Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2014) xxx–xxx
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Binding interaction between sorafenib and calf thymus DNA: Spectroscopic methodology, viscosity measurement and molecular docking Jie-Hua Shi a,b,⇑, Jun Chen a, Jing Wang a, Ying-Yao Zhu a a b
College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032, China State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, Zhejiang University of Technology, Hangzhou 310032, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Sorafenib binds to DNA via minor
It was confirmed that sorafenib interacts with ct-DNA via minor groove binding mode through spectroscopic methods (such as UV–vis absorption spectroscopy, and fluorescence emission spectroscopy) and molecular doching. CF3
O
Cl
O
O
H
Molecular docking H
H Sorafenib 0.30 1 ( Csorafenib =0)
0.25 0.20
Absorbance
groove binding and forms 1:1 complex with it. The main interaction forces were van der Waals and hydrogen bonding interactions. There was slight change of the secondary structure of DNA due to binding sorafenib. The flexibility of sorafenib plays an important role in increasing the sorafenib–DNA stability.
7 (C sorafenib = 5.52×10
-5
M)
0.15 0.10 0.05 0.00 250
300
350
400
wavelength (nm)
UV-vis absorption spectroscopy DNA Note: all solutions conclude fixed concentration of DNA and Hoechst 33258
Fluorescence intensity (a.u.)
350 300
1 ( C sorafenib =0)
250 200
-5
6 (Csorafenib=3×10 M)
150
λ ex = 365 nm
100 50 0 400
450
500
550
600
650
Wavelength (nm)
Sorafenib - DNA complex
a r t i c l e
i n f o
Article history: Received 18 June 2014 Received in revised form 27 August 2014 Accepted 18 September 2014 Available online xxxx Keywords: Sorafenib Calf thymus-DNA UV–vis spectroscopy Circular dichroism Fluorescence spectroscopy Molecular docking
Fluorescence emission spectroscopy
a b s t r a c t The binding interaction of sorafenib with calf thymus DNA (ct-DNA) was studied using UV–vis absorption spectroscopy, fluorescence emission spectroscopy, circular dichroism (CD), viscosity measurement and molecular docking methods. The experimental results revealed that there was obvious binding interaction between sorafenib and ct-DNA. The binding constant (Kb) of sorafenib with ct-DNA was 5.6 103 M–1 at 298 K. The enthalpy and entropy changes (DH0 and DS0) in the binding process of sorafenib with ct-DNA were –27.66 KJ mol–1 and –21.02 J mol–1 K–1, respectively, indicating that the main binding interaction forces were van der Waals force and hydrogen bonding. The docking results suggested that sorafenib preferred to bind on the minor groove of A-T rich DNA and the binding site of sorafenib was 4 base pairs long. The conformation change of sorafenib in the sorafenib–DNA complex was obviously observed and the change was close relation with the structure of DNA, implying that the flexibility of sorafenib molecule played an important role in the formation of the stable sorafenib–ct-DNA complex. Ó 2014 Elsevier B.V. All rights reserved.
Introduction ⇑ Corresponding author at: College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032, China. Tel./fax: +86 571 8832 0064. E-mail address: [email protected] (J.-H. Shi).
Deoxyribonucleic acid (DNA) plays key physiological roles in the life process because it carries important genetic information
http://dx.doi.org/10.1016/j.saa.2014.09.056 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
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and guides the biological synthesis of proteins and enzymes through the process of duplication, transportation and translation of genetic information. Small molecules can bind to DNA via covalent or non-covalent interactions, resulting in alteration or inhibition of DNA function [1,2]. Therefore, the investigation on the binding interaction of DNA with small molecules is helpful to understand the structural features of DNA, origin of some diseases and action mechanism of some drugs, and design improved drugs that target cellular DNA. The researches on the binding interaction between DNA and small molecules have been considered as one of the key topics in the field of life sciences, chemistry and medicine. Currently, the methods used to investigate the binding interaction of DNA with small molecules mainly contain the experimental methods [3–7] and the molecular docking methods [8–14]. Sorafenib (Fig. 1), which is an inhibitor of several tyrosine protein kinases and Raf kinases, is used for the treatment of primary kidney cancer and advanced primary liver cancer [15–19]. It has also been demonstrated that sorafenib has the preclinical and clinical activity against several tumor types such as renal cell carcinoma, and non small cell lung cancer [18,20–23]. In addition, Tod et al studied that the binding interaction between sorafenib and plasma using the quenching fluorescence method and the influence of albuminemia and bilirubinemia on sorafenib disposition in cancer patients, suggesting that sorafenib is highly bound to plasma (>99.5%) and the major influence of albuminemia on sorafenib clearance [24]. However, to our best knowledge, the study on the intermolecular interactions of sorafenib with DNA using UV–vis absorption spectroscopy, fluorescence emission spectroscopy, circular dichroism (CD) and molecular modeling methods has not been reported. In this work, the binding interaction between sorafenib and calf thymus DNA (ct-DNA) was studied using UV–vis absorption spectroscopy, fluorescence emission spectroscopy, circular dichroism (CD), viscosity measurement and molecular docking in order to obtain the detailed information about the binding interaction of sorafenib with ct-DNA such as specific binding site, binding modes, binding constant, effect of sorafenib on conformation of ct-DNA, interaction forces, among others. The study of the binding interaction of ct-DNA with sorafenib molecule has great significance in helping to elucidate the mechanism of action and pharmacokinetics.
Tris–HCl buffer solution (pH = 7.40) consisted of Tris (0.050 M) and was adjusted to pH = 7.40 by 36% HCl solution. The stock solution of ct-DNA was prepared by dissolving of ct-DNA in 0.050 M of the Tris–HCl buffer (pH = 7.4). The stock solution of ct-DNA was stored at 4 °C in the dark for 5 days only and was stirred at frequent intervals to ensure the formation of a homogenous solution. The stock solution of ct-DNA gave a ratio of UV absorbance at 260 nm and 280 nm of above 1.8, indicating that DNA was sufficiently free from protein [3,25]. The final concentration of DNA in the stock solution was determined by UV absorption spectroscopy using the molar absorption coefficient of 6600 M–1 cm–1. Stock solution of sorafenib (6.28 10–5 mol L–1) was prepared in ethanol due to its very low solubility in water. We found that when the concentration of ethanol was lower than 12% the absorbances of ct-DNA solution and the mixture solution of sorafenib and ct-DNA were about the same, indicating that the concentration of ethanol lower than 12% did not affect on the conformational of DNA, which was consistent with the reference reported [26,27], and on the interaction of DNA with sorafenib. Therefore, the final concentration of ethanol in the test solution of sorafenib and ctDNA was controlled at lower than 10% to avoid the conformational change of ct-DNA and the effect on interaction of ct-DNA with sorafenib. UV–vis absorption spectral measurements The UV–vis spectra for all mixture solutions of ct-DNA and sorafenib were recorded from 230 to 400 nm on UV-1601 spectrophotometer with a 1.0 cm quartz cuvette (Shimadzu corporation, Kyoto, Japan) at different temperatures to quantify the binding constants of sorafenib to DNA and to evaluate the effect of temperature on binding interaction of DNA with sorafenib. The corresponding solution of ct-DNA was measured as reference solution. Fluorescence emission spectral measurements The fluorescence emission spectra of all mixture solutions of Hoechst 33258 and ct-DNA in the absence and presence of sorafenib were recorded on a F96S Spectrofluorimeter with 1.0 cm quartz cell (Shanghai LengGuang Industrial Co., Ltd., Shanghai, China) from 400 to 600 nm at kex = 365 nm at ambient temperature. These fluorescence emission spectra were measured as the average of three scans and the appropriate blanks corresponding to the buffer were subtracted to correct background.
Materials and methods Chemical and reagents
Circular dichroism measurements The highly polymerized ct-DNA and Hoechst 33258 were purchased from Sigma Chemical Co., Ltd. and were used without further purification. Sorafenib tosylate was provided from Nanjing Ange Pharmacetutical Co., Ltd. Tris(hydroxymethyl) aminomethane (Tris) (>99%) was purchased from Shanghai Bobo biotechnology Co., Ltd. Other chemicals were of analytical reagent grade and were used without further purification.
CF3 6
Cl
O 12 5
1 2
4 3
Circular dichroism spectra were recorded on JASCO J-815 Spectrophotometer with 1.0 cm quartz cell (Japan Spectroscopic Company, Tokyo, Japan) at ambient temperature, in which the scan range was from 200 to 350 nm with an interval of 1 nm at a scan rate of 100 nm min–1. Each spectrum was the average of three scans and was corrected by corresponding buffer blanks.
O 7 N H
8
11 9 N
21
O 13
16
14
17
10 15
20
22 N 23
N19
24
H
18
H Fig. 1. The structure of sorafenib.
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Viscosity measurements
Results and discussion
Viscosity measurements were performed using Ubbelohde viscometer which was thermostated at 298 K in a constant temperature bath and the inner diameter of capillary was 0.57 mm. The concentration of ct-DNA in Tris–HCl buffer solution (pH = 7.4) in the absence and presence of sorafenib was fixed at 1.38 10–5 M and the flow time was measured using a digital stop watch. The mean values of three replicated measurements were used to evaluate the relative specific viscosity (g/g0)1/3, where g0 and g are the specific viscosity contributions of DNA in the absence and presence of sorafenib, respectively.
