International Journal of Biological Macromolecules 78 (2015) 122–129
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Spectroscopic and molecular docking studies on the interaction of troxerutin with DNA A. Subastri a , C.H. Ramamurthy a , A. Suyavaran a , R. Mareeswaran a , P. Lokeswara Rao a , M. Harikrishna b , M. Suresh Kumar b , V. Sujatha c , C. Thirunavukkarasu a,d,∗ a
Department of Biochemistry and Molecular Biology, Pondicherry University, Puducherry 605 014, India Centre for Bioinformatics, Pondicherry University, Puducherry 605 014, India c Department of Chemistry, Periyar University, Salem 636 011, India d Department of Medicine – Gastroenterology and Liver diseases, 625, Ullmann Building, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10469, USA b
a r t i c l e
i n f o
Article history: Received 26 September 2014 Received in revised form 18 March 2015 Accepted 19 March 2015 Available online 6 April 2015 Keywords: Spectroscopy Molecular docking DNA Troxerutin
a b s t r a c t Troxerutin (TXER) is a derivative of naturally occurring bioflavonoid rutin. It possesses different biological activities in rising clinical world. The biological activity possessed by most of the drugs mainly targets on macromolecules. Hence, in the current study we have examined the interaction mechanism of TXER with calf thymus DNA (CT-DNA) by using various spectroscopic methods, isothermal titration calorimetry (ITC) and molecular docking studies. Further, DNA cleavage study was carried out to find the DNA protection activity of TXER. UV-absorption and emission spectroscopy showed low binding constant values via groove binding. Circular dichroism study indicates that TXER does not modify native B-form of DNA, and it retains the native B-conformation. Furthermore, no effective positive potential peak shift was observed in TXER–DNA complex during electrochemical analysis by which it represents an interaction of TXER with DNA through groove binding. Molecular docking study showed thymine guanine based interaction with docking score −7.09 kcal/mol. This result was compared to experimental ITC value. The DNA cleavage study illustrates that TXER does not cause any DNA damage as well as TXER showed DNA protection against hydroxyl radical induced DNA damage. From this study, we conclude that TXER interacts with DNA by fashion of groove binding. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Interaction of small ligand molecule and DNA is becoming progressive part in current research field [1,2]. DNA is a great biologically significant macromolecule and plays an essential role in cellular progression including DNA replication, transcription and translation. These processes are very crucial for cell survival and cell proliferation. Therefore, cellular DNA is the most important pharmacological target of several drugs including anti-tumour, anti-viral and anti-bacterial drugs [3–5] that are presently in clinical usage or in advanced pharmaceutical trials. Therefore in target of DNA, the number of biological activity of small ligand molecules binds with DNA and causing inhibition of cellular progression by
∗ Corresponding author at: Department of Biochemistry and Molecular Biology, Pondicherry University, Puducherry 605 014, India. Tel.: +91 413 2654972/+11 347 471 6477; fax: +91 413 2655255. E-mail addresses: [email protected], [email protected] (C. Thirunavukkarasu). http://dx.doi.org/10.1016/j.ijbiomac.2015.03.036 0141-8130/© 2015 Elsevier B.V. All rights reserved.
modulating gene expression in DNA [6]. These synthetic or natural small compounds could act as drugs in conditions where inhibition or modification of cellular DNA function is needed to alleviate or control a disease [7]. Moreover, depending on the mode of interaction, compounds can be classified as covalent binding and non-covalent binding drugs. In covalent binding types, drugs are bounded with DNA through alkylation or inter and intra strand cross linking by complete inhibition of DNA progression. But the non-covalent binding, effect is very weak compared to covalent binding. Furthermore, the non-covalent binding is also classified into three types namely, intercalation, groove binding and external binding [8]. Hence, these DNA–drug interaction studies are very valuable and need of the hour for designing novel and more effectual drugs with fewer side effects. On the other hand, mechanism of interaction between drug and DNA is still comparatively meagerly known. From this conception, in the current study we have focused on bioflavonoid rutin derivative troxerutin (TXER) for DNA interaction. TXER is a bioflavonoid derivative of rutin (Fig. 1) found in many plants, vegetables and fruits, and can be largely extracted
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of TXER solution was added to the final concentration of CT-DNA (100 M) and mixed thoroughly by vortex. For the UV–visible and fluorescent spectroscopy studies, 100 M concentration of CT-DNA solution was used along with varying concentrations (10–100 M) of TXER. Circular dichroism spectroscopy study was carried out using with two different concentration of TXER (0.05 mM and 0.1 mM) with constant CT-DNA concentration of 100 M. 2.3. UV–visible spectroscopy measurements
Fig. 1. Chemical structure of troxerutin.
from Sophora japonica. It has been proclaimed to possess various significant pharmacological activities including antioxidant [9–12], anti-inflammatory activity [13], anti-erythrocytic, antithrombiotic and fibrinolytic activity [14]. In addition recent study of TXER reported that protect the cells against UVB radiation [15]. Lu et al. [16] revealed that TXER has protective effect against cognitive impairment. Furthermore, TXER was shown as a chemopreventive agent against colon carcinogenesis [17]. So, previous reports have clearly showed that TXER is an emerging small molecule for numerous diseases in present clinical scenario. However, there are no studies on interaction of TXER with cellular DNA. Hence, the current study has been determined to investigate the binding mode of TXER with DNA. The basic understanding of binding mode and affinity of small molecule with DNA is crucial to analyze the investigational results procured in toxicology and to design the restorative formulation. In the current study, the interaction of TXER with calf thymus DNA (CT-DNA) has been explored in vitro by using various spectroscopic techniques, isothermal titration calorimetry (ITC), molecular docking and DNA protection studies. The elucidation on the binding mechanism of compound TXER with CT-DNA could help in bringing the awareness of the biological significance of this small molecule and its toxicity. 2. Materials and methods 2.1. Materials CT-DNA sodium salt and TXER were purchased from Sigma–Aldrich chemicals (Sigma–Aldrich, USA). The ratio of the absorption at 260/280 nm was measured to confirm the purity of DNA sample. The ratio was found to be 1.8 which revealed that purity of DNA without any protein contamination [18]. The DNA solution, TXER solution and buffer were freshly prepared from deionized ultra pure water. Other chemicals and reagents used in this study were of analytical grade.
The absorption spectra of CT-DNA alone and TXER-DNA complex were recorded using Shimadzu UV–visible spectrophotometer, (Model – UV-1800). Quartz cuvette with a path length of 1 cm was used for the spectroscopy measurements. The binding constant of CT-DNA–TXER complex was calculated by the method of Kanakis et al. [19]. It was assumed that only one type of interaction occurred between drug and DNA in aqueous solution resulting in the formation of one type of complex. Based on this assumption, Eqs. (1) and (2) were established. DNA + Drug → DNA : Drug
(1)
The equilibrium constant is given by K=
DNA : TXER (DNA)(TXER)
(2)
Eq. (2) can be written as K=
CDT [CD ][CT ]
(3)
where, CDT , CD and CT are the analytical concentration of TXER–DNA complex, DNA alone, and TXER alone in 10 mM Tris–HCl buffer solution (pH 7.4, 25 ◦ C) respectively. Beer–Lambert law for the absorption of light is assumed to be followed by the ligand substrate binding. CD = CD0 − CDT CDT =
(4)
A − A0 εDT · L
(5)
A0 εD · L
(6)
and CD0 =
where, CD0 is the concentration of pure DNA, A0 and A are the absorbance of pure DNA and TXER–DNA complex respectively at 260 nm. εD and εDT are the molar extinction coefficient of DNA alone and TXER–DNA complex respectively. L is the path length of cuvette (1 cm). By putting the values of CD and CDT from above equations into the Eq. (3), following equation can be deduced: A0 εD εD 1 = + × A − A0 εDT CT (εDT · K)
(7)
The double reciprocal plot of 1/(A − A0 ) versus 1/CT (CT is the concentration of TXER) is linear and thus the binding constant (K) can be estimated by calculating the ratio of the intercept to the slope [20].
