Journal of Photochemistry and Photobiology B: Biology 147 (2015) 47–55

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Characterization of interaction of calf thymus DNA with gefitinib: Spectroscopic methods and molecular docking Jie-Hua Shi a,b,⇑, Ting-Ting Liu a, Min Jiang a, Jun Chen a, Qi Wang 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

a r t i c l e

i n f o

Article history: Received 20 December 2014 Received in revised form 4 March 2015 Accepted 11 March 2015 Available online 18 March 2015 Keywords: Gefitinib Calf thymus DNA Interaction Spectroscopy Molecular docking

a b s t r a c t The binding interaction of gefitinib with calf thymus DNA (ct-DNA) under the simulated physiological pH condition was studied employing UV absorption, fluorescence, circular dichroism (CD), viscosity measurement and molecular docking methods. The experimental results revealed that gefitinib preferred to bind to the minor groove of ct-DNA with the binding constant (Kb) of 1.29  104 L mol1 at 298 K. Base on the signs and magnitudes of the enthalpy change (DH0 = 60.4 kJ mol1) and entropy change (DS0 = 124.7 J mol1 K1) in the binding process and the results of molecular docking, it can be concluded that the main interaction forces between gefitinib and ct-DNA in the binding process were van der Waals force and hydrogen bonding interaction. The results of CD experiments revealed that gefitinib did not disturb native B-conformation of ct-DNA. And, the significant change in the conformation of gefitinib in gefitinib–ct-DNA complex was observed from the molecular docking results and the change was close relation with the structure of B-DNA fragments, indicating that the flexibility of gefitinib molecule also plays an important role in the formation of the stable gefitinib–ct-DNA complex. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction Deoxyribonucleic acid (DNA), an important biological macromolecule, consists of two biopolymer strands coiled around each other to form a double helix. The two DNA strands are known as polynucleotides because they are composed of simpler units called nucleotides. Each nucleotide is a nitrogen-containing nucleobase, which is either adenine (A), thymine (T), guanine (G) or cytosine (C). The region where the two strands are close to each other (deep–narrow) is called minor grove while the region where they are away from each other (shallow–wide) is called major groove [1]. Because it carries important genetic information and guides the biological synthesis of proteins and enzymes through duplication, transportation and translation of genetic information, DNA plays an important physiological role in the life process. However, small molecules bound upon DNA may alter or inhibit DNA function [2,3]. Therefore, the investigation on the binding interaction of DNA with small molecules is helpful to understand the mechanism of interaction and to improve new drug designing. Currently, the study of the interaction between small molecules and DNA has become a hot topic in the area of life sciences, ⇑ 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). http://dx.doi.org/10.1016/j.jphotobiol.2015.03.005 1011-1344/Ó 2015 Elsevier B.V. All rights reserved.

chemistry and medicine [4–10]. Numerous studies have proved that small molecules bind to DNA mainly through non-covalent interactions [11,3,12]. The non-covalent interaction mode of small molecules with DNA is further classified into three types: intercalation, groove binding and external binding (electrostatic binding). Some planar heterocyclic molecules act as intercalators which stack between adjacent DNA base pairs resulting in significant p-electron overlap without forming covalent bonds and breaking up the hydrogen bonding interactions between the DNA base pairs. Therefore, DNA-intercalator complex is stabilized by p–p stacking interaction. DNA intercalators are used in chemotherapeutic treatment to inhibit DNA replication in rapidly growing cancer cells. Some molecules with several aromatic rings can bind to the minor groove of DNA via van der Waals interaction and hydrogen bonding interaction. Minor groove binding molecules are generally isohelical to the curve of the minor groove of DNA and facilitate binding through van der Waals interactions. And, these molecules can form hydrogen bonding interaction with base pairs. However, unlike to intercalation, groove binding permits but not require an extensive conformational change of the DNA double helix. Additionally, some organic cationic molecules are also capable of forming non-specific, outside edge stacking interaction with the phosphate backbone of DNA. In the binding process, the condensed counterions such as Na+, or Mg2+, which play an important role in the stability of folded DNA

