Influence of DMβCD on the Interaction of Copper(II) Complex of 6-Hydroxychromone-3-carbaldehyde-3-hydroxybenzoylhydrazine with ctDNA.

The interaction mechanism between a scarcely soluble copper(II) complex of Cu(II)-6-hydroxychromone-3-carbaldehyde-(3'-hydroxy)benzoylhydrazone (CuCHz) in aqueous solution and its DMβCD complex was studied in the presence of ctDNA through spectroscopy and thermodynamic methods. The thermodynamic results indicate that the binding process of the CuCHz-DMβCD inclusion complex is a spontaneous process and the inclusion is enthalpy-driven. The binding constants of CuCHz and CuCHz-DMβCD with ctDNA are 2.69 × 103 and 14.7 × 103 L mol-1, respectively. The stoichiometry of the complex is 1:1, and the determined thermodynamic indicates that the process of binding is spontaneous and entropy-driven. A competitive binding titration with ethidium bromide revealed that CuCHz efficiently displaces EB from the EB-DNA system. In addition to the thermal denaturation experiments and docking studies, we can confirm that the mode of binding of this complex to ctDNA is intercalation mode. The presence of DMβCD enhances the aqueous solubility of CuCHz; nevertheless, the cyclodextrin did not affect the interaction of CuCHz with ctDNA because the inclusion complex breaks down when it binds with ctDNA.

Introduction

Design of small molecules that bind and react at specific sequences of deoxyribonucleic acid ctDNA is very important in the development of new therapeutic reagents. ctDNA is generally the primary intracellular target of anticancer drugs, so the interaction between small molecules and ctDNA can often cause ctDNA damage in cancer cells, blocking their division with concomitant cell death.[1]

Chromone(1-benzopyran-4-one) moiety as a ligand scaffold represents a class of naturally occurring molecules.[2] They have attracted attention in recent years because of their diverse pharmacological properties such as antimycobacterial, antifungal, anticancer, antioxidant, antihypertensive, and anti-inflammatory effects.[3,4] Transition-metal complexes obtained from 3-formylchromone Schiff bases have still received considerable attention, in view of their chelating ability exhibiting efficient ctDNA binding.[5−10] This type of compounds bearing N and O donors has structural similarities to neutral biological systems in which the C=N linkage of azomethine derivatives is essential for their displayed biological activity. Also, it has been demonstrated that this chelating activity is enhanced on chelation with metal atoms due to the increase in the planarity of the intercalators, allowing the insertion of the complexes and their stacking between the base pairs of double-helical ctDNA more easier than the free ligands.[7,10] A number of metal complexes (Cu(II), Zn(II), Pd(II), Pt(II), Ni(II), La(III), Ru(II), Sm(III), and Nd(III)) with Schiff base derivative have been reported, and their ctDNA-binding properties have been studied.[5−13] Nevertheless, these metal complexes have a disadvantage due to their water solubility, which is still unsatisfactory, restricting their use as anticancer agents.[14,15]

Drug carrier vehicles play an important role in the active transport of drugs in both increasing concentration and effectiveness of drugs at the required site. In this context, cyclodextrins (CDs) play a pivotal role in supramolecular delivery applications due to their nature as macrocyclic hosts, as reported in many drug formulations.[16] CDs are cyclic oligosaccharides with a cagelike structure formed by α-1,4-linked d-glucopyranose units. CDs have the shape of a truncated cone with internal cavities ranging from 5 to 8 Å. The C–H bonds (H-3′, H-5′) on the ring point inward, producing a hydrophobic cavity. In this conformation, electron lone pairs belonging to glycosidic oxygens remain toward the interior of the CD cavity, thus increasing the electron density along with the Lewis base character of the latter.[17] Cyclodextrins and modified cyclodextrins form inclusion complexes capable of binding substrates that quickly, selectively, and reversibly act as catalysts in a variety of chemical reactions.[18] It is very well known that hydrophobic molecules and/or some hydrophobic moieties display an increased affinity for CD’s cavity in the aqueous solution. In fact, the capability of cyclodextrins to improve and enhance not only the solubility but also the stability of several drugs is known to be mediated through the formation of inclusion complexes.[19] The stability of these inclusion complexes is determined by the fit of the entire or at least the hydrophobic part of the guest molecule. On the other hand, modified cyclodextrin derivatives like methylation of the hydroxyl groups have attracted interest because they have greater water solubility. In this sense, dimethyl-β-cyclodextrin has been used to include diverse drugs like paclitaxel,[20,21] hydroxymethylnitrofurazone,[22] luteolin,[23] 4-hydroxycoumarin,[24] catechin,[25] (−)-epigallocatechin galate,[26] and morin,[27] improving their complexing ability and stabilization of the incorporated drug. Even though cyclodextrins are recognized by the ability to form such complexes as reported elsewhere (vide supra), reports on drugs that bind to ctDNA and solubilized by cyclodextrin are scanty.[28−35] Also, cyclodextrins are recognized to increase the solubility of complexes and/or to modulate the binding of small molecules to ctDNA by selectively blocking/orienting the drug upon its binding.