2.2. Stock solution preparation
2.4. Fluorescence measurements
CT-DNA stock solution was prepared by dissolving CT-DNA (10 mg/ml) in 10 mM Tris–HCl buffer (pH 7.4) at 4 ◦ C for 24 h with intermittent stirring to ensure the formation of the homogeneous solution. The final concentration of the CT-DNA stock solution was estimated spectrophotometrically at 260 nm using molar extinction coefficient ε260 = 6600 cm−1 M−1 . The TXER stock solution (1 mM) was prepared in ultrapure water. Different concentrations
Fluorescence emission study was carried out using FLUOROLOG – FL3-11 spectrofluorometer equipped with a xenon lamp source. All the readings were obtained by using a 10 mm path length cuvette in a 10 mM Tris–HCl buffer at pH 7.4. In this emission spectral study, different concentration (10–100 M) of TXER was mixed with constant 100 M concentration of CT-DNA in the presence of intercalating DNA dye such as ethidium bromide (2 M EtBr).
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The fluorescence intensity of pure CT-DNA has very low emission spectrum. The fluorescence dye, EtBr is used to examine interaction mechanism of drug–DNA complex [5]. EtBr is an eminent conjugate probe to bind with DNA in intercalation mode [21] and maximum excitation of EtBr is 510 nm [22]. In EtBr competitive assay, the emission spectra for different concentration of TXER with CT-DNA complexes were recorded at wavelength of 510–800 nm with excitation at 520 nm. All spectral readings were carried out at 25 ◦ C and all fluorescence intensities of the sample were expressed in arbitrary units. Finally quenching constant (KSV ) values of TXER–DNA complex can be determined by using following Stern–Volmer equation [23]: F0 = 1 + KSV [Q ] F where, F0 and F are the fluorescence intensities in the absence and presence of the quencher (TXER) in EtBr bound DNA respectively and Q is the concentration of the quencher (TXER), KSV is the Stern–Volmer quenching constant. Hence, KSV value can be calculated from the slope of the linear relationship between [F0 /F] and [Q]. 2.5. Circular dichroism spectral measurements Circular dichroism (CD) spectra of pure DNA and its TREX complexes were recorded with JASCO J-815 CD spectrophotometer [24]. All spectra were measured in far-UV range (200–320 nm). Quartz cuvette of 1 mm path length was used for sampling. Three scans with a scan speed of 50 nm/min were performed and averaged. All the measurements were done at 25 ◦ C. Base correction of spectrum was performed with Tris–HCl buffer at pH 7.4 to obtain the spectrum of CT-DNA alone and TXER–DNA complex. 2.6. Cyclic voltammetric study The cyclic voltammetric (CV) study of DNA was carried out by using autolab type (PGSTAT 302N) electrochemical analyzer [25] through three electrode systems composed of glassy carbon electrode as a working electrode, Ag/AgCl served as a reference electrode and Pt wire served as a counter electrode with scan rate of 100 mV/s at room temperature. Electrochemical analysis of all samples was performed by using 50 ml voltammetric cell. In addition, different concentration of TXER (10–100 M) and constant concentration of DNA (100 M) were used and 10 mM Tris–HCl buffer (pH 7.4) exploited as supporting electrolyte. 2.7. Isothermal titration calorimetry In order to confirm the binding interactions between TXER and DNA, we performed the isothermal titration calorimetry (ITC) experiment using NANO ITC. All the samples for ITC experiment were prepared with degassed buffer (10 mM Tris–HCl buffer pH 7.4) to avoid gas bubbles which may possible to disturb the baseline. All ITC experiment was maintained at 25 ◦ C with stirring speed of 150 rpm. The assay was startup after baseline correction so that the heat generated by stirring can be automatically subtracted. The 1 mM concentration of TXER and 0.5 mM of CT-DNA was used into the sample cell through 25 individual injections programmed at 5 min intervals. The titration curves were corrected for heat of dilution determined by injecting the ligand into the buffer and CT-DNA into the buffer solution. The region under each peak was integrated heats of dilution was subtracted and the thermo-gram for the binding of TXER to DNA has been obtained (Fig. 8a and b). The obtained results were further fitted using NANO ITC software and molar change in enthalpy (H in kJ/mol), binding constant Ka (M−1 ) and binding stoichiometry (n) were obtained to conclude the result. The
binding free energy of the experiment was calculated by using the following formula: G = H − TS = −RT ln Ka where, the G, H, and S stand for the changes in free energy, enthalpy and entropy of binding; T, temperature and R (gas constant) = 1.98 cal/mol K. 2.8. Molecular docking studies Docking study for interaction between DNA and TXER was clarified by using Glide software [26], which is part of the Maestro software suite. The PDB structure of CT-DNA sequence dodecamer d(CGCGAATTCGCG)2 (PDB ID: 1BNA) was retrieved from Protein Data Bank was refined using the protein preparation wizard by removing the hetero atoms and using the Imperf minimization tool to converge the heavy atoms. Likewise ligand such as TXER structure was retrieved from the PubChem database. The ligand structure was prepared for docking using LigPrep application in Maestro. Then the ligand was energy minimized using OPLS-2005 force field implemented in Maestro. After preparation of receptor and ligand files, docking tool was applied to generate an interaction between TXER and CT-DNA and finally, binding energy as well as binding pose of the complex was obtained. 2.9. DNA cleavage study The cleavage of DNA was observed through agarose gel electrophoresis by modified method of Tabassum et al. [27]. In this reaction super-coiled pBR322 circular plasmid DNA (250 ng) was treated with various concentrations of TXER (25, 50 and 100 M) in presence and absence of 5 l Fenton’s reagent (30 mM H2 O2 , 50 mM ascorbic acid, 80 mM FeCl3 ) and made upto a final volume of 25 l with Tris–HCl buffer then samples were incubated at 37 ◦ C for 15 min. After incubation gel loading buffer (6×) was added and mixtures were electrophoresed for 1 h at 80 V on 2% agarose gel in TAE buffer. 3. Results and discussion 3.1. UV–visible spectroscopic analysis UV-absorption spectroscopy is an effectual technique for identifying the binding potency and the mode of interaction of small molecule with DNA. The absorption spectra of CT-DNA alone and TXER–DNA complex with various concentration of TXER is depicted in Fig. 2. The maximum absorption peak of TXER–DNA complexes were 255 nm and 350 nm respectively. Moreover, hyperchromic effect was observed without any noticeable red shift (bathochromic shift) at position of maximum absorption of TXER–DNA complexes. So absence of red shift in the UV spectra analysis indicates that the binding mode is not the intercalative binding [24,28]. From the UV spectrum results of TXER–DNA complex suggest that TXER may interact with DNA through groove binding mode. Furthermore, the effect of hyperchromism may be due to unwinding of DNA helix [29] or electrostatic binding [30]. Then stability of bound TXER–DNA complex was determined through calculating binding constant by using the formula described in materials and methods section (Section 2.3). The double reciprocal plot of TXER–DNA complex’s shows as a linear correlation between 1/(A − A0 ) and 1/C (Fig. 3). From the graph, the binding constant value (K) of TXER–DNA complex is 1.75 × 103 M−1 . This calculated binding constant value shows weak binding of TXER with DNA when compared to results observed in strong binding compounds like EtBr, acridine orange interaction study with DNA [31]. And also this
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Fig. 2. UV absorption spectra of CT-DNA alone and TXER–DNA complex with various concentrations of TXER (0.01–0.1 mM). The arrow represents increasing absorbance of TXER–DNA complexes: (a) CT-DNA alone (100 M CT-DNA in 10 mM Tris–HCl buffer, pH 7.4 at 25 ◦ C); (b) CT-DNA + 0.01 mM TXER; (c) CT-DNA + 0.02 mM TXER; (d) CT-DNA + 0.04 mM TXER; (e) CT-DNA + 0.06 mM TXER; (f) CT-DNA + 0.08 mM TXER; (g) CT-DNA + 0.1 mM TXER.
binding constant value closely agrees with the previous reports of some other weak DNA binders [32]. 3.2. Emission spectroscopic study Fluorescent spectroscopy is one of the trendy techniques and is a highly sensitive method to study the interaction between macromolecules and small molecules. So, the binding mechanism of TREX with DNA was determined by fluorescence emission spectroscopy. At this point competitive binding study was carried out to investigate the binding mode of TXER with DNA. For this study, EtBr was used, since it is a prevalent fluorophore that binds to DNA. The endogenous fluorescence property of DNA is very low. But the fluorescence of DNA was increased in the presence of EtBr due to substantial intercalation between the base pairs of CT-DNA. The
Fig. 3. Double reciprocal plot of TXER–DNA interaction by using UV–visible spectroscopy. A0 is the initial absorption of CT-DNA (100 M in 10 mM Tris–HCl buffer, pH 7.4) and A is the absorption at different concentration (0.01–0.1 mM) of TXER–DNA complex and C is the analytical concentration (0.01–0.1 mM) of TXER in 100 M CT-DNA (100 M CT-DNA in 10 mM Tris–HCl buffer, pH 7.4 at 25 ◦ C).