UV–vis absorption spectroscopy
Molecular docking The starting geometry of sorafenib (NCBI, CID 216239) was obtained from the PubChem Compound Database (http://pubchem. ncbi.nlm.nih.gov/summary/summary.cgi?cid=216239&loc=ec_rcs). The structure of sorafenib was first treated by semi-empirical theory at PM3 level and then was optimized by density functional theory (DFT) at B3lyp/6-31+g(d,p) level using Gaussian 03 software until all eigenvalue of the Hessian matrix were positive [28]. The optimized structure of sorafenib was used for the molecular docking calculations. The ct-DNA is composed of two strands that wrap around each other to form a right-handed double helix with the B-form. The crystal structures of B-DNAs used in molecular docking were extracted from Protein Data Bank (http://www.rcsb.org/pdb/ home/home.do) and listed in Table 1. The binding interactions of sorafenib with B-DNA were simulated by molecular docking method using AutoDock 4.0 program [29]. All of the water molecules were removed from B-DNA. The polar hydrogen atoms were added to B-DNAs and the rotatable bonds of sorafenib were set to 6 using Autodock Tools [30,31]. The partial atomic charges of DNA and sorafenib were calculated using Gasteiger–Marsili [32] and Kollman methods [33], respectively. The grid maps of dimensions 60 60 60 Å with a gridpoint spacing of 0.375 Å were created for six B-DNAs to ensure an appropriate size for sorafenib-accessible space. In this work, the centers of grid boxes were set as shown in Table 1. The number of genetic algorithm runs and the number of evaluations were set to 100 and 2.5 million, respectively. Other miscellaneous parameters were assigned the default values given by Autodock program. Cluster analysis was performed on the results of docking by using a root mean square (RMS) tolerance 2.0 Å. Finally, we obtained the dominating configuration of the binding complex of sorafenib with NDA with minimum binding free energy (DG).
UV–vis absorption spectroscopy, one of the simplest testing techniques, has been widely used in the study of the interaction of DNA with small molecules by monitoring changes of UV–vis absorption bands of DNA or small molecules including the hypochromic effect, hyperchromic effect, bathochromic effect (red shift) and hypsochromic effect (blue shift). Generally, small molecule binding on DNA through intercalative interaction results in hypochromism and bathochromism of absorption bands. Because intercalative interaction is a kind of stacking interaction between base pair of DNA and chromophore of small molecules, the p orbital of base pairs of DNA can couple with the p* orbital of small molecules and form p–p* conjugation, resulting in reducing the difference of the p–p* transition energy, which causes a red shift of absorption band, and the coupling p* orbital is partially filled by electron resulting in decreasing the transition probability. The lower the probability is, the smaller the molar absorption coefficient. So, when small molecule intercalated into DNA, the distance between the chromophore of small molecule and the base pair of DNA will decrease, resulting in hypochromism of absorption band [2,34]. In the case of electrostatic attraction between the DNA and small molecules, hyperchromic effect can be observed, which reflects the corresponding changes of the conformation and structure of DNA when the electrostatic interaction of DNA with small molecules has occurred [2]. However, in the case of the groove binding mode between DNA and small molecules, the hypochromic effect can be observed while the position of the absorption band almost does not change, which can be associated with the overlapping of the electronic states of the chromophore of the complex with the nitrogenous bases in the grooves of DNA [35–37]. The UV spectra of sorafenib in the absence and presence of ctDNA were shown in Fig. 2. It can be seen that there is an absorption band at 264 nm for the sorafenib solution in the absence of ct-DNA, which belongs to B-band of an aromatic moiety in sorafenib molecule. However, the intensity of the absorption band at 264 nm decreases with the gradient addition of ct-DNA to sorafenib solution, while the position of the absorption band almost does not change. Based on above viewpoints, it can be concluded that there is the binding interaction of ct-DNA with sorafenib and the main binding mode may be groove binding interaction. Binding constant and thermodynamic parameters measurements As is well known, the binding constant (Kb) for the 1:1 sorafenib–DNA complex can be calculated according to Benesi-Hildebrand equation [4]:
Table 1 Six DNA sequences used for molecular docking.a
a
DNA ID
PDB ID
Sequences
Unit cell constants
Centers of grid boxes
1
1D32
D(CGCG)2
a = 16.88 Å, b = 26.88 Å, c = 82.60 Å a = 90°, b = 90°, c = 90°
26.789, 12.727, 10.387
2
1ZNA
D(CGCG)2
a = 31.27 Å, b = 64.67 Å, c = 19.50 Å a = 90°, b = 90°, c = 90°
18.02, 8.307, 8.794
3
1K2J
D(CGTACG)2
No data
0.234, 2.024, 7.463
4
1BNA
D(CGCGAATTCGCG)2
a = 24.87 Å, b = 40.39 Å, c = 66.20 Å a = 90°, b = 90°, c = 90°
14.78, 20.976, 8.807
5
1DNE
D(CGCGATATCGCG)2
a = 25.48 Å, b = 41.26 Å, c = 66.88 Å a = 90°, b = 90°, c = 90°
15.296, 21.285, 75.966
6
102D
D(CGCAAATTTGCG)2
a = 24.78 Å, b = 41.16 Å, c = 65.51 Å a = 90°, b = 90°, c = 90°
14.558, 21.582, 75.506
These data were extracted from Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). 1ZNA: DNA without gap; 1D32: DNA with two gap.
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0.25
8.7
1 8.6
7
8.5
0.15
ln (Kb)
Absorbance
0.20
0.10
8.4
8.3
0.05
8.2
0.00 250
275
300
325
350
375
400 0.00324
Wavelength (nm)
0.00332
0.00336
-1
1/T (K )
Fig. 2. UV absorption spectra of sorafenib (6.28 10–6 M) in absence and presence of ct-DNA, the concentrations of ct-DNA from 1 to 7 were 0, 0.689 10–5, 1.38 10–5, 2.07 10–5, 2.76 10–5, 4.14 10–5, 5.52 10–5 M, respectively.
Fig. 4. Van’t Hoff plot for the sorafenib–DNA complex.
0.5
298 K 303 K 310 K
-10
0.4
-20 -30
Absorbance
A0/[A-A0]
0.00328
-40 -50
0.3
0.2
-60 0.1
-70 40000
80000
120000
160000 0.0
-1
1/CDNA (L mol )
0.0
0.1
0.2
0.3
0.4
CNaCl (mol/L)
Fig. 3. Plots of A0/(A A0) versus 1/CDNA for sorafenib–DNA complex in Tris–HCl buffer solution (pH = 7.4) at different temperatures.
Fig. 5. Effect of ionic strength on the absorbance of sorafenib–DNA complex. Concentrations of DNA and sorafenib were 1.38 10–5 M and 6.28 10–6 M, respectively.
A0 eD eD 1 ¼ þ A A0 eDDNA eD eDDNA eD K b C DNA
ð1Þ
where A0 is the absorption of sorafenib at 264 nm in the absence of ct-DNA. A is the corresponding absorbance at different concentration of ct-DNA. eD and eD–DNA are the molar extinction coefficient of free sorafenib and sorafenib–ct-DNA complex, respectively. CDNA is the concentration of ct-DNA. It can be found from Fig. 3 that there is a good linear relationship between A0/(A A0) and 1/CDNA at different temperatures, indicating that the stoichiometry of binding interactions between DNA and sorafenib is 1:1. The apparent binding constants (Kb) at different temperatures are calculated from
intercept and slope obtained from Eq. (1) and listed in Table 2. The estimated values of Kb are in the order of 103 in the range from 298 to 310 K, which is significantly lower than that of the classic intercalation binding like EB-DNA complex (Kb = 1.4 106 M–1) [38]. However, it falls on the range of the groove binding constant of DNA with small molecule [7,39]. It is further indicated that the binding mode may be the groove interaction. In the binding process of biomacromolecule with small molecule, there are mainly four types of non-covalent interactions including hydrogen bonding interaction, van der Waals forces,
Table 2 The results for the apparent binding constants (Kb) of sorafenib–DNA complex and thermodynamic parameters. Temperature (K)
B–H equations obtained 4
298 303 310 a b c
Kb (L mol–1)
DG0exp,
5.58 103 4.75 103 3.63 103
–21.37 –21.33 –21.13
1
b
(kJ mol–1)
DG0exp,
c 2
(kJ mol–1)
DH0 (kJ mol–1)
DS0 (J mol–1 K–1)
–27.66
–21.02
a
Slope (10 )
Intercept
r
–3.10 ± 0.08 –3.56 ± 0.09 –4.54 ± 0.10
–1.73 ± 0.58 –1.69 ± 0.68 –1.65 ± 0.73
0.9983 0.9982 0.9987
–21.40 –21.29 –21.14
r Is the correlation coefficient. DG0exp, 1 = –RT ln Kb. DG0exp, 2 = DH0 TDS0.
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1.10
8 6
1.05
CD [medg]
1/3 0
(η/η )
1
4
1.00
2
2 0 200 -2
220
240
260
280
300
320
340
Wavelength (nm)
0.95 -4 -6 0.90 0.0
0.2
0.4
0.6
0.8
1.0
1.2
Fig. 8. The CD spectra of DNA (5 10–5 M) in the presence of sorafenib in Tris–HCl buffer solution. The concentrations of sorafenib from 1 to 2 were 0, 3 10–5 M, respectively.
r =[sorafenib]/[DNA] Fig. 6. Effect of increasing amounts of sorafenib on the viscosity of ct-DNA (1.38 10–5 M) in the Tris–HCl buffer solution.
hydrophobic interaction and electrostatic interaction [40]. Meanwhile, the signs and magnitudes of the thermodynamic parameters (DG0, DH0 and DS0) in the binding process of biomacromolecule with small molecule can be used to confirm the binding modes. These thermodynamic parameters can be calculated by the van’t Hoff equations [41]:
DH 0 DS0 þ RT R 0 DG ¼ RT ln K b ln K b ¼
ð2Þ ð3Þ
where R is the gas constant. DG0, DH0 and DS0 are the changes in Gibbs free energy, enthalpy and entropy in the binding process of biomacromolecule with small molecule, respectively. It is generally suggested that both DH0 and DS0 are positive, indicating that the main interaction force is hydrophobic interaction. DH0 and DS0 are negative, suggesting that the main interaction force is van der Waals force and/or hydrogen bonding interaction. DH0 is almost zero and DS0 is positive, implying that the main interaction force is electrostatic force [41]. For the effect of temperature on the DH0 and DS0 of the interaction of DNA with sorafenib is very small in the range of temperature studied, the values of DH0 and DS0 can be regarded as constant. The values of DH0 and DS0 for the
350
Fluorescence intensity (a.u.)
