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Multi-spectroscopic and molecular modeling studies on the interaction of antihypertensive drug; methyldopa with calf thymus DNA† Nahid Shahabadi* and Maryam Maghsudi The interaction of methyldopa [(S)-2-amino-3-(3,4-dihydroxyphenyl)-2-methyl propanoic acid] (MDP), antihypertensive drug, with calf thymus DNA (ct-DNA) was investigated by spectroscopic and viscometric techniques. According to the results arising from the fluorescence spectra, viscosity measurements and

Received 11th August 2013, Accepted 31st October 2013

molecular modeling studies; we concluded that MDP is a minor groove binder of ct-DNA and preferentially binds to AT rich regions. Ethidium bromide (EB) displacement studies revealed that MDP did not have any effect on EB bound DNA which is indicative of groove binding. This was substantiated by displacement

DOI: 10.1039/c3mb70340a

studies with Hoechst 33258, a known minor groove binder. In addition, the thermodynamic and docking

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plays a major role in this binding.

parameters showed that hydrophobic interaction via drug aromatic rings inside the DNA minor groove

1. Introduction High blood pressure adds to the workload of the heart and arteries. If it continues for a long time, the heart and arteries may not function properly. This can damage the blood vessels of the brain, heart, and kidneys, resulting in a stroke, heart failure, or kidney failure. High blood pressure may also increase the risk of heart attacks. In recent years, increased attention has been focused on the ways in which drugs interact with DNA, with the goal of understanding the toxic as well as chemotherapeutic effects of these small molecules.1–3 Understanding of drug– receptor interactions is complex but is necessary to study the physicochemical properties of drugs (organic molecules) which can be of importance in the future. The study on the interaction of small molecules (often drugs or ligands) with DNA has been focused on some recent research works in the scope of life science, chemistry and clinical medicine.4–6 DNA is quite often the main molecular target of the chemical substances in the environment, thereby people are facing an increase of diseases and many types of cancer can be partly attributed to dramatic changes of the DNA physiological functions.7,8 Methyldopa (2-amino-3-(3,4-dihydroxyphenyl)-2-methyl-propanoic acid) (MDP) is a catechol derivative (catecholamine) widely

Department of Inorganic Chemistry, Faculty of Chemistry, Razi University, Kermanshah, Iran. E-mail: [email protected]; Fax: +98-831-8360795; Tel: +98-831-8360795 † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3mb70340a

338 | Mol. BioSyst., 2014, 10, 338--347

used as an antihypertensive agent. Catecholamine is a compound that consists of amines attached to a benzene ring bearing two hydroxyl groups (catechol). The main sites of catecholamine production are the brain, chromaffin cells of the adrenal medulla and the sympathetic neurons. It can be converted to 1-methyldopamine and 1-methyl norepinephrine.9,10 This catecholamine contains a chiral centre and can therefore occur either as an S- or R-isomer, and has antihypertensive activity due to the S-isomer. It is a centrally acting 2-adrenoceptor agonist, which reduces the sympathetic tone of muscles and produces a fall in blood pressure. However, to the best of our knowledge, the interaction between MDP and ct-DNA has not been studied. Due to the importance of this study, several analytical methods were used to get more information about the mechanism of interaction. In the present study, the interaction of MDP (Fig. 1) with calf thymus DNA was investigated in vitro using Hoechst 33258 dye as a spectral probe by the application of UV-vis absorption, fluorescence and CD spectroscopy and viscosity measurements. The molecular docking was performed to directly compare the predicted data with the experimental data, and to determine the potential modes of action of MDP. The combined data will thus help us to understand the structural activity relationships between the selected drug (MDP) and its binding site on ct-DNA molecule. We hope that this work can benefit further understanding of the binding mechanism of this drug with DNA for understanding the toxicological action of MDP at molecular level and comprehensive MDPs pharmacological effects as well as the design of the structure of new and efficient drug molecules.

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Fig. 1

Molecular BioSystems

Molecular structure of methyldopa.

2. Experimental 2.1.

Reagents

The stock solution (103 mol L1) of methyldopa was prepared in Tris-HCl buffer solution (0.05 mol L1, pH 7.4), calf thymus DNA (Sigma Chemical Co., St. Louis, MO) was used without further purification, and its stock solution was prepared by dissolving an appropriate amount of ct-DNA in Tris-HCl buffer solution. Then the solution was allowed to stand overnight and be stored at 4 1C in the dark for about a week. The concentration of ct-DNA in stock solution was determined by UV absorption at 260 nm using a molar absorption coefficient e260 = 6600 L mol1 cm1 (expressed as molarity of phosphate groups).11 The purity of the DNA was checked by monitoring the ratio of the absorbance at 260 nm to that at 280 nm. The solution gave a ratio of 41.8 at A260/A280, which indicates that DNA was sufficiently free from protein.11,12 EB stock solution (3  103 mol L1) was prepared by dissolving its crystals (Sigma Chemical Co.) in Tris-HCl buffer solution and storing it in a cool and dark place. 2.2.