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Fig. 4. Fluorescence emission spectra of EtBr–DNA complex alone and in the presence of different concentration of TXER (0.01–0.1 mM). The arrow indicates increasing concentration of TXER with EtBr–DNA complexes: (a) EtBr–DNA alone (100 M CT-DNA in 10 mM Tris–HCl buffer, pH 7.4 with 2 M EtBr at 25 ◦ C); (b) 0.01 mM TXER with EtBr–DNA; (c) 0.02 mM TXER with EtBr–DNA; (d) 0.04 mM TXER with EtBr–DNA; (e) 0.08 mM TXER with EtBr–DNA; (f) 0.1 mM TXER with EtBr–DNA. ex = 520.
emission spectrum of EtBr bound DNA–TXER complex is shown in Fig. 4. The different concentrations of TXER with EtBr bound CTDNA showed fluorescence emission peak at 600 nm upon excitation at 520 nm. The fluorescence intensity of EtBr bound DNA complexes slightly decreases without any major peak shift while adding different concentration of TXER, it showing the groove binding property of TXER with DNA [5]. The minor decrease in fluorescence intensity might be due to some aggregation between TXER and EtBr bound DNA complexes. Furthermore, fluorescence quenching constant was determined by using the Stern–Volmer equation, explained in materials and methods (Section 2.4). The value of Stern–Volmer dynamic quenching constant (Ksv ) was determined as 1.45 × 103 M−1 (Fig. 5) which was much lower than the strong DNA intercalating agents [33]. Therefore this interaction represents lesser potential of small molecule such as TXER for intercalative binding. Consequently, the present study suggests the interaction of ligand such as TXER with CT-DNA through groove binding mode.
Fig. 5. The double reciprocal plot between F0 /F and Q, where F0 is the fluorescene intensity of CT-DNA and F is the intensity of TXER–CT-DNA complexes and Q is the analytical concentration (0.01–0.1 mM) of TXER in CT-DNA (100 M CT-DNA in 10 mM Tris–HCl buffer, pH 7.4 at 25 ◦ C with 2 M EtBr).
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with DNA [43]. This confirmation was supported by UV–visible and fluorescence spectroscopy analysis. 3.5. Isothermal titration calorimetry
Fig. 6. Circular dichroism spectra of CT-DNA alone and TXER–DNA complexes: (a) CT-DNA alone (100 M CT-DNA in 10 mM Tris–HCl buffer, pH 7.4 at 25 ◦ C); (b) 0.05 mM TXER + CT-DNA; (c) 0.1 mM TXER + CT-DNA.
3.3. Circular dichroism spectroscopic study CD spectroscopy is a reliable technique for detecting conformational changes in bio macromolecules. The CD spectrum of CT-DNA alone has four major bands at position 210 nm, 220 nm, 245 nm, 275 nm and these bands are markers for B-conformation of DNA. The positive band occurs at 275 nm due to base stacking and negative band forms at 245 nm due to helicity of DNA [34,35]. During the transition of DNA alone from B-form to A-form, the 210 nm peak intensity will decrease and 275 nm peak intensity will increase with band shifting [36]. Further Vorlickova [37] reported that Aconformation of DNA shows a strong positive CD band at 263 nm and negative band at 212 nm. In our present study, two different concentrations [Molar ratio (R) = TXER/DNA = 0.05 mM/0.1 mM, 0.1 mM/0.1 mM] of TXER–DNA complex were subjected to CD spectroscopy analysis. Outcomes for CD spectra of CT-DNA alone and TXER–DNA complex were shown in Fig. 6. The figure shows four peaks, at position 210 nm (negative band), 220 nm (positive band), 245 nm (negative band) and 275 nm (positive band). From this observation, CD spectra of TXER–DNA complexes did not show any perceptible changes in B-conformation of DNA. However, modest variation observed at 274 nm, 210 nm and 245 nm in shift and intensity of TXER–DNA complexes due to aggregation effect of TREX on DNA [38,39]. Conclusively, CD spectrum results suggest that DNA retains its B-conformation after interaction with TXER. 3.4. Electrochemical study Electrochemical method of CV is a powerful technique for detecting the binding mode of TXER-DNA complex. And also, this technique measures the current that is generated in an electrochemical cell under appropriate conditions where voltage is in excess of that calculated by the Nernst equation. Furthermore, CV was accomplished by potential of functioning electrode and measuring the resulting current [40]. The deviation in peak potential and current can be subjected for the prediction of binding specifications. Fig. 7 shows CV measurements of CT-DNA in presence and absence of TXER at different concentration. From this observation we did not find any considerable positive peak shifts in TXER–DNA complexes compared to intercalating binders [41,42]. Additionally, interaction of TXER with CT-DNA shows moderate peak current. This may be due to interaction of TXER with DNA by groove binding mode. This finding agrees with other small molecule interaction
ITC is a valuable technique for an absolute understanding of drug–DNA interactions. Certainly ITC is the only method that directly determines the binding enthalpy changes in the formation of a complex allow the free energy change to be divided into the enthalpic and entropic involvement to the association process. It discloses the nature of some forces that drive the binding reaction [44]. Thermodynamic analysis was carried out for TREX–DNA complex and titration curve has been depicted in Fig. 8(a and b) which shows the ITC titrations results where each heat curve represents to a single TXER injection to DNA. The area under these curves is determined by integration to provide the associated injection heats. The experimental data exposed that enthalpy change H is 81.99 kJ/mol, entropy change S is 330.1 J/mol/K and Gibbs free energy G is −16.7 kJ/mol or −4.0 kcal/mol and binding stoichiometry n = 1.102. The negative values of G illustrated that TXER–DNA complex is a spontaneous process. Therefore, the negative value of the free energy represents more favorable interaction of TXER–DNA [45]. The enthalpy (H) and entropy (S) were both positive, it may be due to the non-covalent interactions such as hydrogen bonds, van der Waals force and hydrophobic interactions involving in TXER–DNA complex [46]. From ITC data, the interaction of TXER–DNA represents entropy driven endothermic binding with positive enthalpy and entropy change. This result suggests interaction of TXER with DNA through groove binding mode [47,48]. Moreover a favorable enthalpy change is observed through ITC for TXER binding to DNA may be due to groove binding mode. This result agrees with Pagano et al. [49] reported that groove binder shows favorable enthalpy changes (H) which may be due to the result of electrostatic interaction and hydrogen bond formation in the complexes. Furthermore, the value of the binding constant in ITC was exposed as 7.5 × 102 M−1 which was much lower than other strong groove binders [50,51]. The strong groove binders have larger binding constant (1011 M−1 ) than intercalators [52]. Additionally Saha et al. [53] reported that tremendous groove binder Hoechst 33258 showed strong binding affinity on DNA. Therefore from these previous reports, the binding constant value of TXER–DNA complex is very low as compared to strong groove binders [54]. Hence the present ITC experiment suggests interaction of TXER with DNA through groove binding with weak binding affinity. 3.6. Molecular docking The discovery and evaluation of novel therapeutic drugs for the treatment of various emerging diseases without any side effects is the most vital goal in modern medical world. The results obtained from spectroscopy and electrochemical methods were supportive in understanding the mode of DNA–TREX complex. In addition to this molecular docking, which is a powerful in silico technique was performed for clarification of the interaction mechanism. The result of molecular docking analysis of TXER–DNA complexes was shown in Fig. 9. These computed parameters asserted the complex of TXER and DNA. The TXER interacts with the deoxy guanine 16 by forming an H bond between the O and N residues with a bond length of ˚ Similarly, the TXER also interacts with the deoxythiamine 3.24 A. residue 7 forming two H bonds between the O atoms of the phosphate group of the DNA and CO moiety of TXER with bond lengths 2.95 A˚ and 3.23 A˚ respectively. The TXER also shows hydrophobic interactions with the deoxy cytosine and deoxy thymine residues. Hence formation of hydrophobic and hydrogen bond recommends
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Fig. 7. Cyclic voltammogram of CT-DNA alone and TXER–DNA complex with various concentrations of TXER (0.01–0.1 mM). The arrow represents increasing concentration of TXER (0.0–0.1 mM) with constant 100 M CT-DNA: (a) CT-DNA alone (100 M CT-DNA in 10 mM Tris–HCl buffer, pH 7.4 at 25 ◦ C); (b) 0.01 mM TXER + CT-DNA; (c) 0.02 mM TXER + CT-DNA; (d) 0.04 mM TXER + CT-DNA; (e) 0.08 mM TXER + CT-DNA; (f) 0.1 mM TXER + CT-DNA.