1 300 250
6
200 150 100
λ ex= 365 nm 50 0 400
450
500
550
600
650
Wavelength (nm) Fig. 7. Fluorescence emission spectra of the DNA–Hoechst 33258 in the presence of sorafenib in Tris–HCl buffer (pH = 7.4). The concentrations of ct-DNA and Hoechst 33258 were 1 10–5 M, 5 10–7 M, respectively. The concentrations of sorafenib from 1 to 6 were 0, 1 10–5, 1.5 10–5, 2 10–5, 2.5 10–5, 3 10–5 M, respectively.
interaction of ct-DNA with sorafenib are obtained from the linear relationship between ln Kb and the reciprocal absolute temperature as shown in Fig. 4 and the results are listed in Table 2. It can be found from Table 2 that the values of DG0, DH0 and DS0 are negative, suggesting that the binding interaction of sorafenib with ct-DNA is exothermic and spontaneous process. Moreover, the values of DH0 and DS0 are –27.66 kJ mol–1 and –21.02 J mol–1 K–1, respectively, indicating that the binding process is enthalpy-driven and the main interaction forces are hydrogen bonding interaction and van der Waals force. Binding mode Extensive research results revealed that small molecules usually bind to DNA in non-covalent way. Moreover, non-covalent interaction can be classified into three modes: (i) electrostatic binding; (ii) groove binding; and (iii) intercalative binding [1,2]. In order to further elucidate the binding mode of sorafenib with ct-DNA, the effect of ionic strength on UV absorption spectra of mixture solutions of sorafenib and ct-DNA, the effect of sorafenib on the viscosity of ct-DNA solution and the competitive binding of sorafenib with Hoechst 33258 on ct-DNA were further studied. The effects of ionic strength The electrostatic binding mode is one of non-covalent binding modes of small molecule on DNA, which is often served as an auxiliary mode to assist groove binding and intercalation. The small molecule that binds strongly to DNA usually includes the electrostatic component. But, if the electrostatic binding interaction plays a dominant role in the binding interaction of DNA with small molecules, the strength of interaction will decrease with the increase of salt concentration in system [1,25]. The experimental results showed that the absorbances of sorafenib–DNA solutions almost did not change with the increase of the concentration of NaCl (Fig. 5), indicating that there was no significant electrostatic binding interaction between ct-DNA and sorafenib. Viscosity studies Viscosity measurement is often regarded as an effective and accurate method to determine the binding mode between small molecules and DNA. It is generally suggested that a classical intercalative binding mode causes a significant increase of DNA viscosity because the intercalative interaction requires the space of adjacent base pairs to be large enough to accommodate the bound small molecules and elongates the double helix [42,43]. However, for the electrostatic or groove binding, there is little effect on the viscosity of DNA [42,44]. The viscosities of ct-DNA
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Table 3 The various energies and the hydrogen bonding interactions in the formation process of sorafenib–DNAs complexes. DNA PDB ID
DGa kJ mol–1
E1b kJ mol–1
E2c kJ mol–1
E3d kJ mol–1
Hydrogen bonding DNA
Sorafenibe
Bond length (Å)
1D32
–31.09
–39.00
–37.99
–1.00
DG6: H2 (Chain B)
O22
1.95
1ZNA
–26.93
–34.21
–32.99
–1.21
DC7: O3 (Chain B) DG4: H22 (Chain A)
H9 O22
2.23 1.80
1K2J
–41.34
–48.24
–46.90
–1.34
DC11: O4 (Chain B) DC11: O4 (Chain B) DA4: H3 (Chain A) DG6: H3 (Chain A) DG8: H22(Chain B)
H7 H9 O8 O22 N19
1.93 1.92 2.12 1.91 2.01
1BNA
–45.44
–51.09
–51.00
–0.04
DT8: O2 (Chain A) DT8: O2 (Chain A) DA6: H3 (Chain A)
H7 H9 O22
2.18 1.91 1.94
1DNE
–40.63
–48.08
–46.86
–1.21
DA17: H3 (Chain B) DT20: O4 (Chain B) DT20: O4 (Chain B)
O22 H7 H9
1.95 1.82 1.94
102D
–42.64
–50.17
–49.37
–0.75
DA6: H3 (Chain A) DT19: O4 (Chain B) DT19: O4 (Chain B) DT21: O4 (Chain B)
O22 H7 H9 H23
2.03 1.72 1.88 1.90
a
DG is the binding energy in the binding process, which is calculated in water solvent using a scoring function. E1 denotes intermolecular interaction energy, which is a sum of van der Waals energy, hydrogen bonding energy, desolvation free energy and electrostatic energy. c E2 is the sum of van der Waals energy, hydrogen bonding energy and desolvation free energy. d E3 is the electrostatic energy. e H7, H9 and H23 are hydrogen atoms linked with N7, N9 and N23 atoms, respectively, as shown in Fig. 1. O8 and O22 denote oxygen atoms linked with C8, C22, respectively, as shown in Fig. 1. b
Fig. 9. Molecular docking results of sorafenib bound to B-DNAs. Color codes of DNA: deoxy adenine (DA) is red, deoxy cytosine (DC) is green, deoxy guanine (DG) is yellow and deoxy thymine (DT) is blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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in the Tris–HCl buffer solution in the absence and presence of sorafenib were measured and the results were shown in Fig. 6. As shown in Fig. 6, the viscosities of ct-DNA in the Tris–HCl buffer solution (pH = 7.4) almost do not change with the increasing concentration of sorafenib, indicating that the binding mode of sorafenib with ct-DNA is not the intercalative binding mode. Therefore, the binding mode of ct-DNA with sorafenib may be the groove binding. Competitive binding of sorafenib with Hoechst 33258 To further affirm the binding mode of sorafenib on ct-DNA, a competitive binding experiment using Hoechst 33258 as probe molecule was performed. Hoechst 33258, which can strongly bind to AT-rich regions of DNA via minor groove binding, is a classical fluorescent probe [45]. In this work, the fluorescence emission spectra of the fixed amount of ct-DNA and Hoechst in Tris–HCl buffer solution (pH = 7.4) were measured with gradually increasing amounts of sorafenib and the results were shown in Fig. 7. It can be found that the fluorescence intensities of ct-DNA–Hoechst solutions decreased with the gradually increasing concentration of sorafenib. Meanwhile, it was not observed that sorafenib reacted with Hoechst 33258 resulting in the change of the fluorescence intensity of Hoechst 33258. Based on above experimental results, it can be concluded that there is competitive binding between Hoechst 33258 and sorafenib on ct-DNA. Therefore, sorafenib binds to AT-rich regions of ct-DNA via minor groove binding interaction. Circular dichroism studies As is well known, the circular dichroism (CD) spectroscopy is a powerful way in determining the secondary structure changes of DNA after binding with small molecules and has widely been used to investigate the interaction between small molecules and DNA [46–48]. The CD spectra of ct-DNA in Tris–HCl buffer solution in the absence and presence of sorafenib were shown in Fig. 8. It can be seen that there are four main characteristic peaks in the range from 200 to 350 nm for the free ct-DNA solution, which are two negative peaks at 211 and 244 nm and two positive peaks at 220 and 275 nm, respectively. These bands are consistent with CD spectrum of double helix DNA in the B conformation [47]. However, the negative peak at 244 nm belongs to helical geometry of BDNA while the positive peak at 275 nm is assigned to stacking of DNA bases. When sorafenib was added to the ct-DNA solution, the change of CD spectrum of ct-DNA was observed. The intensity of the positive band at near 275 nm decreased, while that of the negative band at near 244 nm increased. This revealed that the conformation of ct-DNA had slightly changed due to the binding interaction between sorafenib and ct-DNA. The increase of the negative peak intensity (244 nm) showed that the interaction of sorafenib with ct-DNA made the double helix structure of ct-DNA become tight [11]. The decrease of the intensity of the positive band (275 nm) was likely to be due to a transition from the extended nucleic acid right-handed double helix to more compact form (w structure) [49]. Molecular docking Molecular docking has gained growing interests in the investigation of binding interaction mechanism of biological macromolecule with small molecules. It plays a more and more important role in drug discovery and development [8–14]. In this work, the molecular docking of sorafenib with DNA was carried out using Autodock 4.0 in order to further clarify the binding mode of sorafenib on DNA and the binding structure of DNA-sorafenib complex. Sorafenib, kept as flexible molecule, was docked into the six types of rigid DNAs to search the preferential binding site of sorafenib on
Fig. 10. Conformations of free sorafenib (a) and the sorafenib bound with DNA (1BNA) (b) or DNA (1DNE) (c).
