Electrochemical, spectroscopic, and theoretical studies on the interaction between azathioprine and DNA

Accepted Manuscript Title: Electrochemical, spectroscopic, and theoretical studies on the interaction between azathioprine and DNA Author: Fahimeh Jalali Gelareh Rasaee PII: DOI: Reference:

S0141-8130(15)00560-7 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.08.025 BIOMAC 5293

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

18-6-2015 8-8-2015 10-8-2015

Please cite this article as: F. Jalali, G. Rasaee, Electrochemical, spectroscopic, and theoretical studies on the interaction between azathioprine and DNA, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.08.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Highlights (for review)

Highlights:  The interaction between azathioprine (AZT) and ct-DNA was investigated by different spectroscopic, electrochemical and molecular docking methods.

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 AZT binds to DNA via minor grooves.

 Binding constants and the number of binding sites were calculated.

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 The interaction was enthalpy and entropy driven.

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 Electrostatic interactions were suggested to be the main forces involved.

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*Manuscript

Electrochemical, spectroscopic, and theoretical studies on the interaction between azathioprine and DNA

Department of Chemistry, Razi University, 67346 Kermanshah, Iran

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Fahimeh Jalali* and Gelareh Rasaee

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* Corresponding author. Email address: [email protected]; [email protected]

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Fax: +98 8334274559

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Abstract Possible interaction between immunosuppressive drug, azathioprine, and calf thymus DNA was explored by cyclic voltammetry, spectrophotometry, competitive spectrofluorimetry,

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circular dichroism spectroscopy (CD), and viscosity measurements.Cyclic voltammetry showed negative shift in the reduction peak of azathioprine in the presence of DNA, and large

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decrease in peak current, referring to the predominance of electrostatic forces. The binding

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constant was calculated to be 1.22 × 103 M-1. Absorption hyperchromism without shift in wavelength was observed when DNA was added to azathioprine solution. Competitive

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fluorescence experiments were conducted by using Hoechst 33258 and methylene blue as probes for minor groove and intercalation binding modes, respectively. The studies showed

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that azathioprine could release Hoechst 33258, while negligible effect was detected in the case of methylene blue. Stern-Volmer quenching constant (KSV) and complex formation

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constant (Kf) were obtained from the fluorescence measurements to be 7.6 × 103 M-1 and 7.76

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× 104 M-1, respectively, at 298 K. Enthalpy and entropy changes during the interaction

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between azathioprine and DNA were calculated from Van't Hoff plot (∆H= -20.2 kJ mol-1; ∆S= 26.11 J mol-1 K-1 at 298 K) which showed an exothermic spontaneous reaction, and involvement of electrostatic forces in the complex formation with DNA. Moreover, circular dichroism studies revealed that azathioprine induced detectable changes in the negative band of DNA spectrum. Viscosity of DNA solution decreased in the presence of azathioprine, showed a non-intercalative mode of interaction. Finally, molecular docking calculations showed that in the lowest energy level of drug-DNA complex, azathioprine approaches the minor grooves of DNA. Keywords: Azathioprine; DNA; Interaction; Groove binding; Electrostatic forces

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1. Introduction Azathioprine (AZT), Scheme 1, is an immune-suppressing drug following kidney transplantation [1, 2]. It is also administrated in various conditions such as Crohn's disease

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and ulcerative colitis. Since 2009, the United States Food and Drug Administration (FDA) has required warnings to be put on packaging AZT with respect to increased risks of

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certain cancers [3].

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Scheme 1: Chemical structure of azathioprine

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Deoxyribonucleic acid (DNA) is a target in cellular damage. Small molecules (like

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drugs, pesticides, fungicides, etc.) can interact with DNA and harm it via covalent or noncovalent bindings. There are several sites in the DNA molecule where non-covalent binding can occur: (i) between two base pairs (full intercalation), (ii) in the minor groove, iii) in the major groove, and (iv) outside of the helix with the phosphate groups [4]. It is important to understand, at a molecular level, the kind and strength of interaction of small molecules with DNA in order to explore the probable toxicity, the mechanism of drug action, and for the design of specific DNA – targeted drugs. Different techniques have been employed to study the interaction of small molecules with DNA, including fluorescence spectroscopy [5], UV-Visible spectrophotometry [6-8], circular dichroism [9], mass spectrometry [10], and electrochemical methods [11].