interaction between TXER and DNA. Conclusively TXER interacts in the major groove of the DNA with a glide docking score of −7.097 kcal/mol. This docking free energy was compared to experimental ITC free energy value (G) which is −4.0 kcal/mol. The variation in the free energy values obtained from experimental (ITC) and computational (Molecular docking) results occurs due to many factors. In experiment of ITC, binding free energy contains
the flexibility of DNA with several other energy terms like translational energy, entropy, hydration free energy, conformational free energy, etc. But the molecular docking calculations absolutely considered DNA as a rigid molecule and small molecule (drug) as a flexible molecule. Additionally, the free energy from ITC also gains the effects from counter ion of buffer solution but molecular docking do not consider the counter ions in the interaction process [55].
Fig. 8. (a) ITC profile for the titration of CT-DNA (0.5 mM) with solution of TXER (1 mM) in 10 mM Tris–HCl buffer pH 7.4 at 25 ◦ C. (b) Isothermal plot of heat change verses molar ratio of TXER–DNA.
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Fig. 9. Molecular docking structural representation of TXER–DNA interaction by using Glide software [26]. (a) Molecular docked model of TXER showing groove binding with DNA dodecamer duplex of sequence d(CGCGAATTCGCG)2 (PDB id: 1BNA); (b) Surface view of molecular docked TXER on DNA dodecamer; (c) Docked model of TXER–DNA complex indicating intermolecular H-bonds.
3.7. DNA protection activity The DNA cleavage property of TXER was analyzed by gel electrophoresis using pBR322 plasmid DNA. When a circular plasmid DNA is subjected to electropheresis, the supercoiled form (Form I – shown in Fig. 10) usually migrate faster. If incision occurs on one strand (nicked circular) supercoil unwinds to create a slow moving open circular form (Form II – shown in Fig. 10). When both strands are cleaved, linear form (Form III – shown in Fig. 10) is generated that migrates between form I and form II [56]. The results of DNA cleavage study after incubation with various concentration of TXER in presence and absence of Fenton’s reagent is shown in Fig. 10. Various concentrations of TXER alone treated plasmid DNA (Lane 3–5) did not show any changes (Form III) in electophoretic band pattern of DNA but other condition of DNA i.e., pretreatment with Fenton’s reagent, depicted significant changes in DNA
Fig. 10. Agarose gel electrophoresis pattern of the pBR322 plasmid DNA cleavage by TXER in presence and absence of Fenton’s regent (30 mM H2 O2 , 50 mM ascorbic acid, 80 mM FeCl3 ). [Lane 1: DNA alone; Lane 2: Fenton’s reagent (5 l) + DNA; Lane 3: DNA + TXER (25 M); Lane 4: DNA + TXER (50 M); Lane 5: DNA + TXER (100 M); Lane 6: DNA + TXER (25 M) + Fenton’s reagent (5 l); Lane 7: DNA + TXER (50 M) + Fenton’s reagent (5 l); Lane 8: DNA + TXER (100 M) + Fenton’s reagent (5 l)].
migration pattern. The lane 2 (5 l Fenton’s reagent alone) expressed more intensity of band in linear form (Form I) due to oxidative DNA cleavage by Fenton’s reagent [57]. But on other hand higher concentration of TXER (Lane7: 50 M TXER + 5 l Fenton’s reagent, Lane 8: 100 M TXER + 5 l Fenton’s reagent) diminished the DNA damage caused by hydroxyl radicals (generated through Fenton’s reagent). But Lane 6 (25 M TXER + 5 l Fenton’s reagent) showed very less activity compared to high concentration. From this observation, we suggest that TXER strongly protects the DNA from oxidative damage as well as did not cause any DNA damage. 4. Conclusion In the current study we explored the interaction of small ligand molecule such as TXER with CT-DNA by using various spectroscopic and docking techniques. The results of binding studies suggest that the TXER binds with DNA through groove binding manner. This interaction existence is supported by following findings. The UV absorption spectroscopy showed weak binding of TXER with binding constant value 1.75 × 103 M−1 which suggests weak interaction compared to other strong binders. Emission study also is supportive of the interaction by showing less binding KSV value 1.45 × 103 M−1 . Furthermore, CD results demonstrate that the binding of TXER did not induce any significant conformational changes in B-form of DNA. Electrochemical CV study confirms groove binding of TXER which is visualized through absence of any noticeable positive shift in potential peaks of complex. The thermodynamic analysis of TXER–DNA showed that complex is driven by entropy through groove binding. Additionally, molecular docking study shows interactions of TXER in the groove of DNA via formation of hydrogen bond and hydrophobic bond with docking score −7.09 kcal/mol.