Copper(II) complexes have attracted considerable attention owing to their high affinity for nucleobases and their capability of interacting directly with ctDNA that leads to cell cycle arrest and in turn to cell death.[36] Therefore, this study contributes to improving the aqueous solubility of the copper(II)-6-hydroxychromone-3-carbaldehyde-(3′-hydroxy)benzoylhydrazone (CuCHz) complex through inclusion in DMβCD and also leads to a better understanding of the effect of cyclodextrins on the properties and functionality of drug–ctDNA complexes. Different methods were employed to investigate the ctDNA-binding ability of CuCHz. This included fluorescence titration at different temperatures to determine thermodynamic parameters, fluorescence displacement assays, and thermal denaturation and molecular docking studies.

Results and Discussion

One-Pot Synthesis of Copper(II) Complex (CuCHz)

The mixture of copper(II) chloride dihydrate (4 mmol), 6-hydroxy-chromone-3-carboxaldehyde (2 mmol), and 3-hydroxybenzoylhydrazine (2 mmol) in ethanol was stirred and heated under reflux for 2 h (Scheme ). The resulting green precipitate, copper(II) complex, was collected by filtration, washed several times with ethanol, and dried overnight at 80 °C. Yield, 82%; mp, 277 °C (decomp.). FTIR (cm–1) 3264 (ν NH), 3436–3092 (ν OH), 1638 (ν C=O chromone), 1605 (ν C=O hydrazone), 1569 (ν C=N). HRMS (C17H12Cl2CuN2O5): [M–H]− = 457.9292. Anal. calcd for C17H12Cl2CuN2O5: C, 44.51%; H, 2.64%; N, 6.11%. Found: C, 43.93%; H, 2.56%; N, 5.91% (Figures S1–S3).

Scheme 1

One-Pot Synthesis of the Copper(II) Complex of 6-Hydroxychromone-3-carbaldehyde-3-hydroxybenzoylhydrazine (CuCHz)

The copper(II) complex is easily soluble in DMF and DMSO; slightly soluble in ethanol, methanol, water, and acetone; and insoluble in benzene and diethyl ether. The UV spectra had a strong band at λmax = 260 nm, a medium band at λmax = 372 nm, and a weak band at λmax = 432 nm. Fluorescence spectra have a maximum at 440 nm with an excitation wavelength of 335 nm with bandwidths for excitation and emission of 5 and 12 nm, respectively. Even though the complex was formed by one-pot synthesis, characterization of CHz (6-hydroxychromone-3-carbaldehyde-(3′-hydroxy)-benzoylhydrazone) and the NMR titration curve with Cu(II) was recorded to confirm the complex formation (Figures S4–S9).

The stability of CuCHz in the aqueous solution has been studied by observing the UV–vis spectrum and remained unaltered for 24 h. The molar conductivity of the Cu(II) complex is 88.3 S cm2 mol–1 in DMSO, showing that it is 1:1 electrolyte, indicating that the chloride anion is out of the coordination sphere as a counter ion.[37] The thermogram show no weight loss up to 250 °C for CHz and 200 °C for CuCHz, confirming the stability of the copper(II) complex as well as the absence of any water molecule in/out of the coordination sphere (Figure S3). The magnetic moment of the complex CuCHz is 1.70 BM, giving an indication of a one-electron paramagnetic d9–Cu(II) center that suggests a square planar complex.[38] The characterization of the obtained complex indicates that the structure is [Cu(CHz)Cl]Cl.