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Moreover, theoretical calculations based on energy minimization of drug-DNA mixture, provide some insight into the probable spatial structure of the aggregate formed, which may confirm the experimental results. Binding energies, geometry, electron distribution, hydrogen

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bonding properties, hydrophobicity and polarizability of the interacting species are some of the results of molecular docking studies [12].

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In this work, we have investigated the interaction between AZT and calf thymus DNA

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by cyclic voltammetry, UV absorption, competitive fluorescence spectroscopy and circular dichroism (CD), as well as viscosity measurements. Docking calculations were also used in

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order to compare the theoretical results with experimental findings. The study revealed that, at physiological pH (7.4), AZT approaches the minor grooves of DNA.

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2. Experimental 2.1.Materials and methods

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The highly polymerized double stranded calf thymus DNA (DNA), Hoechst 33258

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and Tris–HCl were purchased from Sigma Chem. Co. AZT powder was a gift kindly

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provided by Bakhtar-Bioshimi Pharm. Co. (Kermanshah, Iran). Methylene blue (MB) was purchased from Merck. All other chemicals were of highest purity available and were used without further purification.

The buffer solution was prepared from Tris–(hydroxymethyl)-aminomethane–

hydrogen chloride (Tris), and its pH was adjusted to 7.4. A stock solution of DNA (1.3 × 102

M) was prepared by dissolving appropriate solid DNA in Tris buffer (0.05 M, pH 7.4). It

could be stored for about one week at 4°C in the dark. The concentration of DNA was determined by absorption spectrometry, using the molar absorptivity ε260 =6600 L mol-1 cm1

[13, 14]. Purity of DNA was checked by monitoring the ratio of its absorbance at 260 nm to

that at 280 nm. The solution gave a ratio of A260 ⁄A280=1.82, indicating that DNA was sufficiently free from protein[15].Stock solution of AZT (1.0 ×10-3M) was prepared by 4 Page 5 of 35

dissolving an appropriate amount of the drug in Tris buffer(0.05 M, pH 7.4) by addition of few drops of NaOH (0.1 M).

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2.2.Instrumentation and procedures Cyclic voltammetric experiments were performed in a conventional three electrode

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cell using a µ-Autolab system (Utrecht, The Netherlands). The system was run using

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NOVA 1.10 software. The three electrode system consisted of a glassy carbon electrode (GCE, surface area of 0.0314 cm2 ) as working electrode, Ag/AgCl (KCl, 3 M) as the

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reference and a platinum wire as the auxiliary electrodes.

Before each measurement, GCE was polished mechanically using Al2O3 slurry. After

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polishing, it was rinsed thoroughly with deionized water. Then, it was placed in buffer supporting electrolyte and voltammograms were recorded in the range of –1.2 to +0.1 V until

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a steady state baseline voltammogram was obtained (6 – 8 cycles). This procedure ensured

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reproducible experimental results. Cyclic voltammograms for AZT, DNA, and their various

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mixtures were recorded in the range of −1.2 to +0.1 V vs. Ag/AgClat a scan rate of 100 mV s-1 .

UV-Visible absorption spectra were obtained using a double beam Agilent UV-

Visible spectrophotometer model 8453 with diode array detector. A quartz cell (1 cm path length) was used for absorption measurements. The experiments were carried out by keeping the concentration of drug constant (5.0 ×10-5 M), while varying DNA concentration from 2.5 × 10-6 to 1.4 × 10-4 M (ri = [DNA] / [drug] = 0.05–2.8). The spectra were recorded after each successive addition of DNA to AZT and buffer solutions after equilibration (ca. on time). In order to obtain the changes in absorption spectra of AZT in the mixture, the corresponding spectrum of DNA was subtracted.