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Finally, TXER has DNA protection activity against hydroxy radical induced DNA damage. The present experimental observations contribute to understanding the interaction mechanism of TXER with DNA and may be very worthful for the researchers in emerging drug development. Acknowledgements The authors duly acknowledge the funding support from Indian Council of Medical Research (ICMR Ref: 52/13/2007) and Department of Science and Technology (NO.SR/FT/LS-63/2011 & DST-FIST), New Delhi, India. The first author also acknowledges the UGC, India for the fellowship in the form of BSR-fellow. The authors also thank Central Instrumentation Facility in Pondicherry University. In addition to authors thank DBT-IPLS in Pondicherry University for providing the ITC instrument facility. References [1] S. Rauf, J.J. Gooding, K. Akhtar, M.A. Ghauri, M. Rahman, M.A. Anwar, A.M. Khalid, J. Pharma. Biomed. Anal. 37 (2005) 205–217. [2] J. Li, S. Shuang, C. Dong, Talanta 77 (2009) 1043–1049. [3] L.H. Hurley, Nat. Rev. Cancer 2 (2002) 188–200. [4] Y.J. Liu, X.Y. Wei, F.H. Wu, W.J. Mei, L.X. He, Spectrochim. Acta A: Mol. Biomol. Spectrosc. 70 (2008) 171–176. [5] S.U. Rehman, Z. Yaseen, A.R. Husain, T. Sarwar, H.M. Ishqi, M. Tabish, PLOS ONE 9 (2014) e93913. [6] D. Gibson, Pharmacogenomics J. 2 (2002) 275–276. [7] O. Kennard, Pure Appl. Chem. 65 (1993) 1213–1222. [8] M. Sirajuddin, S. Ali, A. Badshah, J. Photochem. Photobiol. B 124 (2013) 1–19. [9] I.E. Blasig, H. Loewe, B. Elbert, Biomed. Biochim. Acta 47 (1987) S539–S544. [10] I.E. Blasig, H. Loewe, B. Elbert, Biomed. Biochim. Acta 47 (1988) S252–S255. [11] C. Wenisch, P.M. Biffignandi, Curr. Med. Res. Opin. 17 (2001) 123–127. [12] M. Kessler, G. Ubeaud, T. Walter, F. Sturm, L. Jung, J. Dermatolog. Treat 13 (2002) 133–141. [13] S.H. Fan, Z.F. Zhang, Y.L. Zheng, J. Lu, D.M. Wu, Q. Shan, B. Hu, Y.Y. Wang, Int. Immunopharmacol. 9 (2009) 91–96. [14] M. Biosseau, G. Freyburger, C. Beylot, M. Busquet, Arteres Veines 5 (1986) 231–234. [15] K.S. Lee, H.J. Cha, G.T. Lee, K.K. Lee, J.T. Hong, K.J. Ahn, I.S. An, S. An, S. Bae, Int. J. Mol. Med. 33 (2014) 934–942. [16] J. Lu, D.M. Wu, Z.H. Zheng, Y.L. Zheng, B. Hu, Z.F. Zhang, Brain 134 (2011) 783–797. [17] R. Vinothkumar, M. Sudha, P. Viswanathan, T. Balasubramanian, N. Nalini, Exp. Mol. Pathol. 96 (2014) 15–26. [18] J.A. Glasel, Biotechniques 18 (1995) 62–63. [19] C.D. Kanakis, P.A. Tarantilis, C. Pappas, J. Bariyanga, H.A. Tajmir-Riahi, M.G. Polissiou, J. Photochem. Photobiol. B 95 (2009) 204–212. [20] M. Purcell, J.F. Neault, H.A. Tajmir-Riahi, Biochim. Biophys. Acta 1478 (2000) 61–68. [21] Y. Song, J. Kang, J. Zhou, Z. Wang, X. Lu, L. Wang, J. Gao, Spectrochim. Acta A: Mol. Biomol. Spectrosc. 56 (2000) 2491–2497. [22] J. Janockova, Z. Gulasova, J. Plsikova, K. Musilek, K. Kuca, J. Mikes, L. Culka, P. Fedorocko, M. Kozurkova, Int. J. Biol. Macromol. 64 (2014) 53–62.
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Spectroscopic and molecular docking studies on the interaction of troxerutin with DNA A. Subastri a , C.H. Ramamurthy a , A. Suyavaran a , R. Mareeswaran a , P. Lokeswara Rao a , M. Harikrishna b , M. Suresh Kumar b , V. Sujatha c , C. Thirunavukkarasu a,d,∗ a
Department of Biochemistry and Molecular Biology, Pondicherry University, Puducherry 605 014, India Centre for Bioinformatics, Pondicherry University, Puducherry 605 014, India c Department of Chemistry, Periyar University, Salem 636 011, India d Department of Medicine – Gastroenterology and Liver diseases, 625, Ullmann Building, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10469, USA b
a r t i c l e
i n f o
Article history: Received 26 September 2014 Received in revised form 18 March 2015 Accepted 19 March 2015 Available online 6 April 2015 Keywords: Spectroscopy Molecular docking DNA Troxerutin
a b s t r a c t Troxerutin (TXER) is a derivative of naturally occurring bioflavonoid rutin. It possesses different biological activities in rising clinical world. The biological activity possessed by most of the drugs mainly targets on macromolecules. Hence, in the current study we have examined the interaction mechanism of TXER with calf thymus DNA (CT-DNA) by using various spectroscopic methods, isothermal titration calorimetry (ITC) and molecular docking studies. Further, DNA cleavage study was carried out to find the DNA protection activity of TXER. UV-absorption and emission spectroscopy showed low binding constant values via groove binding. Circular dichroism study indicates that TXER does not modify native B-form of DNA, and it retains the native B-conformation. Furthermore, no effective positive potential peak shift was observed in TXER–DNA complex during electrochemical analysis by which it represents an interaction of TXER with DNA through groove binding. Molecular docking study showed thymine guanine based interaction with docking score −7.09 kcal/mol. This result was compared to experimental ITC value. The DNA cleavage study illustrates that TXER does not cause any DNA damage as well as TXER showed DNA protection against hydroxyl radical induced DNA damage. From this study, we conclude that TXER interacts with DNA by fashion of groove binding. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Interaction of small ligand molecule and DNA is becoming progressive part in current research field [1,2]. DNA is a great biologically significant macromolecule and plays an essential role in cellular progression including DNA replication, transcription and translation. These processes are very crucial for cell survival and cell proliferation. Therefore, cellular DNA is the most important pharmacological target of several drugs including anti-tumour, anti-viral and anti-bacterial drugs [3–5] that are presently in clinical usage or in advanced pharmaceutical trials. Therefore in target of DNA, the number of biological activity of small ligand molecules binds with DNA and causing inhibition of cellular progression by
∗ Corresponding author at: Department of Biochemistry and Molecular Biology, Pondicherry University, Puducherry 605 014, India. Tel.: +91 413 2654972/+11 347 471 6477; fax: +91 413 2655255. E-mail addresses: [email protected], [email protected] (C. Thirunavukkarasu). http://dx.doi.org/10.1016/j.ijbiomac.2015.03.036 0141-8130/© 2015 Elsevier B.V. All rights reserved.
modulating gene expression in DNA [6]. These synthetic or natural small compounds could act as drugs in conditions where inhibition or modification of cellular DNA function is needed to alleviate or control a disease [7]. Moreover, depending on the mode of interaction, compounds can be classified as covalent binding and non-covalent binding drugs. In covalent binding types, drugs are bounded with DNA through alkylation or inter and intra strand cross linking by complete inhibition of DNA progression. But the non-covalent binding, effect is very weak compared to covalent binding. Furthermore, the non-covalent binding is also classified into three types namely, intercalation, groove binding and external binding [8]. Hence, these DNA–drug interaction studies are very valuable and need of the hour for designing novel and more effectual drugs with fewer side effects. On the other hand, mechanism of interaction between drug and DNA is still comparatively meagerly known. From this conception, in the current study we have focused on bioflavonoid rutin derivative troxerutin (TXER) for DNA interaction. TXER is a bioflavonoid derivative of rutin (Fig. 1) found in many plants, vegetables and fruits, and can be largely extracted
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of TXER solution was added to the final concentration of CT-DNA (100 M) and mixed thoroughly by vortex. For the UV–visible and fluorescent spectroscopy studies, 100 M concentration of CT-DNA solution was used along with varying concentrations (10–100 M) of TXER. Circular dichroism spectroscopy study was carried out using with two different concentration of TXER (0.05 mM and 0.1 mM) with constant CT-DNA concentration of 100 M. 2.3. UV–visible spectroscopy measurements
Fig. 1. Chemical structure of troxerutin.
from Sophora japonica. It has been proclaimed to possess various significant pharmacological activities including antioxidant [9–12], anti-inflammatory activity [13], anti-erythrocytic, antithrombiotic and fibrinolytic activity [14]. In addition recent study of TXER reported that protect the cells against UVB radiation [15]. Lu et al. [16] revealed that TXER has protective effect against cognitive impairment. Furthermore, TXER was shown as a chemopreventive agent against colon carcinogenesis [17]. So, previous reports have clearly showed that TXER is an emerging small molecule for numerous diseases in present clinical scenario. However, there are no studies on interaction of TXER with cellular DNA. Hence, the current study has been determined to investigate the binding mode of TXER with DNA. The basic understanding of binding mode and affinity of small molecule with DNA is crucial to analyze the investigational results procured in toxicology and to design the restorative formulation. In the current study, the interaction of TXER with calf thymus DNA (CT-DNA) has been explored in vitro by using various spectroscopic techniques, isothermal titration calorimetry (ITC), molecular docking and DNA protection studies. The elucidation on the binding mechanism of compound TXER with CT-DNA could help in bringing the awareness of the biological significance of this small molecule and its toxicity. 2. Materials and methods 2.1. Materials CT-DNA sodium salt and TXER were purchased from Sigma–Aldrich chemicals (Sigma–Aldrich, USA). The ratio of the absorption at 260/280 nm was measured to confirm the purity of DNA sample. The ratio was found to be 1.8 which revealed that purity of DNA without any protein contamination [18]. The DNA solution, TXER solution and buffer were freshly prepared from deionized ultra pure water. Other chemicals and reagents used in this study were of analytical grade.