The IR spectra of Cu(II)-6-hydroxychromone-3-carbaldehyde-(3′-hydroxy)benzoylhydrazone (CuCHz) show a broad band at around 3000 cm–1 due to the presence of OH and NH moieties. Also, a strong band at 1569 cm–1 is observed due to the stretching vibration of the ν(C=N) group. This signal was shifted to a lower frequency compared to ν(C=N) of CHz, which indicates the coordination of azomethine nitrogen to the metal ion. This shift may be the result of a transfer of the electron density from the donor atom (N) toward the metal center, leading to weakening of the ν(C=N) absorption band. As well, two characteristic carbonyl stretching frequencies are observed at 1638 and 1605 cm–1 that are assigned to ν(C=O)chromone and ν(C=O)hydrazone, moieties, respectively. The latter signals are shifted to a lower wavenumber compared to the vibration of CHz, indicating that both carbonyl oxygen and azomethine nitrogen atoms could participate as an ONO-tridentate ligand[39,40] in the presence of copper. Besides different tautomeric forms that can exist for CHz, there is only one structural copper complex that takes place and is definitively that poses two carbonyl groups.

When increased concentrations of DMβCD are added to the reaction medium containing CuCHz, the recorded absorption spectra show only minor changes; however, the effect of DMβCD on the fluorescence spectra is more pronounced. At pH 7.4, the emission maximum is at 440 nm and the addition of increasing concentrations of DMβCD from 0 to 2.65 mmol L–1 resulted in a corresponding increase in the fluorescence signal with a slight displacement to lower wavenumber (Figure ). One of the results of formation inclusion complexes, in our case CuCHz and DMβCD, is the increase in the observed fluorescence intensity, which could be due to the change in the microenvironment of CuCHz as it entered in the cavity of DMβCD. To determine the stoichiometry of this complex, we analyzed the intensity changes in the emission spectra according to the Benesi–Hildebrand plot,[41] results that confirmed a 1:1 stoichiometry corroborated by a straight line by the continuous variation method (Figure S11).

Figure 1

Fluorescence intensity of CuCHz (1 × 10–5 mol L–1) in the presence of various concentrations of DMβCD (0.2–2.6 × 10–3 mol L–1) in phosphate buffer, pH 7.4. Inset: Double reciprocal plot 1/ΔF versus 1/[DMβCD] obtained from the fluorescence intensities recorded for the CuCHz–DMβCD complexes. λexc, 335 nm; λem, 440 nm. Excitation and emission bandwidths were set at 5 and 12 nm, respectively.

The association constants, Ka, of the inclusion complexes between the Cu(II) complex (CuCHz) and cyclodextrin at different temperatures (298, 308, and 318 K) were calculated from the double reciprocal plot, and the results are summarized in Table . From Table , it is possible to notice that the determined association constant for the CuCHz–DMβCD complex decreases when the temperature increases, in total agreement with an exothermic process. This could be thought of as a decreasing degree of interaction occurring at higher temperatures due to the weakening of hydrogen bonds in the heating process.

Table 1

Association Constant (Ka) of the CuCHz–DMβCD Complex at Different Temperatures and Thermodynamic Parameters

T (K)	Ka (L mol–1)	ΔG (kJ mol–1)	ΔH (kJ mol–1)	ΔS (kJ mol–1 K–1)
298	240 ± 11	–13.60	–11.58	6.60 × 10–3
308	199 ± 37
318	179 ± 33

The thermodynamic parameters (ΔG, ΔH, and ΔS) for the formation of inclusion complexes were determined from the temperature dependence of the apparent formation constants using the classical van’t Hoff equation and plotting ln K versus 1/Twhere K is the associative binding constant corresponding to various temperatures and R is the gas constant (8.314 J K–1 mol–1). The standard-state enthalpy change (ΔH°) can be calculated from the slope of the van’t Hoff relationship, the standard-state entropy change (ΔS°) can be calculated from the intercept, and the standard-state Gibbs energy change (ΔG°) can be estimated from the following relationshipIn CD complex formation, several driving forces must be considered, namely, hydrogen bonding formation among hydroxyl groups of CDs and the guest molecules. Also, van der Waals interactions play an important role in host–guest molecules. Hydrophobic and the expulsion of “high energy waters” from the interior of cyclodextrin toward bulk water must be considered. The result of the changes can be noticed in large negative enthalpy and in entropy values (either negative or slightly positive), which can be attributed to strong van der Waals interactions and hydrogen bond formation at the interior of the hydrophobic cavity, leading to guest inclusion without extensive desolvation.[42] According to the values of ΔH° and ΔS° given in Table , the inclusion process of CuCHz with DMβCD might correspond to an enthalpy-driven process. The negative value of the standard Gibbs energy change (ΔG), which is the result of the interplay of enthalpy and entropy changes, indicates in our case that the formation of the inclusion complex is a spontaneous process.