Apparatus

The UV-Vis spectra for ct-DNA–MDP interaction were obtained using an Agilent 8453 spectrophotometer. CD measurements were recorded on a JASCO (J-810) spectropolarimeter. Viscosity measurements were made using a viscosimeter (SCHOT AVS 450) which termostated at 25  0.5 C by a constant temperature bath. Fluorescence measurements were carried out with a JASCO spectrofluorimeter (FP 6200). 2.3.

Experimental procedures

2.3.1. Fluorescence measurements. A 3.0 mL solution, containing 3  105 mol L1 MDP, was titrated by successive addition of ct-DNA. The solution was allowed to stand for 10 min to equilibrate. The fluorescence emission spectra were then measured at 288, 298 and 310 K in the wavelength range of 290–400 nm with an excitation wavelength at 280 nm. Appropriate blanks corresponding to the buffer solution were subtracted to correct background fluorescence. The competitive interaction between the Hoechst 33258 and MDP with ct-DNA was carried out as follows: fixed amounts of the Hoechst 33258 and DNA (5  106 and 1  104 M1,

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respectively, Ex: 340 nm, Em: 415 nm) were titrated with increasing amounts of MDP solution in the wavelength range of 360–560 nm with an excitation wavelength at 340 nm. In addition, the competitive interaction between EB and MDP with ct-DNA was studied. Different amounts of MDP was added to the EB–DNA solution (5  106 and 5  106 M1, respectively), and the effect of MDP on the emission intensities were measured. The samples were excited at 530 nm and the emissions were observed between 550 and 670 nm. The fluorescence emission measurements were made after 30 min to equilibrate. All fluorescence emission measurements were carried out in 0.05 mol L1 Tris-HCl buffer (pH 7.4) at room temperature. 2.3.2. Iodide quenching experiments. Quenching experiments were conducted by adding stoichiometric small aliquots of potassium iodide stock solution to MDP and ct-DNA–MDP complex solutions, respectively. The fluorescence intensity was recorded, and the quenching constants were calculated.13,14 2.3.3. UV-Vis measurements. Absorption experiments were carried out by keeping the MDP concentration constant (5  105 M) while varying the ct-DNA concentration from 5  106 to 3.5  105 M (ri = [ct-DNA]/[MDP] = 0.1–0.7). Absorbance values were recorded after each successive addition of ct-DNA solution and equilibration (ca. 24 h). 2.3.4. CD studies. The CD spectra of ct-DNA incubated with MDP at molar ratios ([MDP]/[ct-DNA]) of 0, 0.6, and 1 were measured in the wavelength range of 220–320 nm. The changes in CD spectra were monitored against a blank. The optical chamber of the CD spectrometer was deoxygenated with dry nitrogen before use and kept in a nitrogen atmosphere during experiments. All CD measurements were carried out in a Tris-HCl buffer (pH 7.4) at room temperature. 2.3.5. Viscosity measurements. Viscometric titrations were performed using a viscometer, which was kept at 25  0.1 1C by a constant temperature bath. The experiments were conducted by adding appropriate amounts of MDP into the viscometer to give a certain r (r = [MDP]/[ct-DNA]) value while keeping the ct-DNA concentration constant. The mean values of three replicated measurements were used to evaluate the average relative viscosity of the samples. The data were presented as (Z/Z0)1/3 versus the ratios of the concentration of MDP to that of DNA,15 where Z and Z0 represent the viscosity of ct-DNA in the presence and absence of MDP, respectively. Viscosity values were calculated from the observed flow time of ct-DNA containing solutions (t) and corrected for buffer solution (t0), Z = (t  t0)/t0. 2.3.6. Molecular docking study. MGL tools 1.5.4 with AutoGrid4 and AutoDock416,17 were used to set up and perform blind docking calculations between the MDP and DNA sequence. DNA sequence d(CGCGAATTCGCG)2 dodecamer (PDB ID: 1BNA) obtained from the Protein Data Bank. Receptor (DNA) and ligand (drug) files were prepared using AutoDock Tools. The DNA was enclosed in a box with a number of grid points in x  y  z directions, 106  100  76 and a grid spacing of 0.3751 A. Lamarckian genetic algorithms, as implemented in AutoDock, were employed to perform docking calculations. All other parameters were default settings. For each of the docking cases, the lowest energy docked conformation, according to the AutoDock scoring function, was selected as the

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binding mode. The output from AutoDock was rendered with PyMol.18

3. Results and discussion

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3.1.

