently titrated alone and in the presence of copper(II) ion at 0.5:1, 1:1, and 2:1 CCu/CS ratios and the respective equilibrium constants used in the calculations. Backtitrations with a standard HNO3 solution were performed to confirm stabilization of the acquired data. Calculation of Equilibrium Constants. The overall protonation constants, βHi , of the L and studied substrates, the overall stability constants of complexes, βMmHhLl, and the overall association constants of the complexes of L with the substrates, βMmHhLlSs, were calculated by fitting the potentiometric data obtained for all of the performed titrations in the same experimental conditions with the Hyperquad program.32 The hydrolysis constants for copper(II) and zinc(II) were held constant during data refinement.33 The initial computations were obtained in the form of the overall constants, βHhL = [HhLl]/[H]h[L]l, βMmHhLl = [MmHhLl]/[M]m[H]h[L]l or βMmHhLlSs = [MmHhLlSs]/ [M]m[H]h[L]l[S]s. The errors quoted are the standard deviations of the overall constants given directly by the program for the input data, which include all of the experimental points of all titration curves. The errors quoted for the stepwise constant values were calculated using the propagation rules. The HySS program34 was used to calculate the concentration of equilibrium species from the calculated constants from which distribution diagrams were plotted. The species considered in a particular model were those that could be justified by the principles of coordination and supramolecular chemistry.

Table 4. Crystallographic Data and Experimental Details for [Cu2L(Cl)2⊃Cl]Cl·2H2O chemical formula fw temperature (K) wavelength (Å) cryst form, color cryst size (mm) cryst syst space group a (Å) b (Å) c (Å) α (deg) β (deg) γ (deg) V (Å3) Z d (mg cm−3) μ (mm−1) θ range (deg) reflns collected/unique Rint GOF final R indices [I > 2σ(I)]

R1 = ∑||F o | − |F c ||/∑|F o |. ∑[w(Fo2)2]]1/2. a

C36H52O2Cl4Cu2N8 897.73 150(2) 0.71073 block, blue 0.18 × 0.12 × 0.10 triclinic P1̅ 8.8195(7) 14.541(1) 17.658(1) 67.988(4) 83.402(4) 73.402(4) 2015.4(3) 2 1.479 1.364 2.509−26.492 25001/8167 0.0461 1.032 R1 = 0.0439,a wR2 = 0.1031b b

wR2 = [∑[w(F o 2 − F c 2 ) 2 ]/

CCDC 1056038 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/ data_request/cif. Potentiometric Measurements. Reagents and Solutions. All of the solutions were prepared using demineralized water (obtained by a Millipore/Milli-Q system). A stock solution of L·6HNO3 was prepared at ca. 2.0 × 10−3 M by dissolving L in 0.012 M HNO3. Phenyl phosphate and peptide solutions were prepared from the corresponding acids. Stock solutions of Cu(NO3)2 and Zn(NO3)2 (analytical grade) were prepared at about 5.00 × 10−2 M, and the exact concentrations were checked by titration with K2H2edta following standard methods.29 Carbonate-free solutions of the KOH titrant were

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NMR STUDIES H NMR Studies. A stock solution of [Zn2L]4NO3 was prepared by adding 2 equiv of Zn(NO3)2 to 1 equiv of L in water and adjusting the pH to 7.2 with HNO3 or KOH solutions. Solutions of H2pST3 in the presence of 1 equiv of [Zn2L]4+ were prepared in 90:10 (v/v) H2O/D2O at 2 mM at H

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Article

Inorganic Chemistry

nescence. Images were acquired by means of a Chemidoc device (Bio-Rad Laboratories, Inc., Hercules, CA). Cell Number/Viability Assays. In order to evaluate the bioactive properties of the inhibitor in living cells, we determined the cell number and viability by means of 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays, as previously described.36,37 Very briefly, the MTT assay is based on the metabolic reduction of MTT to formazan, producing a colored precipitate proportional to the number of live cells. The LDH release assay measures the activity of LDH in the extracellular medium. LDH is a cytosolic enzyme, and it is only found outside cells when cell membranes break during cell death. Therefore, an increase in the LDH activity indicates cell death. During these procedures, the morphology of the cells was also monitored to ensure that cells actually look healthy. HT22 cells were kindly provided by Dr. David Schubert (The Salk Institute for Biological studies, La Jolla, CA). U251 human glioma cells and C6 rat glioma cells were obtained from ECACC (calalog nos. 09063001 and 9209040, respectively), and HEK293 human kidney cells were purchased from ATCC (CRL-1573).