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Fluorescence measurements were carried out by using a JASCO spectrofluorimeter (FP 6200). Binding location of AZT in DNA was studied in the presence of two probes (Hoechst 33258 and MB) using the fluorescence titration method. The concentration of probe

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and DNA were fixed, and AZT was gradually added to the mixtures ([AZA] = 0.28 – 2.37 ×10-4 M). The fluorescence spectra of different mixtures were recorded over a wavelength

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range of 300-550 nm (λex = 340 nm) in the case of Hoechst 33258, and 600-700 nm (λex =

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630 nm) in the case of MB.

Circular dichroism (CD) measurements were recorded on a JASCO (J-810)

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spectropolarimeter, keeping the concentration of DNA constant (5.0 ×10-5 M) while varying the drug concentration (ri= [drug]/ [DNA] = 0−3).

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Viscosity measurements were made using a viscometer (SCHOT AVS 450) maintained at 25 ± 0.5 °C in a thermostated water bath. The DNA concentration was fixed at

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5.0 ×10-5 M and different amounts of AZT (ri = 0−2) were added. The flow time of each

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solution was measured with a digital stopwatch; mean values of two measurements were used to evaluate the viscosity of the samples. The relative specific viscosity was calculated as

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(η/η0)1/3, where η0 and η are the specific viscosity contributions of DNA in the absenceand in the presence of AZT, respectively. 2.3.Molecular docking study

In order to perform docking calculations, MGL tools 1.5.4 with Auto-Grid4 and AutoDock 4 [16, 17] were used. DNA sequence (CGCGAATTCGCG)2dodecamer was obtained from Protein Data Bank (PDBID: 1BNA). Water was removed from the DNA PDB file. Essential hydrogen atoms and Gasteiger charges were added with the aid of AutoDock tools (ADT). Receptor (DNA) and ligand (AZT) files were prepared using ADT. DNA was enclosed in a box with the number of grid points in x × y × z directions, 106 × 100 × 76 and a grid spacing of 0.3751 Å. Lamarckian genetic algorithms, as implemented in AutoDock, were 6 Page 7 of 35

employed to perform docking calculations. All other parameters were default settings. For each of the docking cases, the lowest energy conformation, according to the AutoDock scoring function, was selected as the binding mode. The output from AutoDock was rendered

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to PyMol [18]. 3. Results and discussion

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3.1.DNA binding study by cyclic voltammetry

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AZT is an electroactive nitro-compound that can be electrochemically reduced on the surface of the electrodes. The electroreduction of –NO2 group in aromatic compounds is

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attributed to reduction to the corresponding hydroxylamine 4 e− [19, 20]. Due to the redox activity of AZT, cyclic voltammetry has been proved as one of the important analytical

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techniques to elucidate the mechanism of drug action, in vivo.

In general, when an electroactive species binds to DNA via intercalation, positive

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shift in potential is observed, while in the case of electrostatic and groove interaction , the

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potential will shift to negative direction[21, 22].

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Cyclic voltammetry was applied to a buffer solution containing AZT from +0.1 to – 1.2 V (vs. Ag/AgCl) with a scan rate of 100 mV/s. The first cathodic peak (C1 at−0.70 V) can be related to the irreversible reduction of the nitro to hydroxylamine group (−NHOH), and peaks A2 (0.08 V) andC2 (−0.2V) are related to the quasi-reversible behavior of the hydroxylamine group (Fig. 1). Scheme 2 is a representation of the mechanism of redox behavior of AZT [23].

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Scheme 2: Mechanism of redox behavior of AZT.