different DNAs and the docking results were listed in Table 3. Generally, the more negative the binding energy is, the stronger the interaction between small molecule and DNA, the most stable the complex formed by small molecule and DNA is. From Table 3, it can be found that the binding free energy (DG) is obviously lower when there are adenine (A) and thymine (T) base pairs in the DNA sequences, indicating that the preferential binding site of sorafenib on the A-T rich sequence of DNA. However, sorafenib prefers to bind on the minor groove of A-T rich DNAs, which is consistent with above experimental results, and the binding site is 4 base pairs long and involves A-T residues as shown in Fig. 9. And, significant change of conformation of sorafenib occurs in the binding process with DNA to orient easily along the minor groove. The conformation change of sorafenib is close relation with the structure of DNA minor groove (Fig. 10). In addition, it can be found from the docking results that there are hydrogen bonding interactions between sorafenib and DNAs (Table 3), suggesting that during the binding process of sorafenib with DNA, the narrower and deeper shape of the minor groove can offer several sites of action, resulting in the close contact with the surface of sorafenib through van der Waals forces and hydrogen bonding interaction. This further illustrates that the main forces of the interaction of sorafenib with DNAs are hydrogen bonds and van der Waals in binding process of sorafenib with DNA, which is consistent with the result of the thermodynamic parameter analysis, and the flexibility of sorafenib molecule plays an important role in the binding process of DNA with sorafenib. From Table 3, it can be also seen that the electrostatic energy is very much lower than the sum of van der Waals energy, hydrogen bonding energy and desolvation free energy in the binding process
Please cite this article in press as: J.-H. Shi et al., Binding interaction between sorafenib and calf thymus DNA: Spectroscopic methodology, viscosity measurement and molecular docking, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), http://dx.doi.org/10.1016/ j.saa.2014.09.056
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of sorafenib with DNAs, indicating that the main interaction mode between sorafenib and DNAs is not electrostatic binding mode, which is also consistent with the results of the effects of ionic strength on the UV–vis absorbance of sorafenib–ct-DNA solution. Conclusion In this work, UV–vis absorption spectroscopy, fluorescence emission spectroscopy, circular dichroism (CD), viscosity measurement and molecular docking were carried out to research the binding interaction between sorafenib and ct-DNA. It can be found that sorafenib interacts with ct-DNA via minor groove binding mode with the binding constant (Kb) of 5.4 103 at 298 K. In the binding process of sorafenib with ct-DNA, the main interaction forces were van-der Waals force and hydrogen bonding force. Additionally, the conformation change of sorafenib is obvious, indicating that the flexibility of sorafenib molecule plays an important role in the formation of the stable sorafenib–ct-DNA complex. The present study reveals the details of binding affinity, mode of binding interaction, main interaction forces of sorafenib with ct-DNA and structure of sorafenib–ct-DNA complex. Therefore, this study of the interaction mechanisms of sorafenib with ct-DNA would provide useful information in further understanding the mechanism of action and pharmacokinetics. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]
L. Strekowski, B. Wilson, Mutat. Res. 623 (2007) 3–13. M. Sirajuddin, S. Ali, A. Badshah, J. Photochem. Photobiol. B 124 (2013) 1–19. N. Shahabadi, N.H. Moghadam, Spectrochim. Acta A 99 (2012) 18–22. R. Hajian, T.G. Huat, J. Spectrosc. (2013). Article ID 380352. S. Agarwal, D.K. Jangir, R. Mehrotra, J. Photochem. Photobiol. B 120 (2012) 177–182. N. Shahabadi, N.H. Moghadam, J. Lumin. 134 (2013) 629–634. N. Shahabadi, S. Hadidi, Spectrochim. Acta A 96 (2012) 278–283. Y. Guo, Q. Yue, B. Gao, Int. J. Biol. Macromol. 49 (2011) 55–61. S. Charak, M. Shandilya, G. Tyagi, R. Mehrotra, Int. J. Biol. Macromol. 51 (2012) 406–411. R. Bera, B.K. Sahoo, K.S. Ghosh, S. Dasgupta, Int. J. Biol. Macromol. 42 (2008) 14–21. H. Li, X. Bu, J. Lu, C. Xu, X. Wang, X. Yang, Spectrochim. Acta A 107 (2013) 227– 234. F. Perveen, R. Qureshi, F.L. Ansari, S. Kalsoom, S. Ahmed, J. Mol. Struct. 1004 (2011) 67–73. P. Zhu, G. Zhang, Y. Ma, Y. Zhang, H. Miao, Y. Wu, Spectrochim. Acta A 112 (2013) 7–14. M.M. Islam, M. Chakraborty, P. Pandya, A.A. Masum, N. Gupta, S. Mukhopadhyay, Dyes Pigments 99 (2013) 412–422. G.M. Keating, A. Santoro, Drugs 69 (2009) 223–240. R.A. Smith, J. Dumas, L. Adnane, S.A. Wilhelm, Curr. Top. Med. Chem. 6 (2006) 1071–1089. K.S.M. Smalley, M. Xiao, J. Villanueva, T.K. Nguyen, K.T. Flaherty, R. Letrero, P. Van Belle, D.E. Elder, Y. Wang, K.L. Nathanson, M. Herlyn, Oncogene 28 (2009) 85–94.
[18] C.H. Takimoto, A. Awada, Cancer Chemother. Pharm. 61 (2008) 535–548. [19] S.M. Wilhelm, L. Adnane, P. Newell, A. Villanueva, J.M. Llovet, M. Lynch, Mol. Cancer Therapy 7 (2008) 3129–3140. [20] A. Dasari, L. Gore, W.A. Messersmith, S. Diab, A. Jimeno, C.D. Weekes, K.D. Lewis, H.A. Drabkin, T.W. Flaig, D.R. Camidge, Invest. New Drugs 31 (2013) 115–125. [21] M. Chaparro, L. GonzaLez-Moreno, M. Trapero-Marugan, J. Medina, R. MorenoOtero, Aliment. Pharm. Therapy 28 (2008) 1269–1277. [22] J. Tabernero, R. Garcia-Carbonero, J. Cassidy, A. Sobrero, E.V. Cutsem, C.-H. Kohno, S. Tajpar, O. Gladkov, I. Davidenko, R. Salazar, L. Vladimirova, S. Cheporov, O. Burdaeva, F. Rivera, L. Samuel, I. Bulavina, V. Potter, F. Rivera, L. Samuel, I. Bulavina, V. Potter, Y.-L. Chang, N.A. Lokker, P.J. O’Dwyer, Clin. Cancer Res. 19 (2013) 2541–2550. [23] T. Morisaki, M. Umebayashi, A. Kiyota, N. Koya, H. Tanaka, H. Onishi, M. Katano, Anticancer Res. 33 (2013) 1387–1395. [24] M. Tod, O. Mir, N. Bancelin, R. Coriat, A. Thomas-Schoemann, F. Taieb, P. Boudou-Rouquette, S. Ropert, J. Michels, H. Abbas, J.P. Durand, A. Dauphin, M. Vidal, F. Goldwasser, B. Blanchet, Pharm. Res. 28 (2011) 3199–3207. [25] D. Sarkar, P. Das, S. Basak, N. Chattopadhyay, J. Phys. Chem. B 112 (2008) 9243–9249. [26] V.N. Potaman, Y.A. Bannikov, L.S. Shlyachtenko, Nucleic Acids Res. 8 (1980) 635–642. [27] K.C.J. Nejedly´, M. Vorlickov, I. Hrabcova, J. Kypr, Nucleic Acids Res. 33 (2005) e5. [28] J.B. Foresman, A. Frisch, Gaussian Inc., Pittsburgh, PA, USA, 1966. [29] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew, A.J. Olson, Autodock, version 4.0.1, The Scripps Research Institute, LaJolla, CA, USA, 2007. [30] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew, A.J. Olson, J. Comput. Chem. 19 (1998) 1639–1662. [31] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.J. Olson, J. Comput. Chem. 30 (2009) 2785–2791. [32] J. Gasteiger, M. Marsili, Tetrahedron 36 (1980) 3219–3228. [33] R. Tiwari, K. Mahasenan, R. Pavlovicz, Ch. Li, W. Tjarks, J. Chem. Inf. Model. 49 (2009) 1581–1589. [34] X. Xu, D. Wang, X. Sun, S. Zeng, L. Li, D. Sun, Thermochim. Acta 493 (2009) 30– 36. [35] A. Shah, E. Nosheen, S. Munir, A. Badshah, R. Qureshi, Z. Rehman, N. Muhammad, H. Hussain, J. Photochem. Photobiol. B 120 (2013) 90–97. [36] F.A. Tanious, J.M. Veal, H. Buczak, L.S. Ratmeyer, W.D. Wilson, Biochemistry 31 (1992) 3103–3112. [37] R. Fukuda, S. Takenaka, M. Takagi, J. Chem. Soc. – Chem. Commun. 15 (1990) 1028–1030. [38] M.J. Waring, J. Mol. Biol. 13 (1965) 269–282. [39] S. Kashanian, S. Javanmardi, Ar. Chitsazan, K. Omidfar, M. Paknejad, DNA Cell Biol. 31 (2012) 1349–1355. [40] I.M. Klotz, Ann. N.Y. Acad. Sci. 226 (1973) 18–35. [41] J.-H. Shi, Y.-Y. Zhu, J. Wang, J. Chen, Y.-J. Shen, Spectrochim. Acta A 103 (2013) 287–294. [42] J.M. Kelly, A.B. Tossi, D.J. McConnel, C. OhUigin, Nucleic Acids Res. 13 (1985) 6017–6034. [43] L.S. Lerman, J. Mol. Biol. 3 (1963) 18–30. [44] S. Satyanarayana, J.C. Dabrowiak, J.B. Chairers, Biochemistry 31 (1992) 9319– 9324. [45] A.H. Hegde, S.N. Prashanth, J. Seetharamappa, J. Pharm. Biomed. Anal. 63 (2012) 40–46. [46] N.R. Barnes, A.F. Schreiner, M.A. Dolan, J. Inorg. Biochem. 72 (1998) 1–12. [47] R. Marty, C.N. N’soukpoe–Kossi, D. Charbonneau, C.M. Weinert, L. Kreplak, H.A. Tajmir-Riahi, Nucleic Acids Res. 37 (2008) 849–857. [48] D.K. Jangir, S. Charak, R. Mehrotra, S. Kundu, J. Photochem. Photobiol. B 105 (2011) 143–148. [49] C.F. Jordan, L.S. Lerman, J.H. Venable, Nat. New Biol. 236 (1972) 67–70.