Electronic absorption spectroscopy

Electronic absorption spectroscopy is an efficient method to examine the binding mode of DNA with drugs. The absorption spectra were recorded for a fixed concentration of free MDP (5  105 M) with increasing concentrations of ct-DNA (0–3.5  105 M) (Fig. 2). A strong ‘‘hyperchromic effect’’ was observed for increasing concentration of ct-DNA, although no appreciable change in the position of the MDP band was observed. Hyperchromism has been attributed to the presence of synergic non-covalent interactions: external contact (electrostatic binding), hydrogen bonding and groove surface binding (major or minor) along outside of ct-DNA helix. As there is no change in the position of absorption bands (bathochromic or hypsochromic shift), it can be inferred that MDP exhibit groove binding interactions to ct-DNA.19 The intrinsic binding constant, Kb, was calculated from the Wolfe–Shimmer equation (eqn (1)) through a plot of [ct-DNA]/ea  ef vs. [ct-DNA], where [ct-DNA] is the concentration of ct-DNA in the nucleotides, and the apparent extinction coefficients ea, ef and eb correspond to (Aobs/[M]), the extinction coefficient for free MDP and that of compound when fully bound to DNA, respectively. Kb was found to be 4  104.20 ½DNA ½DNA 1 ¼ þ ea  ef eb  ef Kb ðeb  ef Þ

(1)

3.2.

Emission spectral studies

3.2.1. Fluorescence spectra. Fluorescence spectroscopy is one of the most accurate and reliable methods for studying the relative binding of small molecules to ct-DNA as fluorescence quenching measurements can be used to evaluate binding.21 At excitation wavelength of 280 nm, the fluorescence quenching spectra of MDP with growing amounts of ct-DNA are shown in Fig. 3. The interaction between naproxen and ds-DNA in a groove-binding mode22 causes a strong fluorescence quenching. The fluorescence intensity of MDP at around 318 nm regularly decreased and the maximum emission wavelength did not apparently shift with the increase of ct-DNA concentration. The quenching in the long wavelength band suggests the occurrence of ligand–ligand excitation interactions in the presence of DNA or changes in ligand environment, such as hydrophobic interactions with the DNA binding sites versus hydrophilic interactions with the solvent. This also contributes to the observed fluorescence quenching of the 318 nm emission band relative to the free ligand. This phenomenon indicated that the binding of MDP to ct-DNA was protected from water molecules as solvent by the hydrophobic environment inside the DNA helix.23 A variety of molecular interactions can result in quenching, including excited-state reactions, molecular rearrangement, energy transfer, ground-state complex formation, and collisional quenching. Quenching normally refers to nonradiative energy transfer from excited species to other molecules. In order to study the quenching process systematically and to distinguish the possible quenching mechanism, fluorescence quenching tests were performed at different temperatures. Quantitative estimation of the quenching in terms of the fluorescence quenching data was performed using the Stern–Volmer eqn (2):24 F0/F = 1 + KSV[Q] = 1 + kqt0[Q]

Fig. 2 UV absorption spectra of MDP varying with concentrations of ct-DNA at pH 7.4 and room temperature. C[MDP] = 5  105 mol L1, and C[ct-DNA] = 0.0, 5.0, 15.0, 25.0 and 35.0  106 mol L1 1 for curves a–e, respectively. Inset: plots of [ct-DNA]/(ea  ef) vs. [ct-DNA] for the titration of MDP with ct-DNA.

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(2)

Fig. 3 Influence of ct-DNA concentration on the fluorescence intensity of MDP. Conditions: C[MDP] = 3  105 mol L1; 1–22, C[ct-DNA] = 0, 0.22, 0.45, 0.67, 0.89, 1.2, 1.6, 1.8, 2.00, 2.4, 3.00, 3.3, 4.00, 4.5, 5.1, 6.5, 7.5, 9.00, 10, 12  105 mol L1.

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Fig. 4 Stern–Volmer plots for the fluorescence quenching of MDP by ct-DNA at different temperatures.