The absorption spectra of CT-DNA alone and TXER-DNA complex were recorded using Shimadzu UV–visible spectrophotometer, (Model – UV-1800). Quartz cuvette with a path length of 1 cm was used for the spectroscopy measurements. The binding constant of CT-DNA–TXER complex was calculated by the method of Kanakis et al. [19]. It was assumed that only one type of interaction occurred between drug and DNA in aqueous solution resulting in the formation of one type of complex. Based on this assumption, Eqs. (1) and (2) were established. DNA + Drug → DNA : Drug
(1)
The equilibrium constant is given by K=
DNA : TXER (DNA)(TXER)
(2)
Eq. (2) can be written as K=
CDT [CD ][CT ]
(3)
where, CDT , CD and CT are the analytical concentration of TXER–DNA complex, DNA alone, and TXER alone in 10 mM Tris–HCl buffer solution (pH 7.4, 25 ◦ C) respectively. Beer–Lambert law for the absorption of light is assumed to be followed by the ligand substrate binding. CD = CD0 − CDT CDT =
(4)
A − A0 εDT · L
(5)
A0 εD · L
(6)
and CD0 =
where, CD0 is the concentration of pure DNA, A0 and A are the absorbance of pure DNA and TXER–DNA complex respectively at 260 nm. εD and εDT are the molar extinction coefficient of DNA alone and TXER–DNA complex respectively. L is the path length of cuvette (1 cm). By putting the values of CD and CDT from above equations into the Eq. (3), following equation can be deduced: A0 εD εD 1 = + × A − A0 εDT CT (εDT · K)
(7)
The double reciprocal plot of 1/(A − A0 ) versus 1/CT (CT is the concentration of TXER) is linear and thus the binding constant (K) can be estimated by calculating the ratio of the intercept to the slope [20].
2.2. Stock solution preparation
2.4. Fluorescence measurements
CT-DNA stock solution was prepared by dissolving CT-DNA (10 mg/ml) in 10 mM Tris–HCl buffer (pH 7.4) at 4 ◦ C for 24 h with intermittent stirring to ensure the formation of the homogeneous solution. The final concentration of the CT-DNA stock solution was estimated spectrophotometrically at 260 nm using molar extinction coefficient ε260 = 6600 cm−1 M−1 . The TXER stock solution (1 mM) was prepared in ultrapure water. Different concentrations
Fluorescence emission study was carried out using FLUOROLOG – FL3-11 spectrofluorometer equipped with a xenon lamp source. All the readings were obtained by using a 10 mm path length cuvette in a 10 mM Tris–HCl buffer at pH 7.4. In this emission spectral study, different concentration (10–100 M) of TXER was mixed with constant 100 M concentration of CT-DNA in the presence of intercalating DNA dye such as ethidium bromide (2 M EtBr).
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The fluorescence intensity of pure CT-DNA has very low emission spectrum. The fluorescence dye, EtBr is used to examine interaction mechanism of drug–DNA complex [5]. EtBr is an eminent conjugate probe to bind with DNA in intercalation mode [21] and maximum excitation of EtBr is 510 nm [22]. In EtBr competitive assay, the emission spectra for different concentration of TXER with CT-DNA complexes were recorded at wavelength of 510–800 nm with excitation at 520 nm. All spectral readings were carried out at 25 ◦ C and all fluorescence intensities of the sample were expressed in arbitrary units. Finally quenching constant (KSV ) values of TXER–DNA complex can be determined by using following Stern–Volmer equation [23]: F0 = 1 + KSV [Q ] F where, F0 and F are the fluorescence intensities in the absence and presence of the quencher (TXER) in EtBr bound DNA respectively and Q is the concentration of the quencher (TXER), KSV is the Stern–Volmer quenching constant. Hence, KSV value can be calculated from the slope of the linear relationship between [F0 /F] and [Q]. 2.5. Circular dichroism spectral measurements Circular dichroism (CD) spectra of pure DNA and its TREX complexes were recorded with JASCO J-815 CD spectrophotometer [24]. All spectra were measured in far-UV range (200–320 nm). Quartz cuvette of 1 mm path length was used for sampling. Three scans with a scan speed of 50 nm/min were performed and averaged. All the measurements were done at 25 ◦ C. Base correction of spectrum was performed with Tris–HCl buffer at pH 7.4 to obtain the spectrum of CT-DNA alone and TXER–DNA complex. 2.6. Cyclic voltammetric study The cyclic voltammetric (CV) study of DNA was carried out by using autolab type (PGSTAT 302N) electrochemical analyzer [25] through three electrode systems composed of glassy carbon electrode as a working electrode, Ag/AgCl served as a reference electrode and Pt wire served as a counter electrode with scan rate of 100 mV/s at room temperature. Electrochemical analysis of all samples was performed by using 50 ml voltammetric cell. In addition, different concentration of TXER (10–100 M) and constant concentration of DNA (100 M) were used and 10 mM Tris–HCl buffer (pH 7.4) exploited as supporting electrolyte. 2.7. Isothermal titration calorimetry In order to confirm the binding interactions between TXER and DNA, we performed the isothermal titration calorimetry (ITC) experiment using NANO ITC. All the samples for ITC experiment were prepared with degassed buffer (10 mM Tris–HCl buffer pH 7.4) to avoid gas bubbles which may possible to disturb the baseline. All ITC experiment was maintained at 25 ◦ C with stirring speed of 150 rpm. The assay was startup after baseline correction so that the heat generated by stirring can be automatically subtracted. The 1 mM concentration of TXER and 0.5 mM of CT-DNA was used into the sample cell through 25 individual injections programmed at 5 min intervals. The titration curves were corrected for heat of dilution determined by injecting the ligand into the buffer and CT-DNA into the buffer solution. The region under each peak was integrated heats of dilution was subtracted and the thermo-gram for the binding of TXER to DNA has been obtained (Fig. 8a and b). The obtained results were further fitted using NANO ITC software and molar change in enthalpy (H in kJ/mol), binding constant Ka (M−1 ) and binding stoichiometry (n) were obtained to conclude the result. The
binding free energy of the experiment was calculated by using the following formula: G = H − TS = −RT ln Ka where, the G, H, and S stand for the changes in free energy, enthalpy and entropy of binding; T, temperature and R (gas constant) = 1.98 cal/mol K. 2.8. Molecular docking studies Docking study for interaction between DNA and TXER was clarified by using Glide software [26], which is part of the Maestro software suite. The PDB structure of CT-DNA sequence dodecamer d(CGCGAATTCGCG)2 (PDB ID: 1BNA) was retrieved from Protein Data Bank was refined using the protein preparation wizard by removing the hetero atoms and using the Imperf minimization tool to converge the heavy atoms. Likewise ligand such as TXER structure was retrieved from the PubChem database. The ligand structure was prepared for docking using LigPrep application in Maestro. Then the ligand was energy minimized using OPLS-2005 force field implemented in Maestro. After preparation of receptor and ligand files, docking tool was applied to generate an interaction between TXER and CT-DNA and finally, binding energy as well as binding pose of the complex was obtained. 2.9. DNA cleavage study The cleavage of DNA was observed through agarose gel electrophoresis by modified method of Tabassum et al. [27]. In this reaction super-coiled pBR322 circular plasmid DNA (250 ng) was treated with various concentrations of TXER (25, 50 and 100 M) in presence and absence of 5 l Fenton’s reagent (30 mM H2 O2 , 50 mM ascorbic acid, 80 mM FeCl3 ) and made upto a final volume of 25 l with Tris–HCl buffer then samples were incubated at 37 ◦ C for 15 min. After incubation gel loading buffer (6×) was added and mixtures were electrophoresed for 1 h at 80 V on 2% agarose gel in TAE buffer. 3. Results and discussion 3.1. UV–visible spectroscopic analysis UV-absorption spectroscopy is an effectual technique for identifying the binding potency and the mode of interaction of small molecule with DNA. The absorption spectra of CT-DNA alone and TXER–DNA complex with various concentration of TXER is depicted in Fig. 2. The maximum absorption peak of TXER–DNA complexes were 255 nm and 350 nm respectively. Moreover, hyperchromic effect was observed without any noticeable red shift (bathochromic shift) at position of maximum absorption of TXER–DNA complexes. So absence of red shift in the UV spectra analysis indicates that the binding mode is not the intercalative binding [24,28]. From the UV spectrum results of TXER–DNA complex suggest that TXER may interact with DNA through groove binding mode. Furthermore, the effect of hyperchromism may be due to unwinding of DNA helix [29] or electrostatic binding [30]. Then stability of bound TXER–DNA complex was determined through calculating binding constant by using the formula described in materials and methods section (Section 2.3). The double reciprocal plot of TXER–DNA complex’s shows as a linear correlation between 1/(A − A0 ) and 1/C (Fig. 3). From the graph, the binding constant value (K) of TXER–DNA complex is 1.75 × 103 M−1 . This calculated binding constant value shows weak binding of TXER with DNA when compared to results observed in strong binding compounds like EtBr, acridine orange interaction study with DNA [31]. And also this
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Fig. 2. UV absorption spectra of CT-DNA alone and TXER–DNA complex with various concentrations of TXER (0.01–0.1 mM). The arrow represents increasing absorbance of TXER–DNA complexes: (a) CT-DNA alone (100 M CT-DNA in 10 mM Tris–HCl buffer, pH 7.4 at 25 ◦ C); (b) CT-DNA + 0.01 mM TXER; (c) CT-DNA + 0.02 mM TXER; (d) CT-DNA + 0.04 mM TXER; (e) CT-DNA + 0.06 mM TXER; (f) CT-DNA + 0.08 mM TXER; (g) CT-DNA + 0.1 mM TXER.