Please cite this article in press as: J.-H. Shi et al., Binding interaction between sorafenib and calf thymus DNA: Spectroscopic methodology, viscosity measurement and molecular docking, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), http://dx.doi.org/10.1016/ j.saa.2014.09.056
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Binding interaction between sorafenib and calf thymus DNA: Spectroscopic methodology, viscosity measurement and molecular docking Jie-Hua Shi a,b,⇑, Jun Chen a, Jing Wang a, Ying-Yao Zhu a a b
College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032, China State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, Zhejiang University of Technology, Hangzhou 310032, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Sorafenib binds to DNA via minor
It was confirmed that sorafenib interacts with ct-DNA via minor groove binding mode through spectroscopic methods (such as UV–vis absorption spectroscopy, and fluorescence emission spectroscopy) and molecular doching. CF3
O
Cl
O
O
H
Molecular docking H
H Sorafenib 0.30 1 ( Csorafenib =0)
0.25 0.20
Absorbance
groove binding and forms 1:1 complex with it. The main interaction forces were van der Waals and hydrogen bonding interactions. There was slight change of the secondary structure of DNA due to binding sorafenib. The flexibility of sorafenib plays an important role in increasing the sorafenib–DNA stability.
7 (C sorafenib = 5.52×10
-5
M)
0.15 0.10 0.05 0.00 250
300
350
400
wavelength (nm)
UV-vis absorption spectroscopy DNA Note: all solutions conclude fixed concentration of DNA and Hoechst 33258
Fluorescence intensity (a.u.)
350 300
1 ( C sorafenib =0)
250 200
-5
6 (Csorafenib=3×10 M)
150
λ ex = 365 nm
100 50 0 400
450
500
550
600
650
Wavelength (nm)
Sorafenib - DNA complex
a r t i c l e
i n f o
Article history: Received 18 June 2014 Received in revised form 27 August 2014 Accepted 18 September 2014 Available online xxxx Keywords: Sorafenib Calf thymus-DNA UV–vis spectroscopy Circular dichroism Fluorescence spectroscopy Molecular docking
Fluorescence emission spectroscopy
a b s t r a c t The binding interaction of sorafenib with calf thymus DNA (ct-DNA) was studied using UV–vis absorption spectroscopy, fluorescence emission spectroscopy, circular dichroism (CD), viscosity measurement and molecular docking methods. The experimental results revealed that there was obvious binding interaction between sorafenib and ct-DNA. The binding constant (Kb) of sorafenib with ct-DNA was 5.6 103 M–1 at 298 K. The enthalpy and entropy changes (DH0 and DS0) in the binding process of sorafenib with ct-DNA were –27.66 KJ mol–1 and –21.02 J mol–1 K–1, respectively, indicating that the main binding interaction forces were van der Waals force and hydrogen bonding. The docking results suggested that sorafenib preferred to bind on the minor groove of A-T rich DNA and the binding site of sorafenib was 4 base pairs long. The conformation change of sorafenib in the sorafenib–DNA complex was obviously observed and the change was close relation with the structure of DNA, implying that the flexibility of sorafenib molecule played an important role in the formation of the stable sorafenib–ct-DNA complex. Ó 2014 Elsevier B.V. All rights reserved.
Introduction ⇑ Corresponding author at: College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032, China. Tel./fax: +86 571 8832 0064. E-mail address: [email protected] (J.-H. Shi).
Deoxyribonucleic acid (DNA) plays key physiological roles in the life process because it carries important genetic information
http://dx.doi.org/10.1016/j.saa.2014.09.056 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: J.-H. Shi et al., Binding interaction between sorafenib and calf thymus DNA: Spectroscopic methodology, viscosity measurement and molecular docking, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), http://dx.doi.org/10.1016/ j.saa.2014.09.056
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and guides the biological synthesis of proteins and enzymes through the process of duplication, transportation and translation of genetic information. Small molecules can bind to DNA via covalent or non-covalent interactions, resulting in alteration or inhibition of DNA function [1,2]. Therefore, the investigation on the binding interaction of DNA with small molecules is helpful to understand the structural features of DNA, origin of some diseases and action mechanism of some drugs, and design improved drugs that target cellular DNA. The researches on the binding interaction between DNA and small molecules have been considered as one of the key topics in the field of life sciences, chemistry and medicine. Currently, the methods used to investigate the binding interaction of DNA with small molecules mainly contain the experimental methods [3–7] and the molecular docking methods [8–14]. Sorafenib (Fig. 1), which is an inhibitor of several tyrosine protein kinases and Raf kinases, is used for the treatment of primary kidney cancer and advanced primary liver cancer [15–19]. It has also been demonstrated that sorafenib has the preclinical and clinical activity against several tumor types such as renal cell carcinoma, and non small cell lung cancer [18,20–23]. In addition, Tod et al studied that the binding interaction between sorafenib and plasma using the quenching fluorescence method and the influence of albuminemia and bilirubinemia on sorafenib disposition in cancer patients, suggesting that sorafenib is highly bound to plasma (>99.5%) and the major influence of albuminemia on sorafenib clearance [24]. However, to our best knowledge, the study on the intermolecular interactions of sorafenib with DNA using UV–vis absorption spectroscopy, fluorescence emission spectroscopy, circular dichroism (CD) and molecular modeling methods has not been reported. In this work, the binding interaction between sorafenib and calf thymus DNA (ct-DNA) was studied using UV–vis absorption spectroscopy, fluorescence emission spectroscopy, circular dichroism (CD), viscosity measurement and molecular docking in order to obtain the detailed information about the binding interaction of sorafenib with ct-DNA such as specific binding site, binding modes, binding constant, effect of sorafenib on conformation of ct-DNA, interaction forces, among others. The study of the binding interaction of ct-DNA with sorafenib molecule has great significance in helping to elucidate the mechanism of action and pharmacokinetics.
Tris–HCl buffer solution (pH = 7.40) consisted of Tris (0.050 M) and was adjusted to pH = 7.40 by 36% HCl solution. The stock solution of ct-DNA was prepared by dissolving of ct-DNA in 0.050 M of the Tris–HCl buffer (pH = 7.4). The stock solution of ct-DNA was stored at 4 °C in the dark for 5 days only and was stirred at frequent intervals to ensure the formation of a homogenous solution. The stock solution of ct-DNA gave a ratio of UV absorbance at 260 nm and 280 nm of above 1.8, indicating that DNA was sufficiently free from protein [3,25]. The final concentration of DNA in the stock solution was determined by UV absorption spectroscopy using the molar absorption coefficient of 6600 M–1 cm–1. Stock solution of sorafenib (6.28 10–5 mol L–1) was prepared in ethanol due to its very low solubility in water. We found that when the concentration of ethanol was lower than 12% the absorbances of ct-DNA solution and the mixture solution of sorafenib and ct-DNA were about the same, indicating that the concentration of ethanol lower than 12% did not affect on the conformational of DNA, which was consistent with the reference reported [26,27], and on the interaction of DNA with sorafenib. Therefore, the final concentration of ethanol in the test solution of sorafenib and ctDNA was controlled at lower than 10% to avoid the conformational change of ct-DNA and the effect on interaction of ct-DNA with sorafenib. UV–vis absorption spectral measurements The UV–vis spectra for all mixture solutions of ct-DNA and sorafenib were recorded from 230 to 400 nm on UV-1601 spectrophotometer with a 1.0 cm quartz cuvette (Shimadzu corporation, Kyoto, Japan) at different temperatures to quantify the binding constants of sorafenib to DNA and to evaluate the effect of temperature on binding interaction of DNA with sorafenib. The corresponding solution of ct-DNA was measured as reference solution. Fluorescence emission spectral measurements The fluorescence emission spectra of all mixture solutions of Hoechst 33258 and ct-DNA in the absence and presence of sorafenib were recorded on a F96S Spectrofluorimeter with 1.0 cm quartz cell (Shanghai LengGuang Industrial Co., Ltd., Shanghai, China) from 400 to 600 nm at kex = 365 nm at ambient temperature. These fluorescence emission spectra were measured as the average of three scans and the appropriate blanks corresponding to the buffer were subtracted to correct background.
Materials and methods Chemical and reagents
Circular dichroism measurements The highly polymerized ct-DNA and Hoechst 33258 were purchased from Sigma Chemical Co., Ltd. and were used without further purification. Sorafenib tosylate was provided from Nanjing Ange Pharmacetutical Co., Ltd. Tris(hydroxymethyl) aminomethane (Tris) (>99%) was purchased from Shanghai Bobo biotechnology Co., Ltd. Other chemicals were of analytical reagent grade and were used without further purification.
CF3 6
Cl
O 12 5
1 2
4 3
Circular dichroism spectra were recorded on JASCO J-815 Spectrophotometer with 1.0 cm quartz cell (Japan Spectroscopic Company, Tokyo, Japan) at ambient temperature, in which the scan range was from 200 to 350 nm with an interval of 1 nm at a scan rate of 100 nm min–1. Each spectrum was the average of three scans and was corrected by corresponding buffer blanks.
O 7 N H
8
11 9 N
21
O 13
16
14
17
10 15
20
22 N 23
N19
24
H
18
H Fig. 1. The structure of sorafenib.
Please cite this article in press as: J.-H. Shi et al., Binding interaction between sorafenib and calf thymus DNA: Spectroscopic methodology, viscosity measurement and molecular docking, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), http://dx.doi.org/10.1016/ j.saa.2014.09.056
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Viscosity measurements
Results and discussion
Viscosity measurements were performed using Ubbelohde viscometer which was thermostated at 298 K in a constant temperature bath and the inner diameter of capillary was 0.57 mm. The concentration of ct-DNA in Tris–HCl buffer solution (pH = 7.4) in the absence and presence of sorafenib was fixed at 1.38 10–5 M and the flow time was measured using a digital stop watch. The mean values of three replicated measurements were used to evaluate the relative specific viscosity (g/g0)1/3, where g0 and g are the specific viscosity contributions of DNA in the absence and presence of sorafenib, respectively.