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Fig. 1

As the concentration of DNA increased, the peak currents decreased rapidly, which shows the

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slower mass transfer of AZT to the electrode surface. The diffusion coefficient was calculated

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using the following equation for a diffusion-controlled irreversible system [24] : ip= 2.99 ×105 n (αnα)1/2 A C0 D1/2 ν1/2

(1)

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where ip (A) is the peak current, A is the surface area of the electrode (0.034 cm2), C is the bulk concentration of the electroactive species (1.0 × 10-6mol/cm3), D (cm2 s-1) is the

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diffusion coefficient, ν (Vs-1) is the scan rate, nα is the number of electrons in the rate-

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determining step (nα = 1) and α is the transfer coefficient (α was calculated as 0.66 for AZT and 0.674 for AZT-DNA). The diffusion-control behavior was evident from the linear plot of

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the voltammetric response as a function of ν1/2. From the slope of the obtained line, diffusion

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coefficient for free AZT (Df) was calculated as 5.86× 10-5 cm2 s-1, whereas for the AZT– DNA complex, Db was reduced to 2.90 × 10-5 cm2 s-1. The negative shift in potential of C1 during successive additions of DNA, confirms the nonintercalative nature of the interaction, indeed it refers to electrostatic interactions with DNA [21, 25-28].

The effect of scan rate (ν) on peak current (iC1) was tested before and after

addition of DNA. Both peak currents of AZT and AZT–DNA complex were linearly dependent on the square root of scan rate, suggesting that the oxidation process was controlled by diffusion of the electroactive species to the electrode surface [24].

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In order to explore the effect of the macromolecule on the viscosity of the solution and on the effective surface area of the electrode, cyclic voltammetry was conducted in a solution of 1.0 × 10-3 M K4Fe(CN)6, in the absence and presence of DNA (up to 1 × 10-4

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M). The negligible change in the peak current of Fe(CN)6-4 confirmed that the presence of low concentrations of DNA has a minor effect on viscosity of the solution as well

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as on the effective surface area of the electrode.

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According to the following equation [29], the binding constant of AZT to DNA was calculated from voltammetric data: 

 



(2)

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log   log   log 

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where [DNA] is the molar concentration of DNA, i0 and i are the cathodic peak currents (at C2) of the total and free AZT, respectively. From the intercept of the linear plot of  

(Fig. 1, inset), Kf was obtained to be 1.22 × 103 at 25 ºC.

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3.2.



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log  against log 

Study of the interaction between DNA and AZT by UV-Visible spectroscopy

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UV–Visible absorption spectroscopy has been widely used in DNA binding studies as

it can give information concerning the binding strength and binding mode of small molecules to DNA.

The absorption spectra of AZT in the absence and presence of DNA (pH 7.4) are

shown in Fig. 2. A comparison was made (inset) between the recorded spectra of DNA (curve a), AZT (curve b), sum of the two spectra (curve c) and AZT-DNA mixture (curve d). In the absence of interaction, the sum of spectra of DNA and AZT (curve c) was much more intense than that experimentally obtained for their mixture. Thus, it was concluded that there exists some interaction between DNA and the drug. When the drug was titrated with DNA, a hyperchromic effect centered at 280 nm was observed. 9 Page 10 of 35

Fig. 2

In order to calculate Kf from absorption data, the following equation was used:

[ DNA] [ DNA] 1 = + ε a − ε f ε b − ε f K f (ε b − ε f )

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(3)

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where [DNA] is the concentration of DNA; εa, εf and εb correspond to the apparent, free drug and DNA-bound absorption coefficients in 280 nm, respectively. In the plots of

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[DNA]/ (εa- εf ) versus [DNA], Kf was obtained by the ratio of the slope to intercept. The

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value for Kf was obtained to be 2.1×105 M-1. This Kf value is similar to those reported for well-established groove binding agents [30], although different from the value obtained from

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voltammetric data. It may be due to the large difference between the methods used.

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3.3. Study of the interaction between AZT and DNA by spectrofluorimetry

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The changes in fluorescence spectra of molecules could be used as a sensitive optical tool to follow the interaction of chemical species. Studies of interaction of drugs with DNA

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require the presence of a fluorophore in the drug, due to the negligible emission of DNA molecule. Competitive method, on the other hand, could be useful in situations that the drug is not fluorescent, neither in the absence nor presence of DNA. In this method, a fluorescing molecule, with well-known location on DNA, was added to the DNA solution. If the drug binds to DNA through the same location, the probe will be replaced (completely or partially), causing an observable change in the fluorescence; otherwise, the emission signal would not change significantly. In our study of AZT-DNA interaction, the competitive fluorescence method was used, due to the weak emission of AZT. Two probes were used as competing agents for minor