The electronic structure of the Cu(II) complex was obtained by means of density functional theory (DFT) calculations. The molecular geometry was fully optimized using Gaussian 09 at the B3LYP level. Nonmetal atoms were described with B3LYP/6-31G(d), and Cu(II) atom was treated by B3LYP/LANL2DZ basis sets. The optimized structure is shown in Figure a. With this conformation at hand, we used a previously optimized DMβCD[28] to carried out molecular docking studies using the AutoDock 4.2 version program to determine the most stable structure of the Cu(II) complex. Then, the most stable conformation for the Cu(II) complex was chosen according to the best docking score. According to this result, only one orientation of the guest complex and the host molecule (DMβCD) was found where the chromone moiety of CuCHz remains toward the primary ring and the benzoyl ring toward the secondary rim. In this attained conformation, it is possible to observe three hydrogen bonds namely, a hydrogen bond between the OH group of benzoyl and 2-OCH3 in the secondary face at 2.2 Å. Another hydrogen bond remains with the anomeric oxygen of the chromone moiety at 1.9 Å toward the primary face of DMβCD. The N–H group in that conformation forms a hydrogen bond with the opposite anomeric oxygen at 1.8 Å. In that actual conformation, the complex remains completely embedded in the CD hydrophobic cavity (Figure b).

Figure 2

(a) Fully optimized molecular geometry of CuCHz and (b) molecular structure of the most stable CuCHz–DMβCD complex, as calculated by docking studies.

Interaction studies of metal complexes with ctDNA are significant for designing new drugs. Spectral changes in absorbance or fluorescence are important to find out the possible mechanism of binding of the compound with ctDNA. The absorption spectrum of CuCHz forming an inclusion complex with DMβCD presents two bands corresponding to 268 and 423 nm. In the presence of an increasing concentration of ctDNA, a marked decrease in the absorbance at 423 nm (approximately 33.16%) and a progressive blue shift of 8 nm (Figure S12) are observed, indicative of an interaction between CuCHz–DMβCD and ctDNA, which could be due to a stacking interaction between the aromatic chromophore and the base pair of ctDNA.[35,43]

We used fluorescence spectroscopy to analyze the interactions occurring between CuCHz with ctDNA, considering the presence (or the absence) of DMβCD at fixed concentrations of CuCHz and increased concentrations of ctDNA (Figures S13 and S14). In the case of fluorescence, a significant increase in the fluorescence emission is normally observed for intercalation modes of interaction.[44] The degree of freedom of guest molecules, especially rotations, favors the deactivation of excited states. However, when molecule CuCHz, that is used in this study, is bound to ctDNA, it becomes rigid, favoring the emission fluorescence with an observed concomitant increase in the emission. Other interactions, namely, electrostatic, hydrogen bonding, or hydrophobic, known to occur in groove binding agents result in a decrease in the fluorescence intensity in the presence of ctDNA mainly due to their closeness to the sugar–phosphate backbone.[45] The Cu(II) complex emits weak fluorescence in Tris buffer with a maximum wavelength of about 440 nm. Upon addition of increasing concentrations of ctDNA, there is an enhancement in the emission intensity, indicative of a certain type of interaction with ctDNA (Figure ). In the presence of cyclodextrin, the behavior is to some extent alike; there is still an enhancement of fluorescence in the presence of ctDNA without a significant shift of the emission wavelength maximum in the aqueous solution. This increase in the emission intensity agrees well with that observed for other intercalators,[46] suggesting that CuCHz interacts with ctDNA and is protected from solvent water molecules by the hydrophobic environment inside the ctDNA helix. Nevertheless, the cyclodextrin complex interacts with ctDNA more efficiently, which could be due to the enhancement of the solubility of the Cu(II) complex.