UV–vis absorption spectroscopy
Molecular docking The starting geometry of sorafenib (NCBI, CID 216239) was obtained from the PubChem Compound Database (http://pubchem. ncbi.nlm.nih.gov/summary/summary.cgi?cid=216239&loc=ec_rcs). The structure of sorafenib was first treated by semi-empirical theory at PM3 level and then was optimized by density functional theory (DFT) at B3lyp/6-31+g(d,p) level using Gaussian 03 software until all eigenvalue of the Hessian matrix were positive [28]. The optimized structure of sorafenib was used for the molecular docking calculations. The ct-DNA is composed of two strands that wrap around each other to form a right-handed double helix with the B-form. The crystal structures of B-DNAs used in molecular docking were extracted from Protein Data Bank (http://www.rcsb.org/pdb/ home/home.do) and listed in Table 1. The binding interactions of sorafenib with B-DNA were simulated by molecular docking method using AutoDock 4.0 program [29]. All of the water molecules were removed from B-DNA. The polar hydrogen atoms were added to B-DNAs and the rotatable bonds of sorafenib were set to 6 using Autodock Tools [30,31]. The partial atomic charges of DNA and sorafenib were calculated using Gasteiger–Marsili [32] and Kollman methods [33], respectively. The grid maps of dimensions 60 60 60 Å with a gridpoint spacing of 0.375 Å were created for six B-DNAs to ensure an appropriate size for sorafenib-accessible space. In this work, the centers of grid boxes were set as shown in Table 1. The number of genetic algorithm runs and the number of evaluations were set to 100 and 2.5 million, respectively. Other miscellaneous parameters were assigned the default values given by Autodock program. Cluster analysis was performed on the results of docking by using a root mean square (RMS) tolerance 2.0 Å. Finally, we obtained the dominating configuration of the binding complex of sorafenib with NDA with minimum binding free energy (DG).
UV–vis absorption spectroscopy, one of the simplest testing techniques, has been widely used in the study of the interaction of DNA with small molecules by monitoring changes of UV–vis absorption bands of DNA or small molecules including the hypochromic effect, hyperchromic effect, bathochromic effect (red shift) and hypsochromic effect (blue shift). Generally, small molecule binding on DNA through intercalative interaction results in hypochromism and bathochromism of absorption bands. Because intercalative interaction is a kind of stacking interaction between base pair of DNA and chromophore of small molecules, the p orbital of base pairs of DNA can couple with the p* orbital of small molecules and form p–p* conjugation, resulting in reducing the difference of the p–p* transition energy, which causes a red shift of absorption band, and the coupling p* orbital is partially filled by electron resulting in decreasing the transition probability. The lower the probability is, the smaller the molar absorption coefficient. So, when small molecule intercalated into DNA, the distance between the chromophore of small molecule and the base pair of DNA will decrease, resulting in hypochromism of absorption band [2,34]. In the case of electrostatic attraction between the DNA and small molecules, hyperchromic effect can be observed, which reflects the corresponding changes of the conformation and structure of DNA when the electrostatic interaction of DNA with small molecules has occurred [2]. However, in the case of the groove binding mode between DNA and small molecules, the hypochromic effect can be observed while the position of the absorption band almost does not change, which can be associated with the overlapping of the electronic states of the chromophore of the complex with the nitrogenous bases in the grooves of DNA [35–37]. The UV spectra of sorafenib in the absence and presence of ctDNA were shown in Fig. 2. It can be seen that there is an absorption band at 264 nm for the sorafenib solution in the absence of ct-DNA, which belongs to B-band of an aromatic moiety in sorafenib molecule. However, the intensity of the absorption band at 264 nm decreases with the gradient addition of ct-DNA to sorafenib solution, while the position of the absorption band almost does not change. Based on above viewpoints, it can be concluded that there is the binding interaction of ct-DNA with sorafenib and the main binding mode may be groove binding interaction. Binding constant and thermodynamic parameters measurements As is well known, the binding constant (Kb) for the 1:1 sorafenib–DNA complex can be calculated according to Benesi-Hildebrand equation [4]:
Table 1 Six DNA sequences used for molecular docking.a
a
DNA ID
PDB ID
Sequences
Unit cell constants
Centers of grid boxes
1
1D32
D(CGCG)2
a = 16.88 Å, b = 26.88 Å, c = 82.60 Å a = 90°, b = 90°, c = 90°
26.789, 12.727, 10.387
2
1ZNA
D(CGCG)2
a = 31.27 Å, b = 64.67 Å, c = 19.50 Å a = 90°, b = 90°, c = 90°
18.02, 8.307, 8.794
3
1K2J
D(CGTACG)2
No data
0.234, 2.024, 7.463
4
1BNA
D(CGCGAATTCGCG)2
a = 24.87 Å, b = 40.39 Å, c = 66.20 Å a = 90°, b = 90°, c = 90°
14.78, 20.976, 8.807
5
1DNE
D(CGCGATATCGCG)2
a = 25.48 Å, b = 41.26 Å, c = 66.88 Å a = 90°, b = 90°, c = 90°
15.296, 21.285, 75.966
6
102D
D(CGCAAATTTGCG)2
a = 24.78 Å, b = 41.16 Å, c = 65.51 Å a = 90°, b = 90°, c = 90°
14.558, 21.582, 75.506
These data were extracted from Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). 1ZNA: DNA without gap; 1D32: DNA with two gap.
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0.25
8.7
1 8.6
7
8.5
0.15
ln (Kb)
Absorbance
0.20
0.10
8.4
8.3
0.05
8.2
0.00 250
275
300
325
350
375
400 0.00324
Wavelength (nm)
0.00332
0.00336
-1
1/T (K )
Fig. 2. UV absorption spectra of sorafenib (6.28 10–6 M) in absence and presence of ct-DNA, the concentrations of ct-DNA from 1 to 7 were 0, 0.689 10–5, 1.38 10–5, 2.07 10–5, 2.76 10–5, 4.14 10–5, 5.52 10–5 M, respectively.
Fig. 4. Van’t Hoff plot for the sorafenib–DNA complex.
0.5
298 K 303 K 310 K
-10
0.4
-20 -30
Absorbance
A0/[A-A0]
0.00328
-40 -50
0.3
0.2
-60 0.1
-70 40000
80000
120000
160000 0.0
-1
1/CDNA (L mol )
0.0
0.1
0.2
0.3
0.4
CNaCl (mol/L)
Fig. 3. Plots of A0/(A A0) versus 1/CDNA for sorafenib–DNA complex in Tris–HCl buffer solution (pH = 7.4) at different temperatures.
Fig. 5. Effect of ionic strength on the absorbance of sorafenib–DNA complex. Concentrations of DNA and sorafenib were 1.38 10–5 M and 6.28 10–6 M, respectively.
A0 eD eD 1 ¼ þ A A0 eDDNA eD eDDNA eD K b C DNA
ð1Þ
where A0 is the absorption of sorafenib at 264 nm in the absence of ct-DNA. A is the corresponding absorbance at different concentration of ct-DNA. eD and eD–DNA are the molar extinction coefficient of free sorafenib and sorafenib–ct-DNA complex, respectively. CDNA is the concentration of ct-DNA. It can be found from Fig. 3 that there is a good linear relationship between A0/(A A0) and 1/CDNA at different temperatures, indicating that the stoichiometry of binding interactions between DNA and sorafenib is 1:1. The apparent binding constants (Kb) at different temperatures are calculated from
intercept and slope obtained from Eq. (1) and listed in Table 2. The estimated values of Kb are in the order of 103 in the range from 298 to 310 K, which is significantly lower than that of the classic intercalation binding like EB-DNA complex (Kb = 1.4 106 M–1) [38]. However, it falls on the range of the groove binding constant of DNA with small molecule [7,39]. It is further indicated that the binding mode may be the groove interaction. In the binding process of biomacromolecule with small molecule, there are mainly four types of non-covalent interactions including hydrogen bonding interaction, van der Waals forces,
Table 2 The results for the apparent binding constants (Kb) of sorafenib–DNA complex and thermodynamic parameters. Temperature (K)
B–H equations obtained 4
298 303 310 a b c
Kb (L mol–1)
DG0exp,
5.58 103 4.75 103 3.63 103
–21.37 –21.33 –21.13
1
b
(kJ mol–1)
DG0exp,
c 2
(kJ mol–1)
DH0 (kJ mol–1)
DS0 (J mol–1 K–1)
–27.66
–21.02
a
Slope (10 )
Intercept
r
–3.10 ± 0.08 –3.56 ± 0.09 –4.54 ± 0.10
–1.73 ± 0.58 –1.69 ± 0.68 –1.65 ± 0.73
0.9983 0.9982 0.9987
–21.40 –21.29 –21.14
r Is the correlation coefficient. DG0exp, 1 = –RT ln Kb. DG0exp, 2 = DH0 TDS0.
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1.10
8 6
1.05
CD [medg]
1/3 0
(η/η )
1
4
1.00
2
2 0 200 -2
220
240
260
280
300
320
340
Wavelength (nm)
0.95 -4 -6 0.90 0.0
0.2
0.4
0.6
0.8
1.0
1.2
Fig. 8. The CD spectra of DNA (5 10–5 M) in the presence of sorafenib in Tris–HCl buffer solution. The concentrations of sorafenib from 1 to 2 were 0, 3 10–5 M, respectively.
r =[sorafenib]/[DNA] Fig. 6. Effect of increasing amounts of sorafenib on the viscosity of ct-DNA (1.38 10–5 M) in the Tris–HCl buffer solution.
hydrophobic interaction and electrostatic interaction [40]. Meanwhile, the signs and magnitudes of the thermodynamic parameters (DG0, DH0 and DS0) in the binding process of biomacromolecule with small molecule can be used to confirm the binding modes. These thermodynamic parameters can be calculated by the van’t Hoff equations [41]:
DH 0 DS0 þ RT R 0 DG ¼ RT ln K b ln K b ¼
ð2Þ ð3Þ
where R is the gas constant. DG0, DH0 and DS0 are the changes in Gibbs free energy, enthalpy and entropy in the binding process of biomacromolecule with small molecule, respectively. It is generally suggested that both DH0 and DS0 are positive, indicating that the main interaction force is hydrophobic interaction. DH0 and DS0 are negative, suggesting that the main interaction force is van der Waals force and/or hydrogen bonding interaction. DH0 is almost zero and DS0 is positive, implying that the main interaction force is electrostatic force [41]. For the effect of temperature on the DH0 and DS0 of the interaction of DNA with sorafenib is very small in the range of temperature studied, the values of DH0 and DS0 can be regarded as constant. The values of DH0 and DS0 for the
350
Fluorescence intensity (a.u.)