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grooves (Hoechst 33258) and intercalation (MB). AZT was added to a mixture of DNA-probe and fluorescence spectra were recorded. It is well-known that Hoechst 33258 binds strongly to the minor grooves of double-

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stranded DNA with specificity for adenine-thymine-rich sequences [31]. Figure 3A represents the fluorescence spectrum of Hoechst 33258 in Tris buffer solution (pH 7.4), and

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different amounts of DNA. An emission peak is observed centered at about 410 nm (λex =

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340 nm). Addition of DNA to this solution increased the fluorescence intensity, due to the localization of Hoechst 33258 in grooves of DNA, thus, the non-emissive deactivation

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processes were reduced, effectively [32]. Significant quenching of fluorescence was observed by addition of AZT to the above mixture (Figure 3B), which shows the liberation of Hoechst

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to the solvent medium. The direct interaction between AZT and Hoechst was excluded by addition of AZT to a Hoechst solution, and the absence of significant changes in the

Fig. 3

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fluorescence spectrum.

In principle, two mechanisms for quenching are known: dynamic (collisional) and

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static. Dynamic quenching occurs when the quencher (AZT) come into contact to the fluorophore (Hoechst-DNA) in the excited state. The energy of the excited fluorophore is lost by collision without the formation of a new chemical species. Static quenching, on the other hand, refers to the formation of a complex between the quencher and the fluorophore in the ground state.

Static and dynamic quenching can be differentiated by several methods such as, studying the effect of temperature on the quenching constants, and study of temperature dependence of fluorescence lifetime and viscosity [33]. Stability of the products of static quenching will be reduced by temperature augmentation while, due to the increase in collisions at higher temperatures, dynamic quenching will proceed. Moreover, the lifetime of the excited state of

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the fluorophore is not affected by increased temperature in the case of static quenching (due to the interaction with the ground state of the fluorophore), while life time is largely reduced in dynamic quenching.

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Based on the above discussion, Stern-Volmer equation is usually used to investigate the quenching mechanism [33]: 

= 1     = 1+  [AZT]

(4)

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where F0 and F represent the fluorescence intensities of the fluorophorein theabsence and in the presence of quencher, respectively; kq is the quenching rate constant, τ0 is the lifetime of

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the fluorophore in the absence of quencher (usually τ0 =10-8 s). KSV is the Stern-Volmer

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quenching constant and [AZT] is the concentration of AZT.

The straight lines obtained at different temperatures confirms a single quenching

 

against [AZT].

The results showed that KSV decreased at higher

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linear plot of

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mechanism in experimental conditions used (Fig. 4). KSV was calculated from the slope of the

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temperatures (Table 1), which refers to static quenching, as was mentioned above. Thus, the formation of a non-fluorescing complex between AZT and DNA which liberates Hoechst to the solution is more probable than dynamic collisional quenching. Fig. 4

Kf was calculated from fluorescence quenching results according to the following

equation:

log

F

F

F

log K "  nlogAZT 5

Here, F0 and F are the fluorescence intensities of the fluorophore in the absence and in the presence of different concentrations of the drug, respectively, and n is the number of binding sites of DNA per each drug molecule. 12 Page 13 of 35

From fluorescence experiments at three different temperatures (288, 298, 310K), the plots of log

  

against log [AZT] were recorded (not shown). Table 1 shows Kf values which

are similar to that calculated from absorbance data (2.1×105 M-1). The number of binding

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sites of DNA per each drug molecule (n) was approximately unity at all studied temperatures. Table 1

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In order to investigate the probable intercalating of AZT between base pairs of DNA,

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methylene blue (MB) was used as a proper fluorophore. MB is a phenothiazinium dye that can interact with DNA mostly by intercalation, thus, upon binding to DNA, the fluorescence

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of MB is efficiently changed (in this case quenched). Negligible changes were observed after addition of AZT to the DNA-MB mixture (Fig. S1, Ref. [34]). The results indicate that AZT

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could not compete with MB for intercalation into base pairs of DNA.