Figure 3

Relative changes in the fluorescence intensity (F/F0) of CuCHz (10 μM) and CuCHz–DMβCD (10 μM) with the increased addition of ctDNA (0–22.5 μM) in the buffer solution.

To compare the binding strength of both complexes with ctDNA, the binding constant, Kb, and the number of binding sites, n, from the fluorescence spectral changes on titration with ctDNA were determined using the following equationwhere Kb is the binding constant, n is the number of binding sites, F0 is the fluorescence intensity of the small molecule in the absence of ctDNA, while F is the fluorescence intensity of the small molecule in the presence of ctDNA, and [Q] is the concentration of ctDNA. Kb and n are easily calculated from the double logarithm regression curve of log(F0 – F)/F versus log[Q]. The values of Kb and n were estimated from the intercept and slope of the plot of log ΔF/F versus log[Q], respectively. The binding constant and the number of binding sites for the interaction between CuCHz and ctDNA in the absence and presence of cyclodextrin were determined (Table ). The values of n are approximately 1.3 for both Cu(II) complex (with and without cyclodextrin); this showed a 1:1 stoichiometric interaction of CuCHz and ctDNA, which indicates the existence of just a single binding site in ctDNA for CuCHz. The binding constant, Kb, of CuCHz with ctDNA at 298 K was 2.69 × 103 L mol–1 (correlation coefficient, 0.990), and in the presence of cyclodextrin, the Kb value increases to 14.7 × 103 L mol–1 (correlation coefficient, 0.986). It has been reported that cyclodextrin does not cause any DNA cleavage;[47] therefore, DMβCD besides enhancing the solubility of CuCHz selectively orients the drug molecule upon its binding to ctDNA.[28] From these results, we confirm that cyclodextrin is a good delivery nanovehicle for the supply of a drug to target ctDNA.

Table 2

Binding Constants (Kb) of CuCHz and the CuCHz–DMβCD Complex with ctDNA at Different Temperatures and Thermodynamic Parameters of CuCHz–DMβCD with ctDNA

	T (K)	log Kb	Ka (L mol–1)	n	R	ΔG (kJ mol–1)	ΔH (kJ mol–1)	ΔS (kJ mol–1 K–1)
CuCHz	298	3.43	2690	1.29	0.990
CuCHz–DMβCD	298	4.17	14 791	1.33	0.986	–24.76	3.58	9.20 × 10–2
CuCHz–DMβCD	308	4.19	15 488	1.36	0.991
CuCHz–DMβCD	318	4.21	16 218	1.35	0.985

As depicted in Table , the Kb values of CuCHz on ctDNA in the presence of cyclodextrin increase in the range of temperature from 298 to 318 K. This demonstrated that CuCHz has a good affinity for ctDNA and the stability of the association of CuCHz with ctDNA growth upon an increase in temperature.

The analysis of thermodynamic parameters including ΔG, ΔH, and ΔS is one of the effective methods for judging the mode of association of small molecules with biomolecules. As stated before, several interactions can be identified when it comes to describing the binding of drugs to a pharmaceutical target such as DNA, namely, hydrogen bonds, van der Waals forces, and electrostatic and hydrophobic interactions. Enthalpy and entropy changes can be determined when the dependence of Kb with temperature is analyzed by means of the van’t Hoff equation (eq ). Our results are given in Table . The sign and magnitude of several thermodynamic parameters associated with individual interactions occurring in the association process of drugs and macromolecules have been given elsewhere.[48,49] Using this description, we observed what kinds of interactions were the predominant ones. When ΔH < 0 or ΔH = 0 and ΔS > 0, the mainly acting force is electrostatic; when ΔH < 0, ΔS < 0, van der Waals interactions or hydrogen bonds dominate the reaction, and when ΔH > 0, ΔS > 0, the main force is hydrophobic. Thermodynamic parameters obtained are listed in Table , where the positive small values 3.58 kJ mol–1 for ΔH and 0.092 kJ mol–1 for ΔS between CuCHz–DMβCD and ctDNA indicated that the binding is mainly entropy-driven. This means that the hydrophobic forces play a major role in the binding; nevertheless, due to the low value of the enthalpy change, the other noncovalent interactions cannot be excluded.[50,51] In general, the complex of major/minor groove binding is stabilized by electrostatic, hydrogen bonding, and/or hydrophobic interactions;[52] however, the complex of intercalation, where a planar aromatic chromophore is inserted between two adjacent base pairs in a ctDNA helix, is a process that starts with the transfer of the intercalating molecule from a hydrophobic cavity (cyclodextrin) to the hydrophobic space between two adjacent ctDNA base pairs and is stabilized by hydrophobic interactions and van der Waals forces.[47] This suggests that entropy and enthalpy processes drive the interaction of CuCHz–DMβCD with ctDNA and this binding can be assigned to the intercalation mode.