1 300 250
6
200 150 100
λ ex= 365 nm 50 0 400
450
500
550
600
650
Wavelength (nm) Fig. 7. Fluorescence emission spectra of the DNA–Hoechst 33258 in the presence of sorafenib in Tris–HCl buffer (pH = 7.4). The concentrations of ct-DNA and Hoechst 33258 were 1 10–5 M, 5 10–7 M, respectively. The concentrations of sorafenib from 1 to 6 were 0, 1 10–5, 1.5 10–5, 2 10–5, 2.5 10–5, 3 10–5 M, respectively.
interaction of ct-DNA with sorafenib are obtained from the linear relationship between ln Kb and the reciprocal absolute temperature as shown in Fig. 4 and the results are listed in Table 2. It can be found from Table 2 that the values of DG0, DH0 and DS0 are negative, suggesting that the binding interaction of sorafenib with ct-DNA is exothermic and spontaneous process. Moreover, the values of DH0 and DS0 are –27.66 kJ mol–1 and –21.02 J mol–1 K–1, respectively, indicating that the binding process is enthalpy-driven and the main interaction forces are hydrogen bonding interaction and van der Waals force. Binding mode Extensive research results revealed that small molecules usually bind to DNA in non-covalent way. Moreover, non-covalent interaction can be classified into three modes: (i) electrostatic binding; (ii) groove binding; and (iii) intercalative binding [1,2]. In order to further elucidate the binding mode of sorafenib with ct-DNA, the effect of ionic strength on UV absorption spectra of mixture solutions of sorafenib and ct-DNA, the effect of sorafenib on the viscosity of ct-DNA solution and the competitive binding of sorafenib with Hoechst 33258 on ct-DNA were further studied. The effects of ionic strength The electrostatic binding mode is one of non-covalent binding modes of small molecule on DNA, which is often served as an auxiliary mode to assist groove binding and intercalation. The small molecule that binds strongly to DNA usually includes the electrostatic component. But, if the electrostatic binding interaction plays a dominant role in the binding interaction of DNA with small molecules, the strength of interaction will decrease with the increase of salt concentration in system [1,25]. The experimental results showed that the absorbances of sorafenib–DNA solutions almost did not change with the increase of the concentration of NaCl (Fig. 5), indicating that there was no significant electrostatic binding interaction between ct-DNA and sorafenib. Viscosity studies Viscosity measurement is often regarded as an effective and accurate method to determine the binding mode between small molecules and DNA. It is generally suggested that a classical intercalative binding mode causes a significant increase of DNA viscosity because the intercalative interaction requires the space of adjacent base pairs to be large enough to accommodate the bound small molecules and elongates the double helix [42,43]. However, for the electrostatic or groove binding, there is little effect on the viscosity of DNA [42,44]. The viscosities of ct-DNA
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Table 3 The various energies and the hydrogen bonding interactions in the formation process of sorafenib–DNAs complexes. DNA PDB ID
DGa kJ mol–1
E1b kJ mol–1
E2c kJ mol–1
E3d kJ mol–1
Hydrogen bonding DNA
Sorafenibe
Bond length (Å)
1D32
–31.09
–39.00
–37.99
–1.00
DG6: H2 (Chain B)
O22
1.95
1ZNA
–26.93
–34.21
–32.99
–1.21
DC7: O3 (Chain B) DG4: H22 (Chain A)
H9 O22
2.23 1.80
1K2J
–41.34
–48.24
–46.90
–1.34
DC11: O4 (Chain B) DC11: O4 (Chain B) DA4: H3 (Chain A) DG6: H3 (Chain A) DG8: H22(Chain B)
H7 H9 O8 O22 N19
1.93 1.92 2.12 1.91 2.01
1BNA
–45.44
–51.09
–51.00
–0.04
DT8: O2 (Chain A) DT8: O2 (Chain A) DA6: H3 (Chain A)
H7 H9 O22
2.18 1.91 1.94
1DNE
–40.63
–48.08
–46.86
–1.21
DA17: H3 (Chain B) DT20: O4 (Chain B) DT20: O4 (Chain B)
O22 H7 H9
1.95 1.82 1.94
102D
–42.64
–50.17
–49.37
–0.75
DA6: H3 (Chain A) DT19: O4 (Chain B) DT19: O4 (Chain B) DT21: O4 (Chain B)
O22 H7 H9 H23
2.03 1.72 1.88 1.90
a
DG is the binding energy in the binding process, which is calculated in water solvent using a scoring function. E1 denotes intermolecular interaction energy, which is a sum of van der Waals energy, hydrogen bonding energy, desolvation free energy and electrostatic energy. c E2 is the sum of van der Waals energy, hydrogen bonding energy and desolvation free energy. d E3 is the electrostatic energy. e H7, H9 and H23 are hydrogen atoms linked with N7, N9 and N23 atoms, respectively, as shown in Fig. 1. O8 and O22 denote oxygen atoms linked with C8, C22, respectively, as shown in Fig. 1. b
Fig. 9. Molecular docking results of sorafenib bound to B-DNAs. Color codes of DNA: deoxy adenine (DA) is red, deoxy cytosine (DC) is green, deoxy guanine (DG) is yellow and deoxy thymine (DT) is blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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J.-H. Shi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2014) xxx–xxx
7
in the Tris–HCl buffer solution in the absence and presence of sorafenib were measured and the results were shown in Fig. 6. As shown in Fig. 6, the viscosities of ct-DNA in the Tris–HCl buffer solution (pH = 7.4) almost do not change with the increasing concentration of sorafenib, indicating that the binding mode of sorafenib with ct-DNA is not the intercalative binding mode. Therefore, the binding mode of ct-DNA with sorafenib may be the groove binding. Competitive binding of sorafenib with Hoechst 33258 To further affirm the binding mode of sorafenib on ct-DNA, a competitive binding experiment using Hoechst 33258 as probe molecule was performed. Hoechst 33258, which can strongly bind to AT-rich regions of DNA via minor groove binding, is a classical fluorescent probe [45]. In this work, the fluorescence emission spectra of the fixed amount of ct-DNA and Hoechst in Tris–HCl buffer solution (pH = 7.4) were measured with gradually increasing amounts of sorafenib and the results were shown in Fig. 7. It can be found that the fluorescence intensities of ct-DNA–Hoechst solutions decreased with the gradually increasing concentration of sorafenib. Meanwhile, it was not observed that sorafenib reacted with Hoechst 33258 resulting in the change of the fluorescence intensity of Hoechst 33258. Based on above experimental results, it can be concluded that there is competitive binding between Hoechst 33258 and sorafenib on ct-DNA. Therefore, sorafenib binds to AT-rich regions of ct-DNA via minor groove binding interaction. Circular dichroism studies As is well known, the circular dichroism (CD) spectroscopy is a powerful way in determining the secondary structure changes of DNA after binding with small molecules and has widely been used to investigate the interaction between small molecules and DNA [46–48]. The CD spectra of ct-DNA in Tris–HCl buffer solution in the absence and presence of sorafenib were shown in Fig. 8. It can be seen that there are four main characteristic peaks in the range from 200 to 350 nm for the free ct-DNA solution, which are two negative peaks at 211 and 244 nm and two positive peaks at 220 and 275 nm, respectively. These bands are consistent with CD spectrum of double helix DNA in the B conformation [47]. However, the negative peak at 244 nm belongs to helical geometry of BDNA while the positive peak at 275 nm is assigned to stacking of DNA bases. When sorafenib was added to the ct-DNA solution, the change of CD spectrum of ct-DNA was observed. The intensity of the positive band at near 275 nm decreased, while that of the negative band at near 244 nm increased. This revealed that the conformation of ct-DNA had slightly changed due to the binding interaction between sorafenib and ct-DNA. The increase of the negative peak intensity (244 nm) showed that the interaction of sorafenib with ct-DNA made the double helix structure of ct-DNA become tight [11]. The decrease of the intensity of the positive band (275 nm) was likely to be due to a transition from the extended nucleic acid right-handed double helix to more compact form (w structure) [49]. Molecular docking Molecular docking has gained growing interests in the investigation of binding interaction mechanism of biological macromolecule with small molecules. It plays a more and more important role in drug discovery and development [8–14]. In this work, the molecular docking of sorafenib with DNA was carried out using Autodock 4.0 in order to further clarify the binding mode of sorafenib on DNA and the binding structure of DNA-sorafenib complex. Sorafenib, kept as flexible molecule, was docked into the six types of rigid DNAs to search the preferential binding site of sorafenib on
Fig. 10. Conformations of free sorafenib (a) and the sorafenib bound with DNA (1BNA) (b) or DNA (1DNE) (c).