where F0 and F are the fluorescence intensities of MDP in the absence and presence of ct-DNA, respectively. KSV is the Stern–Volmer quenching constant, t0 is the average fluorescence lifetime of bimolecular and equals 108 s; kq, which equals KSV/t0, is the apparent bimolecular quenching rate constant. For dynamic quenching, the maximum scattering collisional quenching constant of various quenchers is 2.0  1010 L mol1 s1. The plots of the Stern–Volmer equation at different temperatures are shown in Fig. 4. KSV and kq obtained from this equation are presented in Table 1. It is seen that KSV and kq increase as temperature increases, indicating that the mechanism of the quenching may be a dynamic quenching. However, kq is much larger than 2.0  1010 L mo1 s1, suggesting that the quenching process may be a static quenching. As described, the quenching mechanism can be classified as static quenching and dynamic quenching, and they represent two very different quenching processes.25 These two kinds of quenching mechanism demonstrate some differences that can be distinguished experimentally, such as change in the UV-visible spectrum of DNA and temperature dependence of the quenching constant.25 A complex of DNA and drug forms in static quenching, so there will be some changes in the UV-visible spectrum of DNA, whereas dynamic quenching has no such change. Diffusion is the control step for dynamic quenching,

Table 1 KSV and kq of MDP and ct-DNA interaction system at different temperatures (lex = 295 nm, pH 7.4, T = 298 K)

T (K)

KSV  104

Kq  1012

R2

288 298 310

0.91 1.23 1.38

0.91 1.23 1.38

0.9985 0.9957 0.9976

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so the quenching constant will increase with increasing the temperature.26 As reported by our group and other research groups, most of the fluorescence quenching is in good accordance with this theory and fits the results in the distinguishing experiments.27 Thus, we employed UV-visible absorption spectra to give some more evidences for the actual quenching process. The UV-visible absorption spectra of MDP and the ct-DNA–MDP system were measured. According to the theory mentioned; the UV spectra of MDP would have no detectable change if the quenching was a dynamic mechanism.28 On the other hand, ground-state ct-DNA–MDP complex forms in the static quenching, and the UV spectrum of MDP changes as a direct consequence.29 Therefore, according to the UV spectra, the fluorescence quenching of MDP in our case seems to be primarily caused by complex formation between MDP and ct-DNA. As to the unexpected and adverse temperature dependence, we think it may be untypical static quenching and could be explained by Arrhenius’ theory. As well accepted for dynamic quenching, the number of excited molecules and diffusion limits kq and the probability of collision, limiting it to values less than 2.0  1010 L mol1 s1.28 However, there is no such limitation for static quenching. This theory states that with increasing temperature, viscosity of the solvent decreases, which decrease the chances of collision of the MDP with that ct-DNA molecule. Hence, the dynamic quenching contributes more to the total quenching, and KSV decreases as the temperature increases.28 On the other hand, the value of KSV must increase with increasing temperature according to Arrhenius’ theory. If the extent of the increase caused by the rising temperature is larger than decrease of collision, overall there will be an increase in KSV upon increasing temperature. According to Arrhenius’ theory, the rate constant k is a function of temperature, so the temperature has a crucial impact towards the rate constant k. The higher the temperature, the higher the rate constant is. Meanwhile the activation energy, Ea, is related to the rate constant k. Raising T makes Ea/RT smaller, k will increase and the reaction is faster. The activation energy of the quenching process can be calculated according to the Arrhenius equation (eqn (3)): ln (KSV/t0) = ln kq = Ea/RT + ln A

(3)

kq, which is equal to KSV/t0, is the apparent bimolecular quenching rate constant; Ea is the activation energy of the quenching reaction; A is the pre-exponential factor. From the slopes of ln kq vs. 1/T plots, the value of Ea can be obtained. Fig. 5 shows a good linear relationship. The value of Ea (14.03 kJ mol1) indicates that the impact of temperature on the value of KSV is significant. Thus, the fluorescence quenching is still a static quenching, though untypical to some extent, and MDP forms a complex with ct-DNA in the ground state. 3.2.2. Equilibrium binding titration. Fluorescence titration data were used to determine the binding constant (Kf) and the binding stoichiometry (n) for the complex formation of MDP with ct-DNA. The fluorescence intensity of MDP at 318 nm decreases in the presence of ct-DNA. This change in fluorescence

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Paper Table 2 The binding constants, number of binding sites (n) and thermodynamic parameters of ct-DNA–MDP system at different temperatures (lex = 280 nm, pH 7.4, T = 298 K)

T (K) KF  104 n

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288 1.09 298 1.44 310 1.58

Fig. 5

DG0 (kJ mol1) DH0 (kJ mol1) DS0 (J mol1 K1)

10.2 22.35 1.01 23.57 1.02 25.02

12.53

121.07

Arrhenius plots of methyldopa and calf thymus-DNA system.

intensity was used to estimate Kf and n for the binding of MDP to ct-DNA from the following equation.30 log[(F0  F)/F] = log KF + n log[Q]

(4)