binding constant value closely agrees with the previous reports of some other weak DNA binders [32]. 3.2. Emission spectroscopic study Fluorescent spectroscopy is one of the trendy techniques and is a highly sensitive method to study the interaction between macromolecules and small molecules. So, the binding mechanism of TREX with DNA was determined by fluorescence emission spectroscopy. At this point competitive binding study was carried out to investigate the binding mode of TXER with DNA. For this study, EtBr was used, since it is a prevalent fluorophore that binds to DNA. The endogenous fluorescence property of DNA is very low. But the fluorescence of DNA was increased in the presence of EtBr due to substantial intercalation between the base pairs of CT-DNA. The
Fig. 3. Double reciprocal plot of TXER–DNA interaction by using UV–visible spectroscopy. A0 is the initial absorption of CT-DNA (100 M in 10 mM Tris–HCl buffer, pH 7.4) and A is the absorption at different concentration (0.01–0.1 mM) of TXER–DNA complex and C is the analytical concentration (0.01–0.1 mM) of TXER in 100 M CT-DNA (100 M CT-DNA in 10 mM Tris–HCl buffer, pH 7.4 at 25 ◦ C).
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Fig. 4. Fluorescence emission spectra of EtBr–DNA complex alone and in the presence of different concentration of TXER (0.01–0.1 mM). The arrow indicates increasing concentration of TXER with EtBr–DNA complexes: (a) EtBr–DNA alone (100 M CT-DNA in 10 mM Tris–HCl buffer, pH 7.4 with 2 M EtBr at 25 ◦ C); (b) 0.01 mM TXER with EtBr–DNA; (c) 0.02 mM TXER with EtBr–DNA; (d) 0.04 mM TXER with EtBr–DNA; (e) 0.08 mM TXER with EtBr–DNA; (f) 0.1 mM TXER with EtBr–DNA. ex = 520.
emission spectrum of EtBr bound DNA–TXER complex is shown in Fig. 4. The different concentrations of TXER with EtBr bound CTDNA showed fluorescence emission peak at 600 nm upon excitation at 520 nm. The fluorescence intensity of EtBr bound DNA complexes slightly decreases without any major peak shift while adding different concentration of TXER, it showing the groove binding property of TXER with DNA [5]. The minor decrease in fluorescence intensity might be due to some aggregation between TXER and EtBr bound DNA complexes. Furthermore, fluorescence quenching constant was determined by using the Stern–Volmer equation, explained in materials and methods (Section 2.4). The value of Stern–Volmer dynamic quenching constant (Ksv ) was determined as 1.45 × 103 M−1 (Fig. 5) which was much lower than the strong DNA intercalating agents [33]. Therefore this interaction represents lesser potential of small molecule such as TXER for intercalative binding. Consequently, the present study suggests the interaction of ligand such as TXER with CT-DNA through groove binding mode.
Fig. 5. The double reciprocal plot between F0 /F and Q, where F0 is the fluorescene intensity of CT-DNA and F is the intensity of TXER–CT-DNA complexes and Q is the analytical concentration (0.01–0.1 mM) of TXER in CT-DNA (100 M CT-DNA in 10 mM Tris–HCl buffer, pH 7.4 at 25 ◦ C with 2 M EtBr).
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with DNA [43]. This confirmation was supported by UV–visible and fluorescence spectroscopy analysis. 3.5. Isothermal titration calorimetry
Fig. 6. Circular dichroism spectra of CT-DNA alone and TXER–DNA complexes: (a) CT-DNA alone (100 M CT-DNA in 10 mM Tris–HCl buffer, pH 7.4 at 25 ◦ C); (b) 0.05 mM TXER + CT-DNA; (c) 0.1 mM TXER + CT-DNA.
3.3. Circular dichroism spectroscopic study CD spectroscopy is a reliable technique for detecting conformational changes in bio macromolecules. The CD spectrum of CT-DNA alone has four major bands at position 210 nm, 220 nm, 245 nm, 275 nm and these bands are markers for B-conformation of DNA. The positive band occurs at 275 nm due to base stacking and negative band forms at 245 nm due to helicity of DNA [34,35]. During the transition of DNA alone from B-form to A-form, the 210 nm peak intensity will decrease and 275 nm peak intensity will increase with band shifting [36]. Further Vorlickova [37] reported that Aconformation of DNA shows a strong positive CD band at 263 nm and negative band at 212 nm. In our present study, two different concentrations [Molar ratio (R) = TXER/DNA = 0.05 mM/0.1 mM, 0.1 mM/0.1 mM] of TXER–DNA complex were subjected to CD spectroscopy analysis. Outcomes for CD spectra of CT-DNA alone and TXER–DNA complex were shown in Fig. 6. The figure shows four peaks, at position 210 nm (negative band), 220 nm (positive band), 245 nm (negative band) and 275 nm (positive band). From this observation, CD spectra of TXER–DNA complexes did not show any perceptible changes in B-conformation of DNA. However, modest variation observed at 274 nm, 210 nm and 245 nm in shift and intensity of TXER–DNA complexes due to aggregation effect of TREX on DNA [38,39]. Conclusively, CD spectrum results suggest that DNA retains its B-conformation after interaction with TXER. 3.4. Electrochemical study Electrochemical method of CV is a powerful technique for detecting the binding mode of TXER-DNA complex. And also, this technique measures the current that is generated in an electrochemical cell under appropriate conditions where voltage is in excess of that calculated by the Nernst equation. Furthermore, CV was accomplished by potential of functioning electrode and measuring the resulting current [40]. The deviation in peak potential and current can be subjected for the prediction of binding specifications. Fig. 7 shows CV measurements of CT-DNA in presence and absence of TXER at different concentration. From this observation we did not find any considerable positive peak shifts in TXER–DNA complexes compared to intercalating binders [41,42]. Additionally, interaction of TXER with CT-DNA shows moderate peak current. This may be due to interaction of TXER with DNA by groove binding mode. This finding agrees with other small molecule interaction
ITC is a valuable technique for an absolute understanding of drug–DNA interactions. Certainly ITC is the only method that directly determines the binding enthalpy changes in the formation of a complex allow the free energy change to be divided into the enthalpic and entropic involvement to the association process. It discloses the nature of some forces that drive the binding reaction [44]. Thermodynamic analysis was carried out for TREX–DNA complex and titration curve has been depicted in Fig. 8(a and b) which shows the ITC titrations results where each heat curve represents to a single TXER injection to DNA. The area under these curves is determined by integration to provide the associated injection heats. The experimental data exposed that enthalpy change H is 81.99 kJ/mol, entropy change S is 330.1 J/mol/K and Gibbs free energy G is −16.7 kJ/mol or −4.0 kcal/mol and binding stoichiometry n = 1.102. The negative values of G illustrated that TXER–DNA complex is a spontaneous process. Therefore, the negative value of the free energy represents more favorable interaction of TXER–DNA [45]. The enthalpy (H) and entropy (S) were both positive, it may be due to the non-covalent interactions such as hydrogen bonds, van der Waals force and hydrophobic interactions involving in TXER–DNA complex [46]. From ITC data, the interaction of TXER–DNA represents entropy driven endothermic binding with positive enthalpy and entropy change. This result suggests interaction of TXER with DNA through groove binding mode [47,48]. Moreover a favorable enthalpy change is observed through ITC for TXER binding to DNA may be due to groove binding mode. This result agrees with Pagano et al. [49] reported that groove binder shows favorable enthalpy changes (H) which may be due to the result of electrostatic interaction and hydrogen bond formation in the complexes. Furthermore, the value of the binding constant in ITC was exposed as 7.5 × 102 M−1 which was much lower than other strong groove binders [50,51]. The strong groove binders have larger binding constant (1011 M−1 ) than intercalators [52]. Additionally Saha et al. [53] reported that tremendous groove binder Hoechst 33258 showed strong binding affinity on DNA. Therefore from these previous reports, the binding constant value of TXER–DNA complex is very low as compared to strong groove binders [54]. Hence the present ITC experiment suggests interaction of TXER with DNA through groove binding with weak binding affinity. 