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3.4.Thermodynamic parameters and binding forces for AZT-DNA complexation

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The enthalpy and entropy changes during the interaction between AZT and DNA (∆H and ∆S) were obtained by using Eq. 6: ∆)



∆,

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ln 

*+

(6)

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The calculated Kf values in different temperatures from Table 1 were used. The plot of lnKf against 1/T (Fig. 5) allowed to acquire ∆H and ∆S, assuming that ∆H is nearly constant over the range of applied temperatures [35, 36].The Gibbs’ free energy change of the reaction (∆G) was obtained by using van’t Hoff equation: ∆- ∆.

/∆0 (7)

An exothermic (∆H < 0), spontaneous reaction (∆G <0) was resulted for the binding of AZT to DNA (Table 2). Therefore, the interaction between AZT and DNA was both enthalpy and entropy (∆S > 0) driven. 13 Page 14 of 35

Fig. 5 Table 2

Moreover, Ross and Subramanian [37] showed that the sign and magnitude of the

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thermodynamic parameters (∆H and ∆S) are related to the involved binding forces. The binding forces between a small molecule and a macromolecule in aqueous solution mainly

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include van der Waals, electrostatic, and the hydrophobic forces. Based on these suggestions

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[36], if both ∆H and ∆S are positive, the main interaction force is hydrophobic interaction. ∆H and ∆S are negative, suggesting that the main interaction force is van der Waals force

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and/or hydrogen bonding interaction. ∆H is almost zero or negative and ∆S is positive, implying that the main interaction force is electrostatic force From Table 2, ∆H <0 and ∆S >

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0, shows the predominance of the electrostatic interaction, although hydrophobic forces could not be excluded. Regarding the positive charge on AZT (pKa = 8.07) at experimental

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conditions (pH 7.4), and negative charges of phosphate groups on DNA, the contribution of

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electrostatic forces seems to be rational.

3.5.Circular dichroism spectroscopy Circular dichroism spectroscopy (CD) is a strong tool for probing the asymmetry of

molecular systems. Chirality of the double helix of DNA results from the coupling of bases, phosphate backbone and chiral sugar units, which could be identified from CD spectral measurements[38] .

Different conformations of DNA, namely A, B, Z, etc. are recognizable from its CD spectrum. The characteristics of CD spectrum of DNA consist of a positive band at 275 nm, due to base stacking, anda negative band at 245 nm, due to its helicity(indicative of DNA in the right-handed B form)[39]. The predominant physiological conformation of DNA has been regarded as the B form. The "A" conformation is some 20° tilted with a fairly stiff 14 Page 15 of 35

backbone, which occurs under certain physiological conditions. Another interesting conformer of DNA is the Z conformer which is left-handed, opposite to A and B [40, 41]. During the interaction of DNA with exogenous substances, its conformation may be altered,

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the degree of which depends on the kind and extent of interaction [42-44]. In the present study, CD spectra of DNA were recorded in the absence and presence of AZT

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(Fig. 6). The intensity of the negative band at 245 nm decreased in the presence of AZT, with

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a weak red shift (≈3 nm), while the positive band at 275 nm increased. This observation is consistent with a conformational change from B to A, which usually takes place during

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groove binding mode of interaction [45]. Fig. 6

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3.6.Viscosity study

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Hydrodynamic methods, such as viscometry, are sensitive to the change of length of

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DNA. These methods may be the most effective means to study the binding mode of complexes to DNA in the absence of X-ray or NMR data. A classical intercalation mode

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demands the separation of base pairs to accommodate the binding species, so the DNA helix lengthens, leading to a substantial increase in viscosity. Groove binders, on the other hand, typically cause less pronounced changes (positive or negative) in DNA solution viscosity [46, 47].

Figure 7 shows the relative specific viscosity, (η/η0)1/3 against the mole ratio (ri =

[AZT]/[DNA]). At lower ri values, the viscosity decreased (as in electrostatic interactions) and then increased at higher mole ratios up to the initial viscosity (as in groove binding). It may be concluded that, at low amounts of AZT, it interacts with the exterior phosphate groups of DNA by electrostatic forces; while at higher concentrations of the drug, it enters the minor grooves of macromolecule.