To test whether the copper complex binds to ctDNA via intercalation, ethidium bromide was employed. To check whether the metal complexes have the ability to achieve complexation with ctDNA, competitive binding experiments are a valid method.[53] EB was selected as a fluorescence probe because of its known spectral properties, and it is widely used as a fluorescence chromophore marker for ctDNA. The fluorescence intensity of the EB–ctDNA system is much better than that of free EB due to the strong intercalation between the adjacent ctDNA base pairs. If the Cu(II) complex has the same binding mode with ctDNA as that of EB, the fluorescence-based competition technique can provide indirect evidence for the ctDNA-binding mode. This means that the fluorescence of the EB–ctDNA system can be quenched by the addition of a second species that is able to displace the EB molecules.

Competitive binding studies using ethidium bromide bound to ctDNA were carried out. Figure shows the variation of emission spectra of ctDNA pretreated with EB ([DNA]/[EB] = 1:1) with increasing concentration of the Cu(II) complex in the absence and presence of DMβCD. A fluorescence decrease of EB–ctDNA was observed at the maximum of 593 nm as the concentration of the copper(II) complex increases in the absence and presence of cyclodextrin. The results revealed the quenching of the initial fluorescence intensity about 10 and 18% for CuCHz with and without DMβCD, respectively, and in both situations, the displacement of EB occurs, which indicates that CuCHz in both forms could displace EB from the EB–ctDNA system.

Figure 4

Emission spectra of EB (0.33 μmol L–1) bound to ctDNA (15 μmol L–1) in the presence of (a) CuCHz and (b) CuCHz–DMβCD at different concentrations (0.5–2.5 × 10–5 mol L–1). Inset: fluorescence quenching curves of EB–ctDNA.

To further exclude the possible presence of other processes, thermal denaturation properties are investigated. The effect of denatured ctDNA was studied by heating a native ctDNA solution in a water bath at 100 °C for 10 min and then cooling in an ice–water bath immediately, accordingly to Wang et al.[54] Double-stranded ctDNA splits into two single-stranded ctDNA molecules with the opening of its double helix, so the interaction of CuCHz and CuCHz–DMβCD with denatured ctDNA will be different in comparison to that with double-stranded ctDNA. If the interaction is via intercalation, ctDNA accommodates the copper(II) complex in the helix, and on denaturation of the ctDNA helix, the intercalated molecules are released in the solution, leading an alteration in the fluorescence behavior. Table displays the intensity fluorescence ratios (F/F0) for CuCHz and CuCHz–DMβCD when is in the presence of different amounts of native and denatured ctDNA. From the table, it can be noticed a diminution of the fluorescence in denaturated ctDNA, which in turn supports CuCHz and CuCHz–DMβCD ctDNA intercalation.

Table 3

F/F0 Data for CuCHz and CuCHz–DMβCD Complexes in Different Amounts of Native and Denatured ctDNA

compound	concentration ctDNA (mol L–1)	F/F0 native ctDNA	F/F0 denatured ctDNA
CuCHz	8.0 × 10–5	1.037	1.005
	1.6 × 10–4	1.059	1.012
	2.4 × 10–4	1.067	1.023
	3.2 × 10–4	1.104	1.058
	4.0 × 10–4	1.142	1.104
CuCHz–DMβCD	8.0 × 10–5	1.012	1.007
	1.6 × 10–4	1.055	1.048
	2.4 × 10–4	1.097	1.072
	3.2 × 10–4	1.159	1.112
	4.0 × 10–4	1.226	1.147