3.6. Molecular docking The discovery and evaluation of novel therapeutic drugs for the treatment of various emerging diseases without any side effects is the most vital goal in modern medical world. The results obtained from spectroscopy and electrochemical methods were supportive in understanding the mode of DNA–TREX complex. In addition to this molecular docking, which is a powerful in silico technique was performed for clarification of the interaction mechanism. The result of molecular docking analysis of TXER–DNA complexes was shown in Fig. 9. These computed parameters asserted the complex of TXER and DNA. The TXER interacts with the deoxy guanine 16 by forming an H bond between the O and N residues with a bond length of ˚ Similarly, the TXER also interacts with the deoxythiamine 3.24 A. residue 7 forming two H bonds between the O atoms of the phosphate group of the DNA and CO moiety of TXER with bond lengths 2.95 A˚ and 3.23 A˚ respectively. The TXER also shows hydrophobic interactions with the deoxy cytosine and deoxy thymine residues. Hence formation of hydrophobic and hydrogen bond recommends
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Fig. 7. Cyclic voltammogram of CT-DNA alone and TXER–DNA complex with various concentrations of TXER (0.01–0.1 mM). The arrow represents increasing concentration of TXER (0.0–0.1 mM) with constant 100 M CT-DNA: (a) CT-DNA alone (100 M CT-DNA in 10 mM Tris–HCl buffer, pH 7.4 at 25 ◦ C); (b) 0.01 mM TXER + CT-DNA; (c) 0.02 mM TXER + CT-DNA; (d) 0.04 mM TXER + CT-DNA; (e) 0.08 mM TXER + CT-DNA; (f) 0.1 mM TXER + CT-DNA.
interaction between TXER and DNA. Conclusively TXER interacts in the major groove of the DNA with a glide docking score of −7.097 kcal/mol. This docking free energy was compared to experimental ITC free energy value (G) which is −4.0 kcal/mol. The variation in the free energy values obtained from experimental (ITC) and computational (Molecular docking) results occurs due to many factors. In experiment of ITC, binding free energy contains
the flexibility of DNA with several other energy terms like translational energy, entropy, hydration free energy, conformational free energy, etc. But the molecular docking calculations absolutely considered DNA as a rigid molecule and small molecule (drug) as a flexible molecule. Additionally, the free energy from ITC also gains the effects from counter ion of buffer solution but molecular docking do not consider the counter ions in the interaction process [55].
Fig. 8. (a) ITC profile for the titration of CT-DNA (0.5 mM) with solution of TXER (1 mM) in 10 mM Tris–HCl buffer pH 7.4 at 25 ◦ C. (b) Isothermal plot of heat change verses molar ratio of TXER–DNA.
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Fig. 9. Molecular docking structural representation of TXER–DNA interaction by using Glide software [26]. (a) Molecular docked model of TXER showing groove binding with DNA dodecamer duplex of sequence d(CGCGAATTCGCG)2 (PDB id: 1BNA); (b) Surface view of molecular docked TXER on DNA dodecamer; (c) Docked model of TXER–DNA complex indicating intermolecular H-bonds.
3.7. DNA protection activity The DNA cleavage property of TXER was analyzed by gel electrophoresis using pBR322 plasmid DNA. When a circular plasmid DNA is subjected to electropheresis, the supercoiled form (Form I – shown in Fig. 10) usually migrate faster. If incision occurs on one strand (nicked circular) supercoil unwinds to create a slow moving open circular form (Form II – shown in Fig. 10). When both strands are cleaved, linear form (Form III – shown in Fig. 10) is generated that migrates between form I and form II [56]. The results of DNA cleavage study after incubation with various concentration of TXER in presence and absence of Fenton’s reagent is shown in Fig. 10. Various concentrations of TXER alone treated plasmid DNA (Lane 3–5) did not show any changes (Form III) in electophoretic band pattern of DNA but other condition of DNA i.e., pretreatment with Fenton’s reagent, depicted significant changes in DNA
Fig. 10. Agarose gel electrophoresis pattern of the pBR322 plasmid DNA cleavage by TXER in presence and absence of Fenton’s regent (30 mM H2 O2 , 50 mM ascorbic acid, 80 mM FeCl3 ). [Lane 1: DNA alone; Lane 2: Fenton’s reagent (5 l) + DNA; Lane 3: DNA + TXER (25 M); Lane 4: DNA + TXER (50 M); Lane 5: DNA + TXER (100 M); Lane 6: DNA + TXER (25 M) + Fenton’s reagent (5 l); Lane 7: DNA + TXER (50 M) + Fenton’s reagent (5 l); Lane 8: DNA + TXER (100 M) + Fenton’s reagent (5 l)].
migration pattern. The lane 2 (5 l Fenton’s reagent alone) expressed more intensity of band in linear form (Form I) due to oxidative DNA cleavage by Fenton’s reagent [57]. But on other hand higher concentration of TXER (Lane7: 50 M TXER + 5 l Fenton’s reagent, Lane 8: 100 M TXER + 5 l Fenton’s reagent) diminished the DNA damage caused by hydroxyl radicals (generated through Fenton’s reagent). But Lane 6 (25 M TXER + 5 l Fenton’s reagent) showed very less activity compared to high concentration. From this observation, we suggest that TXER strongly protects the DNA from oxidative damage as well as did not cause any DNA damage. 4. Conclusion In the current study we explored the interaction of small ligand molecule such as TXER with CT-DNA by using various spectroscopic and docking techniques. The results of binding studies suggest that the TXER binds with DNA through groove binding manner. This interaction existence is supported by following findings. The UV absorption spectroscopy showed weak binding of TXER with binding constant value 1.75 × 103 M−1 which suggests weak interaction compared to other strong binders. Emission study also is supportive of the interaction by showing less binding KSV value 1.45 × 103 M−1 . Furthermore, CD results demonstrate that the binding of TXER did not induce any significant conformational changes in B-form of DNA. Electrochemical CV study confirms groove binding of TXER which is visualized through absence of any noticeable positive shift in potential peaks of complex. The thermodynamic analysis of TXER–DNA showed that complex is driven by entropy through groove binding. Additionally, molecular docking study shows interactions of TXER in the groove of DNA via formation of hydrogen bond and hydrophobic bond with docking score −7.09 kcal/mol.
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