pH 7.2. The pH was adjusted to 7.2 by adding small amounts of HNO3 or KOH solutions. Solutions of the substrate in the absence of the receptor were also prepared at the same concentration and at the same pH values. 31 P NMR Studies. Solutions of H2pST3 were prepared in 90:10 (v/v) H2O/D2O at 2 mM at pH 7.2 and titrated by adding aliquots of a stock solution of [Zn2L]4NO3 using a Hamilton syringe. The pH was adjusted to 7.2 by adding small amounts of HNO3 or KOH solutions. In all cases, an internal capillary tube containing D2O and H3PO4 was used for locking and referencing purposes during spectral acquisition. Biological Assays. EMSAs. EMSAs were carried out by means of the LightShift Chemiluminescent EMSA Kit (Life Technologies Ltd., Carlsbad, CA) following manufacturer instructions. Briefly, 6 μg of nuclear extracts from human kidney cells (HEK293) containing STAT3 was incubated with 1 ρmol of a DNA probe containing the STAT3 response element (5′-GATCCTTCTGGGAATTCCTAGATC-3′) and labeled with biotin, in the presence of different concentrations of the inhibitor, for 20 min at room temperature. Samples were then submitted to electrophoresis in a native TBE 1x/ acrylamide 4% gel, transferred to a nylon membrane (Hybond N+, Amersham Biosciences, Piscataway, NJ), and cross-linked with ultraviolet light to fix DNA and DNA/protein associations to the membrane. Membranes were then blocked, incubated with a streptavidin−horseradish peroxidase conjugate, and washed four times before the addition of the detection reagents (luminol/enhancer solution). Chemiluminescence was measured by means of a Chemidoc device (Bio-Rad Laboratories, Inc., Hercules, CA). A free, biotin-labeled DNA probe is smaller and runs faster in the gel, appearing at the bottom of the membrane. The STAT3 dimer/DNA association is heavier and therefore suffers a shift, appearing in the middle of the membrane. The signal is therefore proportional to the amount of STAT3 bound to the DNA probe. The STAT3 dimer/DNA association signals were measured using the ImageJ software35 (NIH, http://imagej.nih.gov/ij/) and plotted as the relative band intensity against inhibitor concentration, from which IC50 was derived. Several assays were conducted to confirm the authenticity of the results, and the IC50 value presented is an average of all values obtained. Control experiments were made in order to verify whether the band really corresponded to the STAT3 dimer/DNA, one in which the reaction mixture had no nuclear extracts and another one with an excess of unlabeled DNA (1:200). Also, to confirm that the dicopper(II) complex of L was the only compound interacting with the STAT3 dimer, reaction mixtures were prepared, one containing 2 μL of a Cu(NO3)2 solution (1.5 mM) and the other containing 2 μL of a L·6HNO3 solution (1.5 mM) (see Figure S21 in the Supporting Information). Western Blotting. U251 human glioma cells were treated with LIF in the presence or absence of the inhibitor, and nuclear proteins were isolated using the NE-PER nuclear extraction kit (Life Technologies Ltd., Carlsbad, CA), following the manufacturer instructions. A total 40 μg of nuclear extracts was submitted to electrophoresis in an acrylamide gel (10% w/ v), transferred to a nitrocellulose membrane, and incubated with a primary antibody against STAT3 (1:1000, Cell Signaling Technologies, Danvers, MA) or Lamin B (1:1000, Santa Cruz Biotechnologies, Paso Robles, CA). Membranes were then washed three times and incubated with a secondary horseradish peroxidase-conjugated antibody for detection by chemilumi-

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b00116. Tables of the overall protonation constants of the peptide and L, overall stability constants of copper(II) and zinc(II) with L, and overall association constants with substrates, of the hydrogen-bonding details of [Cu2L(Cl)2⊃Cl]Cl·2H2O, and of the 1H chemical shifts of free and bound phosphopeptide and figures of the potentiometric titrations of L and pST3, of species distribution, of the X-ray crystal structure of [Cu2L(Cl)2⊃Cl]Cl·2H2O to highlight the conformation adopted, of 1D and 2D NMR and ESI-MS spectra of the L compound, phosphopeptide, and copper(II) complex, and of the EMSA control experiment (PDF) X-ray crystallographic data in CIF format (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (P.M.). *E-mail: [email protected] (R.D.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the Fundaçaõ para a Ciência e a Tecnologia (FCT) for financial support and a fellowship to L.M.M. under Project PTDC/QEQ-SUP/2718/2012. F.H. is supported by Contract IF/00094/2013 from the FCT. The authors also acknowledge support by the FCT (Grant RECI/ BBB-BQB/0230/2012) for the NMR spectrometers as part of the National NMR Facility. The X-ray facilities thank the FCT for funding (Grant RECI/QEQ-QIN/0189/2012). M. C. Almeida from the ITQB Analytical Services Unit is acknowledged for providing elemental analysis and ESI-MS data. The authors thank Dr. Olga Iranzo for supplying the nonphosphorylated peptide. C.V.E., P.M., and V.A. acknowledge I