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Fig. 7

3.7.Effect of ionic strength Electrostatic interactions occur between cationic species and the negatively charged

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phosphate backbone of DNA, so are greatly affected by ionic strength of the solution. Moreover, groove - bound molecules (partially exposed to solvent environment) can be

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infected from the helix with increasing ionic strength, despite intercalating molecules which

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are largely protected from the solvent [48].

The effect of ionic strength was studied on the absorption spectrum of AZT-DNA mixture

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(each 5.0 × 10-5 M). A gradual decrease was observed in the peak intensity without wavelength shift, which is in accordance with electrostatic and groove binding interactions

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(Fig. S2, [34]).

3.8.Theoretical calculations (Molecular docking)

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Theoretical docking studies provide insights into the interactions between the

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macromolecules and small chemical species.

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It is well known that the interactions of chemical species with the minor groove of B-DNA differ from those occurring with the major groove, both in terms of electrostatic 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. In the present work, DNA sequence (PDB id: 8BNA) was obtained from the Protein

Data Bank [49-52]. The calculations resulted in the formation of 3-D docked structures of AZT-DNA complex. These structures were subjected to energy minimization calculations, so that the most stable chemical structure of the complex could be obtained. A part of the run data for the conformers are listed in Table 3. The energetically most favorable conformation (selected rank with maximum frequency, Run No. 65from 100 runs) revealed that AZT approaches the gap between DNA minor grooves mainly through its –NO2 group on 16 Page 17 of 35

imidazolicring (Fig. 8A), and is situated within narrower A–T (10.8 Å) regions compared to G–C (13.2 Å) ones. The preference of A–T regions leads to van der Waals and hydrophobic interactions with DNA which stabilize groove binding mode. The drug molecule exists in a

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crescent shape akin to classical minor groove binders. This shape is the natural curvature of the minor groove of B-DNA.Groove binders usually contain aromatic rings connected by

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single bonds which allows the rotation and nestle of the molecule in the DNA groove,

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properly [4].

A competitive molecular docking was also performed in the presence of Hoechst.

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DNA sequence (PDB ID: 8BNA) was obtained from the Protein Data Bank [46–49]. The energetically most favorable conformation of the docked poserevealed that AZT approached

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the gap inside DNA minor grooves where the Hoechst was (Fig. 8B).The resulted ∆G of interaction from docking optimization, (-26.15 kJ/mol in the case of 8BNA)was very similar

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to experimentalcalculations (-27.89 kJ/mol).

Table 3 Fig. 8

4. Conclusions

Interaction between azathioprine and DNA was studied by different methods. From the results, it was concluded that azathioprine interacts with DNA by electrostatic interactions and minor groove binding, with one binding site per drug molecule. The binding constants were obtained from different methods, which were consistent with groove binding mode. Thermodynamic calculations revealed the involvement of electrostatic forces in the complexation. Energy minimization in molecular docking calculations resulted in a stable

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structure for the mixture of DNA and azathioprine, in which the drug approached the minor

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an

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cr

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groove of DNA from its imidazolic moiety including −NO2 group.

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[1] R.Y. Calne, G. Alexandre, J. Murray, ANYAS, 99 (1962) 743-761. [2] G.B. Elion, Biosci. Rep., 9 (1989) 509-529.

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[3] R. Marty, C.N. N'soukpoé-Kossi, D. Charbonneau, C.M. Weinert, L. Kreplak, H.-A. Tajmir-Riahi, Nucleic Acids Res., 37 (2009) 849-857.

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[4] R.B. Silverman, The organic chemistry of drug design and drug action, Academic press,

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2004.

[5] X. Ling, W. Zhong, Q. Huang, K. Ni, J. Photochem. Photobiol. B: Biol., 93 (2008) 172-

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[6] N. Modukuru, K. Snow, B.S. Perrin Jr, A. Bhambhani, M. Duff, C.V. Kumar, J.