The melting temperature (Tm) of the ctDNA solution, which is defined as the temperature where half of the total base pairs are unbound, is usually measured to study the interaction of an intercalator with the nucleic acid. Small molecules have an impact on the stabilization of the ctDNA double helix, which can be corroborated with an increase in the ctDNA melting temperature, probably due to increased stability of base stacking in the presence of an intercalator.[44] This temperature (Tm) is obtained from the midpoint of ctDNA melting curves, generally increasing ca. 5–8 °C for medium-sized molecules when they bind to ctDNA by intercalation. There is no noticeable change in temperature for nonintercalation binding. In our study, the absence of Cu(II) complexes revealed a Tm of 67.9 °C; however, when Cu(II) complexes were present, the Tm of ctDNA increased to 77.0 and 73.5 °C in the presence or absence of DMβCD, respectively (Figure ). The ΔTm values of 9.1 and 5.6 indicate that melting temperatures correspond to those for classical intercalators.[55] We can infer that the copper(II) complex in the presence of DMβCD binds or intercalates between base pairs of double-helical ctDNA more efficiently, as compared to the Cu(II) complex alone.

Figure 5

Melting curves of ctDNA (■) in the presence of CuCHz (red hexagon) and in the presence of CuCHz–DMβCD (blue triangle up solid). At pH 7.4, [CuCHz] = 25 μM and [DNA] = 50 μM.

Computational docking simulation studies of the copper(II) complex with DNA were carried out to predict the chosen binding site inside the DNA. The docked conformation of the compound with the lowest free energy and pose is shown in Figure . When CuCHz is docked into ctDNA, the planar ligand remains intercalated between the base pairs of DNA, forming π–π interactions with the adjacent nucleotide moieties. As stated, intercalator compounds must display interactions of the π–π type to achieve the expected binding mode. In our case, when the CHz molecule is complexed with Cu(II), it attains a planar conformation around Cu(II), which renders this molecule with the ability of such stacking interactions. Thus, according to our docking results, the chromone moiety of CuCHz remains intercalated to a pair of nucleobases at 3.4 Å at one side of DNA (see Figure ). It is worth mentioning that this pose is cooperatively stabilized by several hydrogen bonds to the DNA structure. The hydroxyl group of the chromone remains to form a hydrogen bond at 1.8 Å with the G base; on the other hand, the hydroxyl group of phenol remains in a hydrogen bonding interaction with the phosphoryl hydrogen. Taking these interactions into account jointly with the strength of hydrogen bonds between G and C bases reinforced the favorable intercalated pose between CuCHz and DNA.

Figure 6

Molecular docked conformation of the copper(II) complex with DNA.

Conclusions

The binding constant and the number of binding sites for the interaction between CuCHz and ctDNA in the absence and the presence of cyclodextrin were determined. In both cases, a single binding site in ctDNA for CuCHz was observed. The binding constant in the presence of cyclodextrin was higher, indicating that DMβCD selectively orients the drug molecule upon its binding to ctDNA. On the other hand, the thermodynamic results suggested that the process that drives the interaction of CuCHz–DMβCD with ctDNA could be assigned to intercalation mode. We examined the interaction of CuCHz to ctDNA and found enough pieces of evidence for its binding mode. The interaction occurrence is supported by the following findings: (i) the fluorescence studies showed an appreciable increase in the CuCHz emission upon the addition of ctDNA. (ii) A competitive reaction monitored among the EB dye, ctDNA, and CuCHz showed that the intercalated EB was displaced from the ctDNA–EB system by CuCHz. (iii) An increase in the melting temperature, Tm, caused by the stabilization of base stacking indicates the intercalation in the base pairs of ctDNA due to the presence of the copper(II) complex and (iv) the docking results of the copper(II) complex with ctDNA. The results presented here show also that, CuCHz displayed a high affinity for ctDNA. Furthermore, the existence of DMβCD did not affect the interaction of CuCHz with ctDNA and the inclusion complex decomposed when it binds to ctDNA.

Materials and Methods

Heptakis-2,6-O-dimethyl-β-cyclodextrin (DMβCD), copper(II) chloride dihydrate, 6-hydroxy-chromone-3-carboxaldehyde, 3-hydroxybenzoylhydrazine, ethidium bromide (EB), and deoxyribonucleic acid sodium salt from calf thymus (ctDNA) were purchased from Sigma-Aldrich. ctDNA was used as received without further purification. The purity of ctDNA was determined from optical measurements (A260/A280 > 1.8, where A represents absorbance). Its concentration was determined spectrophotometrically using the molar coefficient value, ε = 6600 L mol–1 cm–1. The stock solution of ctDNA was prepared in 5 mmol L–1 Tris–HCl/10 mmol L–1 NaCl buffer solution at pH 7.4 and stored in a refrigerator at 4 °C until use. The aqueous solutions used in the experiments were prepared daily from the stock solutions by appropriate dilution in the buffer solution. All solvents employed in the spectrophotometric analyses were of spectroscopic reagent grade. Deionized water from a Milli-Q system apparatus (Millipore Corp., Billerica, MA) was used throughout the experiments.