Here, F0 and F are the fluorescence intensities of the fluorophore in the absence and presence of different concentrations of ct-DNA, respectively. The values of Kf were obtained from the intercept of the plot of log[(F0  F)/F] versus log[Q] (Fig. 6). The corresponding results at different temperatures are shown in Table 2. The value of Kf at 298 K was 1.44  104 L mol1, which agrees well with the Kb value obtained earlier by UV spectroscopy (Kb = 4  104 L mol1) (Section 3.1) and molecular docking (Section 3.6). 3.3.3. Competitive binding between ethidium bromide (EB) and MDP for DNA. Ethidium bromide (EB) is one of the most sensitive fluorescence probes having a planar structure that binds DNA by an intercalative mode.31,32 The fluorescence of EB increases after intercalation into the ct-DNA. If the ligand (in this case MDP) intercalates into ct-DNA, it leads to a decrease in the binding sites of ct-DNA available for EB, which in turn decreases the fluorescence intensity of the EB–DNA system. Fig. 7 shows the fluorescence emission spectra of EB

Fig. 6 Plot of log(F0/F)/F versus log[ct-DNA] at different temperatures (pH = 7.4, lex = 280 nm, lem = 318 nm). C[MDP] = 3  105 mol L1.

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Fig. 7 Fluorescence spectra of the competition between MDP and EB. C[MDP] = 0.2, 0.35, 0.5, 0.74, 1.2, 1.5, 1.7, 2.2 and 2.7  105 mol L1 for curves 2–10, C[EB] = 5.0  106 mol L1 and C[ct-DNA] = 4.9  106 mol L1 at 298 K.

with and without ct-DNA and the effect of the addition of MDP to EB bound ct-DNA. There is no significant displacement of EB by MDP reflecting the absence of an intercalative mode of binding. As there is evidence of complex formation from other experiments, a groove-binding mode could be ascribed to it. Other experimental studies as well as DNA ligand docking have also substantiated this. 3.3.4. Competitive binding studies with Hoechst 33258. To investigate the mode of MDP binding to ct-DNA, a competitive binding experiment was performed. Hoechst 33258, 2-(4-hydroxyphenyl)-5-[5-(4-methylpipera-zine-1-yl) benzimidazo-2-yl]-benzimidazole is a synthetic N-methylpiperazine derivative that binds to the minor groove of B-DNA with different characteristics.33 Hoechst produces weak fluorescence in Tris-HCl buffer due to quenching by the solvent molecules. However, with the addition of increasing concentration of DNA, fluorescence intensity of Hoechst 33258 enhances substantially.34 The fluorescence of Hoechst in DNA grooves increases due to its higher planarity as well as its protection from collisional quenching.34 The displacement of bound Hoechst 33258 competing for the same site on ct-DNA is reflected from a decrease in its fluorescence intensity on addition of the competing MDP. Hoechst 33258 shows an emission maximum at 415 nm on excitation at its absorption maximum of 340 nm. Fig. 8 shows the fluorescence spectral changes of bound Hoechst 33258 to ct-DNA by addition of MDP. Generally, small molecules, such as drugs, organic dyes, etc., bind with DNA in minor groove binding mode for their small

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Fig. 8 Fluorescence spectra of the competition between MDP and Hoechst. C[MDP] = 0.19, 0.46, 1.2, 1.6, 2.3, 2.8  104 mol L1 for curves 2–6, C[Hoechst] = 5.00  106 mol L1, and C[ct-DNA] = 2.5  104 mol L1 at 298 K.

size, while the macromolecules, such as many protein molecules bind with DNA in major groove binding mode. Minor groove drug binding with DNA adopts a characteristic curved shape isohelical with the target groove and it binds in the region of rich A–T base pairs where is narrower than the region of rich G–C base pairs. This is because there are favorable hydrophobic contacts between the C-2 hydrogen atoms and the aromatic rings in drugs,35 so does the MDP. Moreover, it should be noted that there is hydrogen atoms in MDP and the C-2 carbonyl oxygen or N-3 nitrogen in DNA, so, the hydrogen bond might exist between the MDP and base pairs of DNA. The stabilization of DNA–MDP complex may be maintained by hydrophobic interaction and hydrogen bond. 3.3.5. Iodide quenching studies. To obtain an insight into the mode of binding of MDP with DNA, the fluorescence quenching in the ct-DNA environment was studied using potassium iodide (KI) as a quencher, and the correlation between the degree of accessibility of each molecule to the quencher and its steric bulk was examined. A highly negatively charged quencher was expected to be repelled by the negatively charged phosphate backbone of DNA. Accordingly, intercalative bound small molecules should be protected from being quenched by anionic quencher, whereas the free aqueous complexes and groove binding drugs should be quenched readily by anionic quenchers.24,36 The KSV value of the bound small molecule should be lower than that of the free small molecule, if small molecule is intercalated into the helix stack. In contrast, if a small molecule binds to DNA in the groove, the value of KSV of the bound small molecule should be higher than that of the free small molecule.37 Therefore, the negatively charged I was selected to determine the binding mode of MDP to ct-DNA. The fluorescence quenching data were analyzed to obtain the quenching constant by using the well-known Stern–Volmer equation. The values of quenching constants (KSV) of MDP by I ion in the absence and presence of ct-DNA were calculated to be 8.24 and 11.09 mol L1, respectively (Fig. 9). The results displayed almost same iodide quenching effect on the fluorescence