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Photochem. Photobiol. A: Chem., 177 (2006) 43-54.

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Table1.Calculated quenching constants (KSV), binding constants (log Kf), and binding

stoichiometry (n) for the complex AZT-DNA at different temperatures (using the results of fluorescence experiments). T(k)

R2

KSV (L mol-1)

log Kf

R2

288

0.9939

8047.7

5.0355

0.9996

1.3189

298

0.9916

7607.1

4.8897

0.9971

1.2849

310

0.9924

7246.6

4.7747

0.9995

1.2616

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n

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Table 2. Thermodynamic parameters for the binding of AZT to DNA

288

-27.76

298

-27.89

310

-28.33

∆H (kJmol-1)

∆S (Jmol-1K-1)

-20.198

26.11

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∆G (kJ mol-1)

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T(K)

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Table 3: Some of the docking results for DNA (1BNA) − AZT by using the AutoDock program generated different ligand conformers using Genetic Algorithm.

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Ref RMS 30.89 32.41 26.97 28.24 27.49 30.95 28.57 29.07 35.87 22.16 26.4 24.24 34.67 25.34 32.27 29.12 41.45 11.96 26.32 35.13 29.38 30.49 25.19 15.06 28.09 13.65 23.98

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Cl RMS 0 0 0 0 1.88 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

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Inhibconstant 4.51 5.06 8.54 10.08 12.12 14.26 18.39 20.32 23.72 23.92 30.6 31 31.48 35.87 36.95 37.05 46.34 45.73 46.55 49.7 54.2 53.75 55.17 58.79 59.99 60.36 61.31

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Ligandefficiency -0.17 -0.16 -0.15 -0.14 -0.14 -0.13 -0.12 -0.12 -0.12 -0.12 -0.11 -0.11 -0.11 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.09 -0.09 -0.09 -0.09 -0.09 -0.09 -0.09 -0.09

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65 96 92 43 74 98 49 3 62 56 64 88 86 87 78 67 29 70 84 90 30 9 53 13 5 42 10

Bindingenergy -3.2 -3.13 -2.82 -2.72 -2.61 -2.52 -2.37 -2.31 -2.22 -2.21 -2.07 -2.06 -2.05 -1.97 -1.95 -1.95 -1.89 -1.83 -1.82 -1.78 -1.73 -1.73 -1.72 -1.68 -1.67 -1.66 -1.65

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Figures Captions: Figure 1 Cyclic voltammograms of AZT in the presence of DNA. [DNA] = 0 – 1 × 10-3 M,

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Scan rate = 100 mV s-1;[AZT]= 5.0 × 10-4 M. Inset:Plot of log (1/[DNA]) against log [i/(i0-i)] Figure 2 Absorption spectra of azathioprine (5.0 × 10-5 M) in the presence of increasing

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amounts of DNA; (ri = 0.0 – 2.8).Inset:(a) DNA; (b) AZT; (c) Sum of a and b; (d) observed

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spectrum of AZT + DNA mixture.

Figure 3 (A) Fluorescence spectra of Hoechst 33258 in the presence of increasing amounts of

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DNA, [DNA]= 0 – 5×10-4 M. (B)Fluorescence spectra of (A) + increasing amounts of AZT. [Hoechst] = 5.0 ×10-6 M; [AZT]= 0.28 – 2.37 ×10-4 M; T = 288 K,λex = 340 nm.

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Figure 4 Stern–Volmer plots at different temperatures. T=288 , 298 , 310 K.

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Figure 5 Van’t Hoff plot for AZT – DNA complex.

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=0, 0.6, 2.2, 3.0).

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Figure 6 CD spectra of DNA (5×10-5 M) in the presence of increasing amounts of AZT. (ri

Figure 7 Effect of increasing amounts of AZT to DNA on the viscosity of solution. [DNA] =

5×10-5 M; (Ri=0.0 -2.0).

Figure 8 Molecular docked model of AZT with (A) DNA (PDB ID: 1BNA),

d(CGACGCGTCG)2. (B) DNA (PDB ID: 8BNA) in the presence of Hoechst.

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