The infrared spectra were recorded by an FTIR spectrometer (Thermo iS50) equipped with a germanium attenuated total reflection (ATR) accessory, in the range of 4000–600 cm–1, having an average of 20 spectra per analysis. The absorption spectrum was recorded on an Agilent 8453 UV–vis spectrophotometer, which is equipped with a Peltier system ±0.1 °C. Fluorescence measurements were performed on an LS55 PerkinElmer spectrofluorometer equipped with a xenon lamp source and by a Peltier temperature programmer (PerkinElmer PTP-1 Peltier system). For all experiments, 1 cm length cells were used. Mass spectra of the copper complex were recorded by a high-resolution mass spectrometer (Exactive Plus Orbitrap, ThermoFisher Scientific, Bremen, Germany). Thermogravimetric analyses were carried out from room temperature up to 800 °C at a heating rate of 10 °C min–1 on a TA Instruments TGA Q50. The molar conductivities of the complexes in DMSO solutions (10–3 M) were measured at room temperature using a WTW microprocessor conductivity meter LF 539. Magnetic moment measurements of the complex were carried out on a Johnson Matthey magnetic susceptibility balance using Hg[Co(SCN)4] as a calibrant.

Inclusion complexes with DMβCD were obtained as described earlier.[56] For the determination of association constants (Ka), the concentration of CuCHz was 1 × 10–5 mol L–1 and increased buffered solution of DMβCD was added. The resulting mixture was equilibrated in a Precision thermostatic shaking water bath at a determined temperature for 24 h after which the equilibrium was reached and the emission intensity was recorded.

The interaction between ctDNA and CuCHz was carried out as follows: fixed amounts of CuCHz (1 × 10–5 mol L–1) in the absence and presence of DMβCD (1 × 10–3 mol L–1) were titrated with increasing amounts of the ctDNA solution in the Tris–HCl/NaCl buffer solution. While measuring the absorption spectra, an equal amount of ctDNA was added to both the complex solution and the reference solution to eliminate the absorbance of ctDNA itself. In the ctDNA melting experiments, the absorbance of ctDNA (5 × 10–5 mol L–1) at 260 nm was monitored by gradually increasing the temperature from 40 to 95 °C in the presence and absence of CuCHz and CuCHz–DMβCD (5 × 10–5 mol L–1). The absorbance values of ctDNA and CuCHz were normalized and plotted as a function of temperature.

The intercalating effect of the copper(II) complex with the ctDNA–EB was studied by the gradual addition of the complex solution into the solution of ctDNA–EB. The ctDNA–EB complex was prepared by adding 0.33 μ mol L–1 EB and 15 μ mol L–1ctDNA in the Tris–HCl/NaCl buffer solution, pH 7.4. This solution was titrated by the successive addition of 5 × 10–6 mol L–1 stock solution of the copper(II) complex with and without cyclodextrin. The fluorescence spectra of EB bound to ctDNA have a maximum at 593 nm with an excitation wavelength of 500 nm with bandwidths for excitation and emission of 8 and 15 nm, respectively. The emission spectra were recorded in the 530–700 nm range.

Docking studies were carried out using AutoDock[57] (version 4.2) software. Thus, from the RCSB protein data bank, the pdb file for DNA (PDB ID: 1Z3F) was obtained. We used this crystal structure for CuCHz–DNA docking studies. Grid maps were calculated using AutoGrid4 centered on DMβCD defining a volume of 30 Å3 with a 0.375 Å grid spacing. The AutoTors option of AutoDockTools was used to define rotatable bonds. The genetic Lamarckian algorithm was used under the following conditions: population size, 50; maximum number of evaluations, 25 000 000; maximum number of generations, 27 000; rate of mutation, 0.02; and rate of crossover, 0.08. The evaluations were performed with a default dielectric. The most stable pose of CuCHz was chosen according to the best docking score.