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Molecular BioSystems

Fig. 9 Stern–Volmer plots for the quenching of MDP by KI in the absence and presence of ct-DNA. C[MDP] = 3.0  105 mol L1, C[ct-DNA] = 7.5  104 mol L1, pH = 7.5.

of MDP before and after the interaction with ct-DNA, which suggested that MDP binds to ct-DNA through groove binding. 3.3.6. Determination of thermodynamic parameters. The interaction forces between drugs and biomacromolecules may include electrostatic interactions, multiple hydrogen bonds, van der Waals interactions, hydrophobic and steric contacts within the antibody-binding site, and so on.38 If the enthalpy change (DH) does not vary significantly in the temperature range studied, both the enthalpy change (DH) and entropy change (DS) can be evaluated from the van’t Hoff equation:39 lnK ¼ 

DH DS þ RT R

(5)

where Kb is analogous to the associative binding constant at the corresponding temperature and R is the gas constant. The values of DH and DS were obtained from the slope and intercept of the linear plot of eqn (4) based on ln K versus 1/T (Fig. 10). The free energy change (DG) is estimated from the following relationship.40 DG = DH  TDS

(6)

The values of DH, DS and DG calculated are listed in Table 2. The value of DH is greater than 0, which proves that binding

Fig. 10 Van’t Hoff plot for the interaction of ct-DNA and MDP.

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reaction of MDP with ct-DNA is endothermic. Based on the binding constant of MDP with ct-DNA at various temperatures, the binding force of MDP with ct-DNA can be determined. Because DH 4 0 and DS 4 0, ct-DNA–MDP complex is stabilized mainly by hydrophobic interactions.38 The hydrophobic contact occurs between the C-2 hydrogen atoms in DNA and the aromatic ring in MDP, which coincides with the description of Section 3.3.4. Even so, the hydrogen bond should not be excluded in the binding reactions. 3.3.

Circular dichroism (CD) studies

Circular dichroic spectral technique is useful in diagnosing changes in DNA morphology during drug–DNA interactions. The band due to base stacking (275 nm) and that due to righthanded helicity (248 nm) are quite sensitive to the mode of DNA interactions with small molecules.41 The changes in CD signals of DNA observed on the interaction with drugs may often be assigned to the corresponding changes in DNA structure.42 Thus, simple groove binding and electrostatic interaction of small molecules with DNA show less or no perturbation on the base stacking and helicity bands, while intercalation enhances the intensities of both the bands and stabilizing the right-handed B conformation of ct-DNA as observed for the classical intercalator methylene blue.43 The CD spectrum of ct-DNA showed conformational changes upon the addition of MDP without any changes in the wavelengths (Fig. 11). The CD spectrum of DNA in the presence of MDP shows intensity increase and decrease in the positive and negative bands, respectively. The latter being affected slightly more than the other. However, there were not evident shifts in the band positions. A similar observation made for MDP bound to ct-DNA has been ascribed to a conformational conversion from a more B-like to a more A-like structure within the DNA molecule.44 In comparison with ct-DNA–EB sample, the MDP do not bind to ct-DNA by typical intercalation.

Fig. 11 CD spectra of ct-DNA in the absence (blue line) and presence of the MDP (red line r = 0.6 and green line r = 1), r = (r = molar ratio [MDP]/ [ct-DNA]).

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Fig. 12 Effect of increasing amounts of MDP on the relative viscosity of ct-DNA at 298 K. C(ct-DNA) = 5  105 mol L1; pH 7.4.

3.4.

Viscosity measurements

Hydrodynamic properties and especially viscosity can give better indications on the binding mode of a small molecule to DNA. Further information on the nature of the interaction can be obtained through hydrodynamic studies such as viscosity measurements. Classical intercalation results in lengthening of DNA, due to the separation of base pairs at the intercalation site, which produces a concomitant increase in the relative specific viscosity of such solutions. Thus, such studies offer the least ambiguous test of intercalation.30,45,46 Minor positive or negative changes in DNA solution viscosity are observed when binding occurs in the DNA grooves.46 As shown in Fig. 12, MDP exhibited a slight increase in the viscosity of ct-DNA which is not as pronounced as observed for classical intercalators47 and are consistent with substrates that bind to DNA through a groove-binding mode.48 3.5.