Article

Inorganic Chemistry

(18) Addison, A. W.; Rao, T. N.; Reedijk, J.; van Rijn, J.; Verschoor, G. C. J. Chem. Soc., Dalton Trans. 1984, 1349−1356. (19) This peptide was kindly supplied by Dr. Olga Iranzo from the Institut des Sciences Moléculaires de Marseille, Aix Marseille Université, Marseille, France. (20) Ojida, A.; Mito-oka, Y.; Sada, K.; Hamachi, I. J. Am. Chem. Soc. 2004, 126, 2454−2463. (21) Kaneko, S.; Nakatani, Y.; Takezaki, T.; Hide, T.; Yamashita, D.; Ohtsu, N.; Ohnishi, T.; Terasaka, S.; Houkin, K.; Kondo, T. Cancer Res. 2015, 75, 4224−34. (22) Arbuse, A.; Font, M.; Martínez, M. A.; Fontrodona, X.; Prieto, M. J.; Moreno, V.; Sala, X.; Llobet, A. Inorg. Chem. 2009, 48, 11098− 11107. (23) APEX2; Bruker Analytical Systems: Madison,WI, 2005. (24) Altomare, A.; Burla, M. C.; Camalli, M.; Cascarano, G. L.; Giacovazzo, C.; Guagliardi, A.; Moliterni, A. G. G.; Polidori, G.; Spagna, R. J. Appl. Crystallogr. 1999, 32, 115−119. (25) Sheldrick, G. M. Acta Crystallogr., Sect. A: Found. Crystallogr. 2008, 64, 112−122. (26) Farrugia, L. J. J. Appl. Crystallogr. 1999, 32, 837−838. (27) DeLano, W. The PyMOL Molecular Graphics System, version 1.2r3pre, http://www.pymol.org. (28) Spek, A. L. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2009, 65, 148−155. (29) Schwarzenbach, G.; Flaschka, W. Complexometric Titrations; Methuen & Co.: London, 1969; pp 252−268. (30) Rossotti, F. J.; Rossotti, H. J. J. Chem. Educ. 1965, 42, 375−378. (31) Esteves, C. V.; Madureira, J.; Lima, L. M. P.; Mateus, P.; Bento, I.; Delgado, R. Inorg. Chem. 2014, 53, 4371−4386. (32) Gans, P.; Sabatini, A.; Vacca, A. Talanta 1996, 43, 1739−1753. (33) (a) Pettit, L. D.; Powell, H. K. J. IUPAC Stability Constants Database; Academic Software: Timble, Yorks, U.K., 2003. (b) Martell, A. E.; Smith, R. M.; Motekaitis, R. J. Critically selected stability constants of metal complexes database. NIST Standard Reference Database, version 5.0; National Institute of Standards and Technology; Washington, DC, 1998. (34) Alderighi, L.; Gans, P.; Ienco, A.; Peters, D.; Sabatini, A.; Vacca, A. Coord. Chem. Rev. 1999, 184, 311−318. (35) (a) Rasband, W. S. ImageJ; U.S. National Institutes of Health: Bethesda, MD, 1997−2014; http://imagej.nih.gov/ij/. (b) Schneider, C. A.; Rasband, W. S.; Eliceiri, K. W. Nat. Methods 2012, 9, 671−675. (c) Abramoff, M. D.; Magalhaes, P. J.; Ram, S. J. Image Processing with ImageJ; Utrecht University Repository; Utrecht University: Utrecht, The Netherlands, 2004; Vol. 11, p 36−42. (36) Martín, V.; Sainz, R. M.; Antolín, I.; Mayo, J. C.; Herrera, F.; Rodríguez, C. J. Pineal Res. 2002, 33, 204−12. (37) Herrera, F.; Mayo, J. C.; Martín, V.; Sainz, R. M.; Antolín, I.; Rodriguez, C. Cancer Lett. 2004, 211, 47−55.

the FCT for fellowships under Grants SFRH/BD/89501/2012, SFRH/BPD/79518/2011, and SFRH/BPD/78854/2011, respectively. This work was also supported by the FCT through the MOSTMICRO R&D Unit (Grant UID/CBQ/04612/ 2013).

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Inhibition of the STAT3 Protein by a Dinuclear Macrocyclic Complex

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