different DNAs and the docking results were listed in Table 3. Generally, the more negative the binding energy is, the stronger the interaction between small molecule and DNA, the most stable the complex formed by small molecule and DNA is. From Table 3, it can be found that the binding free energy (DG) is obviously lower when there are adenine (A) and thymine (T) base pairs in the DNA sequences, indicating that the preferential binding site of sorafenib on the A-T rich sequence of DNA. However, sorafenib prefers to bind on the minor groove of A-T rich DNAs, which is consistent with above experimental results, and the binding site is 4 base pairs long and involves A-T residues as shown in Fig. 9. And, significant change of conformation of sorafenib occurs in the binding process with DNA to orient easily along the minor groove. The conformation change of sorafenib is close relation with the structure of DNA minor groove (Fig. 10). In addition, it can be found from the docking results that there are hydrogen bonding interactions between sorafenib and DNAs (Table 3), suggesting that during the binding process of sorafenib with DNA, the narrower and deeper shape of the minor groove can offer several sites of action, resulting in the close contact with the surface of sorafenib through van der Waals forces and hydrogen bonding interaction. This further illustrates that the main forces of the interaction of sorafenib with DNAs are hydrogen bonds and van der Waals in binding process of sorafenib with DNA, which is consistent with the result of the thermodynamic parameter analysis, and the flexibility of sorafenib molecule plays an important role in the binding process of DNA with sorafenib. From Table 3, it can be also seen that the electrostatic energy is very much lower than the sum of van der Waals energy, hydrogen bonding energy and desolvation free energy in the binding process
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of sorafenib with DNAs, indicating that the main interaction mode between sorafenib and DNAs is not electrostatic binding mode, which is also consistent with the results of the effects of ionic strength on the UV–vis absorbance of sorafenib–ct-DNA solution. Conclusion In this work, UV–vis absorption spectroscopy, fluorescence emission spectroscopy, circular dichroism (CD), viscosity measurement and molecular docking were carried out to research the binding interaction between sorafenib and ct-DNA. It can be found that sorafenib interacts with ct-DNA via minor groove binding mode with the binding constant (Kb) of 5.4 103 at 298 K. In the binding process of sorafenib with ct-DNA, the main interaction forces were van-der Waals force and hydrogen bonding force. Additionally, the conformation change of sorafenib is obvious, indicating that the flexibility of sorafenib molecule plays an important role in the formation of the stable sorafenib–ct-DNA complex. The present study reveals the details of binding affinity, mode of binding interaction, main interaction forces of sorafenib with ct-DNA and structure of sorafenib–ct-DNA complex. Therefore, this study of the interaction mechanisms of sorafenib with ct-DNA would provide useful information in further understanding the mechanism of action and pharmacokinetics. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]
L. Strekowski, B. Wilson, Mutat. Res. 623 (2007) 3–13. M. Sirajuddin, S. Ali, A. Badshah, J. Photochem. Photobiol. B 124 (2013) 1–19. N. Shahabadi, N.H. Moghadam, Spectrochim. Acta A 99 (2012) 18–22. R. Hajian, T.G. Huat, J. Spectrosc. (2013). Article ID 380352. S. Agarwal, D.K. Jangir, R. Mehrotra, J. Photochem. Photobiol. B 120 (2012) 177–182. N. Shahabadi, N.H. Moghadam, J. Lumin. 134 (2013) 629–634. N. Shahabadi, S. Hadidi, Spectrochim. Acta A 96 (2012) 278–283. Y. Guo, Q. Yue, B. Gao, Int. J. Biol. Macromol. 49 (2011) 55–61. S. Charak, M. Shandilya, G. Tyagi, R. Mehrotra, Int. J. Biol. Macromol. 51 (2012) 406–411. R. Bera, B.K. Sahoo, K.S. Ghosh, S. Dasgupta, Int. J. Biol. Macromol. 42 (2008) 14–21. H. Li, X. Bu, J. Lu, C. Xu, X. Wang, X. Yang, Spectrochim. Acta A 107 (2013) 227– 234. F. Perveen, R. Qureshi, F.L. Ansari, S. Kalsoom, S. Ahmed, J. Mol. Struct. 1004 (2011) 67–73. P. Zhu, G. Zhang, Y. Ma, Y. Zhang, H. Miao, Y. Wu, Spectrochim. Acta A 112 (2013) 7–14. M.M. Islam, M. Chakraborty, P. Pandya, A.A. Masum, N. Gupta, S. Mukhopadhyay, Dyes Pigments 99 (2013) 412–422. G.M. Keating, A. Santoro, Drugs 69 (2009) 223–240. R.A. Smith, J. Dumas, L. Adnane, S.A. Wilhelm, Curr. Top. Med. Chem. 6 (2006) 1071–1089. K.S.M. Smalley, M. Xiao, J. Villanueva, T.K. Nguyen, K.T. Flaherty, R. Letrero, P. Van Belle, D.E. Elder, Y. Wang, K.L. Nathanson, M. Herlyn, Oncogene 28 (2009) 85–94.
[18] C.H. Takimoto, A. Awada, Cancer Chemother. Pharm. 61 (2008) 535–548. [19] S.M. Wilhelm, L. Adnane, P. Newell, A. Villanueva, J.M. Llovet, M. Lynch, Mol. Cancer Therapy 7 (2008) 3129–3140. [20] A. Dasari, L. Gore, W.A. Messersmith, S. Diab, A. Jimeno, C.D. Weekes, K.D. Lewis, H.A. Drabkin, T.W. Flaig, D.R. Camidge, Invest. New Drugs 31 (2013) 115–125. [21] M. Chaparro, L. GonzaLez-Moreno, M. Trapero-Marugan, J. Medina, R. MorenoOtero, Aliment. Pharm. Therapy 28 (2008) 1269–1277. [22] J. Tabernero, R. Garcia-Carbonero, J. Cassidy, A. Sobrero, E.V. Cutsem, C.-H. Kohno, S. Tajpar, O. Gladkov, I. Davidenko, R. Salazar, L. Vladimirova, S. Cheporov, O. Burdaeva, F. Rivera, L. Samuel, I. Bulavina, V. Potter, F. Rivera, L. Samuel, I. Bulavina, V. Potter, Y.-L. Chang, N.A. Lokker, P.J. O’Dwyer, Clin. Cancer Res. 19 (2013) 2541–2550. [23] T. Morisaki, M. Umebayashi, A. Kiyota, N. Koya, H. Tanaka, H. Onishi, M. Katano, Anticancer Res. 33 (2013) 1387–1395. [24] M. Tod, O. Mir, N. Bancelin, R. Coriat, A. Thomas-Schoemann, F. Taieb, P. Boudou-Rouquette, S. Ropert, J. Michels, H. Abbas, J.P. Durand, A. Dauphin, M. Vidal, F. Goldwasser, B. Blanchet, Pharm. Res. 28 (2011) 3199–3207. [25] D. Sarkar, P. Das, S. Basak, N. Chattopadhyay, J. Phys. Chem. B 112 (2008) 9243–9249. [26] V.N. Potaman, Y.A. Bannikov, L.S. Shlyachtenko, Nucleic Acids Res. 8 (1980) 635–642. [27] K.C.J. Nejedly´, M. Vorlickov, I. Hrabcova, J. Kypr, Nucleic Acids Res. 33 (2005) e5. [28] J.B. Foresman, A. Frisch, Gaussian Inc., Pittsburgh, PA, USA, 1966. [29] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew, A.J. Olson, Autodock, version 4.0.1, The Scripps Research Institute, LaJolla, CA, USA, 2007. [30] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew, A.J. Olson, J. Comput. Chem. 19 (1998) 1639–1662. [31] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.J. Olson, J. Comput. Chem. 30 (2009) 2785–2791. [32] J. Gasteiger, M. Marsili, Tetrahedron 36 (1980) 3219–3228. [33] R. Tiwari, K. Mahasenan, R. Pavlovicz, Ch. Li, W. Tjarks, J. Chem. Inf. Model. 49 (2009) 1581–1589. [34] X. Xu, D. Wang, X. Sun, S. Zeng, L. Li, D. Sun, Thermochim. Acta 493 (2009) 30– 36. [35] A. Shah, E. Nosheen, S. Munir, A. Badshah, R. Qureshi, Z. Rehman, N. Muhammad, H. Hussain, J. Photochem. Photobiol. B 120 (2013) 90–97. [36] F.A. Tanious, J.M. Veal, H. Buczak, L.S. Ratmeyer, W.D. Wilson, Biochemistry 31 (1992) 3103–3112. [37] R. Fukuda, S. Takenaka, M. Takagi, J. Chem. Soc. – Chem. Commun. 15 (1990) 1028–1030. [38] M.J. Waring, J. Mol. Biol. 13 (1965) 269–282. [39] S. Kashanian, S. Javanmardi, Ar. Chitsazan, K. Omidfar, M. Paknejad, DNA Cell Biol. 31 (2012) 1349–1355. [40] I.M. Klotz, Ann. N.Y. Acad. Sci. 226 (1973) 18–35. [41] J.-H. Shi, Y.-Y. Zhu, J. Wang, J. Chen, Y.-J. Shen, Spectrochim. Acta A 103 (2013) 287–294. [42] J.M. Kelly, A.B. Tossi, D.J. McConnel, C. OhUigin, Nucleic Acids Res. 13 (1985) 6017–6034. [43] L.S. Lerman, J. Mol. Biol. 3 (1963) 18–30. [44] S. Satyanarayana, J.C. Dabrowiak, J.B. Chairers, Biochemistry 31 (1992) 9319– 9324. [45] A.H. Hegde, S.N. Prashanth, J. Seetharamappa, J. Pharm. Biomed. Anal. 63 (2012) 40–46. [46] N.R. Barnes, A.F. Schreiner, M.A. Dolan, J. Inorg. Biochem. 72 (1998) 1–12. [47] R. Marty, C.N. N’soukpoe–Kossi, D. Charbonneau, C.M. Weinert, L. Kreplak, H.A. Tajmir-Riahi, Nucleic Acids Res. 37 (2008) 849–857. [48] D.K. Jangir, S. Charak, R. Mehrotra, S. Kundu, J. Photochem. Photobiol. B 105 (2011) 143–148. [49] C.F. Jordan, L.S. Lerman, J.H. Venable, Nat. New Biol. 236 (1972) 67–70.
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