Molecular docking analysis

The design of molecules that can recognize specific sequences and structures of nucleic acids plays an important role both for understanding nucleic acid molecular recognition as well as for the development of new chemotherapeutic drugs. Molecular docking technique is an attractive scaffold to understand the drug–DNA interactions for the rational drug design and discovery, as well as in the mechanistic study by placing a small molecule into the binding site of the target specific region of the DNA mainly in a noncovalent fashion,49 which can substantiate the spectroscopic results. It is well known that the interactions of chemical species with the minor groove of B-DNA differ from those occurring in the major groove, both in terms of electrostatic potential and steric effects, because of the narrow shape of the former. Small molecules interact with the minor groove, while large molecules tend to recognize the major groove binding site.50 From the docking calculation, the conformer with minimum binding energy is picked up from the 61 minimum energy conformers from the 200 runs.51,52 The run data for the conformers are listed in Table 3. The energetically most favorable conformation (selected rank with maximum frequency) of the docked pose, (Fig. 13) revealed that MDP approaches towards the gap between

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Table 3 Docking summary of DNA with MDP by the AutoDock program generating different ligand conformers using a Lamarkian GA

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Binding energy Rank Run (kcal M1) Kia 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 a

197 34 149 14 24 8 198 159 10 37 134 155 49 80 181 142 178 98 29 173 68 135 117 64 188 20 160 122 95 76 174 74 139 193 112 88 48 128 59 4 165 44 32 150 182 69 79 13 90 179 54 157 42 161 199 105 118 57 124 53 143

6.41 5.88 5.71 5.42 5.31 5.30 5.26 5.19 5.18 5.15 5.11 5.02 4.94 4.91 4.90 4.85 4.84 4.80 4.74 4.66 4.64 4.63 4.60 4.59 4.57 4.56 4.52 4.51 4.50 4.45 4.44 4.39 4.36 4.34 4.32 4.28 4.26 4.25 4.24 4.22 4.18 4.17 4.02 3.99 3.97 3.97 3.84 3.84 3.79 3.75 3.60 3.60 3.51 3.49 3.48 3.45 3.30 3.29 3.26 3.02 2.89

19.88 mM 49.16 mM 65.10 mM 105.85 mM 105.85 mM 131.21 lM 140.33 mM 157.39 mM 16.53 mM 167.69 mM 180.26 mM 207.68 mM 240.11 mM 253.06 mM 256.70 mM 279.22 mM 283.53 mM 302.72 mM 336.22 mM 385.39 mM 397.40 mM 407.18 mM 427.54 mM 428.39 mM 445.32 mM 455.85 mM 489.41 mM 493.54 mM 503.68 mM 545.88 mM 552.82 mM 603.63 mM 638.09 mM 659.33 mM 675.76 mM 730.76 mM 755.01 mM 763.98 mM 775.23 mM 807.39 mM 864.14 mM 884.46 mM 1.12 mM 1.18 mM 1.24 mM 1.24 mM 1.5 mM 1.5 mM 1.66 mM 914.29 mM 2.29 mM 2.29 mM 2.68 mM 2.78 mM 2.82 mM 2.95 mM 3.80 mM 3.88 mM 4.08 mM 307.14 mM 7.64 mM

Ka (M1) 5.22 2.13 1.59 9.75 8.09 7.96 7.44 6.61 6.49 6.17 5.77 4.95 4.32 4.11 4.04 3.71 3.65 3.41 3.08 2.69 2.60 2.56 2.43 2.39 2.31 2.27 2.12 2.09 2.05 1.88 1.85 1.70 1.62 1.56 1.51 1.41 1.36 1.34 1.32 1.28 1.19 1.17 9.09 8.64 8.35 8.35 6.70 6.70 6.15 5.75 4.46 4.46 3.83 3.70 3.64 3.46 2.68 2.63 2.50 1.67 1.33

                                                            

104 104 104 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 103 102 102 102 102 102 102 102 102 102 102 102 102 102 102 102 102 102 102 102

Cluster Reference rmsd rmsd 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

26.90 27.12 25.52 28.89 28.97 28.59 29.50 25.88 25.99 25.85 28.87 28.27 26.68 29.90 22.75 29.28 28.88 24.71 27.87 29.95 27.11 29.97 26.31 28.75 30.00 28.45 29.28 22.58 28.07 27.33 26.85 27.13 29.06 26.70 29.48 27.83 28.86 28.98 28.72 23.96 35.57 26.06 26.86 24.16 24.36 26.55 27.37 25.20 32.95 29.73 29.91 23.02 30.16 23.82 35.47 25.61 17.84 33.21 31.46 16.50 24.55

Ki is the inhibition constant.

DNA minor groove mainly through benzyl ring and is situated within narrower A–T (10.8 Å) regions compared to G–C (13.2 Å) ones,

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Fig. 13

Minor groove binding in the region of rich A–T base pairs.

due to preferential binding of MDP bearing benzyl moiety to A–T regions compared to the G–C region and leads to van der Waals interaction and hydrophobic contacts with DNA functional groups which define the stability of groove.53 Thus, we can concluded that there is a mutual complement between spectroscopic techniques and molecular docking, which can provide reliable and quick information on the capability of new drugs to interact with DNA. Furthermore, the binding constant obtained by UV-Vis method was correlated with the free binding energy of docked model. Basic formula of binding constant and Gibbs free energy is: DG = RT ln Kb

(7)

where DG is Gibbs free energy, R is gas constant (1.98 cal mol K1), T is temperature at which experiment was done (25 1C i.e. 298 K) and K is binding constant between MDP and free DNA. The free energy change DG1 of the binding of MDP to ct-DNA is 6.25 kcal mol1, and the binding constant is 4.0  104 M1. However, the computational results were different from the experimental results (5.30 kcal mol1, Ka 7.92  103 M1).52 From the docking simulation the observed free energy change of binding (DG) for the DNA–MDP complex is lower than the experimental free energy of binding obtained. This apparent mismatch in the free energy changes could be due to the exclusion of the solvent in the molecular docking studies. To investigate the mode of MDP binding to ct-DNA, a competitive molecular docking was performed. DNA sequence (PDB ID: 8BNA) obtained from the Protein Data Bank.54–57 The run data for the conformers are listed in Table 4. The energetically most favorable conformation (selected rank with maximum frequency) of the docked pose, (Fig. 14) revealed that MDP approaches towards the gap between DNA minor grooves mainly through benzyl ring near Hoechst where the Hoechst is. It can be learned from the results of 12 sets that almost all the binding sites of MDP were located in the groove of double-helix DNAs. From the results, we could find that the DNA residues with ID of T19, T20, A16 and A17 played a major role in the binding site with the optimal energy (Fig. S1, ESI†). Thus, the MDP molecule relatively binds through benzyl ring and is situated within narrower A–T. The computational results

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Table 4 Docking summary of 8BNA with MDP by the AutoDock program generating different ligand conformers using a Lamarkian GA

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Binding energy Rank Run (kcal M1) Kia 1 2 3 4 5 6 7 8 9 10 11 12 a

20 31 44 32 42 12 18 34 17 11 50 8

5.91 5.82 5.12 4.91 4.75 4.67 4.35 4.20 4.20 4.07 4.02 3.57

46.79 mM 54.09 mM 175.48 mM 251.77 mM 330.53 mM 377.19 mM 650.17 mM 831.69 mM 832.67 mM 1.05 mM 1.14 mM 2.40 mM

Ka (M1) 2.16 1.85 5.69 3.99 3.04 2.66 1.55 1.20 1.20 0.97 0.88 0.41

           

104 104 103 103 103 103 103 103 103 103 103 103

Cluster Reference rmsd rmsd 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

27.94 28.82 25.52 28.89 28.97 28.59 29.50 25.88 25.99 25.85 28.87 28.27

Ki is the inhibition constant.

Fig. 14

Competitive molecular docking of MDP-Hoechst.

(5.91 kcal mol1, Ka = 2.16  104 M1) were different from the experimental results (6.25 kcal mol1, Ka = 4.0  104 M1).51 From the docking simulation the observed free energy change of binding (DG) for the DNA–MDP complex is lower than the experimental free energy of binding obtained.

4. Conclusions In the present manuscript, the interaction of MDP with ct-DNA was studied using UV-Vis, fluorescence and CD spectroscopy as well as viscometric measurements and molecular modeling. Here we reported that MDP binds in the minor groove of ct-DNA with no conformational change or unwinding of the double helix. Viscosity, EB and Hoechst 33258 displacement and docking studies reveal a groove binding mode. The preferential binding of the A–T region over the G–C region was confirmed by

346 | Mol. BioSyst., 2014, 10, 338--347

docking and experimental studies. It should be noted that DNA minor groove binders constitute an important class of derivatives in anticancer therapy.58

Acknowledgements Financial support from the Razi University Research Center is gratefully acknowledged.

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Multi-spectroscopic and molecular modeling studies on the interaction of antihypertensive drug; methyldopa with calf thymus DNA.

The interaction of methyldopa [(S)-2-amino-3-(3,4-dihydroxyphenyl)-2-methyl propanoic acid] (MDP), antihypertensive drug, with calf thymus DNA (ct-DNA...
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