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conformations, can be released and these ion release provides an entropic contribution to the binding free energy. Generally, electrostatic binding interaction depends on salt concentration of the solution and is weak under physiologic conditions for monocationic molecules without significant groove binding or intercalation binding. However, electrostatic binding is often served as an auxiliary mode to assist groove binding and intercalation. Gefitinib (Fig. 1) is an epidermal growth factor receptor (EGFR) inhibitor, which interrupts signaling through EGFR in target cells. Since receiving accelerated approval by the United States Food and Drug Administration in 2003 [13], it has been used for the treatment of patients with locally advanced or metastatic nonsmall cell lung cancer [14,15]. Up to now, many studies have mainly focused on its clinical application [16,17]. Many studies results reveal that the adverse effects of gefitinib include acnelike rash, interstitial lung disease, diarrhea, asthenia, stomatitis, skin reactions, nausea, vomiting and so on [18–20]. However, to our best knowledge, the study of the interactions of gefitinib with biological macromolecules such as DNA and serum albumin has not been reported. So, the research in the binding interaction of gefitinib with DNA is very necessary for further understanding the mechanism of action and pharmacokinetics of gefitinib. In order to obtain the detailed information about the binding interaction between gefitinib and ct-DNA like the specific binding site, the binding modes, the binding constant and the interaction forces, the binding interaction of gefitinib with DNA was investigated using UV–vis absorption spectroscopy, fluorescence emission spectroscopy, circular dichroism (CD), viscosity measurement and molecular docking in this work. This study is expected to provide important insight into further elucidating the mode of toxicity and overcome the shortage of information gap on pharmacokinetics.

pH = 7.4). The stock solutions were stored at 4 °C in the dark. Purity of ct-DNA was checked by monitoring the ratio (A260/A280) of the absorbance at 260 nm to that at 280 nm. The solution gave a ratio of above 1.8 at A260/A280, indicating that the DNA was sufficiently protein-free [9]. The stock solution of gefitinib (C = 2.50  103 mol/L) was prepared in ethanol. 2.2. UV–vis absorption spectral measurements The UV spectral measurements of all solutions were performed on UV-1601 spectrophotometer (Shimadzu Corporation, Kyoto, Japan) with a 1.0 cm quartz cuvette. Each solution was allowed to stand for 2 h to ensure complete interaction of DNA with gefitinib, because the absorbance of mixture solution of gefitinib and ctDNA almost did not change when the equilibration time was from 90 to 150 min. The UV spectra of all mixture solutions of ct-DNA and gefitinib were recorded from 200 to 400 nm. The corresponding solutions of ct-DNA or gefitinib were measured as reference solutions. All experiments were performed in triplicate, and the mean values were calculated with RSD of 0.07%. 2.3. Viscosity measurements Viscosity measurements were made using a viscometer maintained at 25 °C in a constant temperature bath. The concentration of ct-DNA in Tris–HCl buffer solution (pH = 7.4) in the absence and presence of gefitinib was fixed at 2.27  105 mol/L. Each sample was measured three times by digital stop watch and an average flow time was calculated. Data were presented as (g/g0)1/3 versus binding ratio r (r = [gefitinib]/[ct-DNA]), where g is the viscosity of ct-DNA in the presence of gefitinib and g0 is the viscosity of ct-DNA alone. Viscosity values were calculated from the observed flow times of ct-DNA-containing solutions (t) corrected for the flow time of buffer alone (t0), g = t  t0 [9,21].

2. Materials and methods 2.4. Fluorescence measurements 2.1. Chemical and reagents ct-DNA, Hoechst 33258 and ethidium bromide (EB) were purchased from Sigma Chemical Co. Ltd and used without further purification. Rhodamine B was purchased from Aladdin Industrial Corporation. Gefitinib (99% purity) was obtained from Guangzhou Eastbang Pharmaceuticals Co., Ltd. Tris (hydroxymethyl) aminomethane (99% purity) was purchased from Shanghai Bobo biotechnology Co., Ltd. All other reagents were of analytical grade and were used without further purification. Tris–HCl buffer solution consisted of Tris (0.050 mol/L) and was adjusted to pH = 7.4 by 36% HCl solution. Stock solutions of ctDNA, EB, Hoechst 33258 and rhodamine B were prepared by dissolving the appropriate amounts of ct-DNA, EB, Hoechst 33258 and rhodamine B in Tris–HCl buffer solution (0.050 mol/L,

18 5'

4'

6'

N 3'

7' O

9' 8'

O 7

8

1 9 N

6

2' 1' O

5

10

2

N3 4

HN 12

17 16

11

15 13

14

The fluorescence emission spectra of all mixture solutions of ctDNA and fluorescence probes such as EB and rhodamine B in the absence and presence of gefitinib were recorded on a F96S Spectrofluorimeter with 1.0 cm quartz cell (Shanghai Leng Guang Industrial Co., Ltd., Shanghai, China) from 500 to 650 nm at room temperature. These fluorescence emission spectra were measured as the average of three scans and the mean values were calculated with RSD of 0.08%. And, the excitation wavelengths for EB and rhodamine B were set at 465 nm. The excitation wavelength for Hoechst 33258 was set at 365 nm. 2.5. Circular dichroism measurements CD spectroscopic measurements were performed with JASCO J815 Spectrophotometer with 1.0 cm quartz cell (Japan Spectroscopic Company, Tokyo, Japan) at room temperature. An average value of three scans was accumulated with scan speed of 100 nm/min from 200 to 340 nm. The RSD of determination was 0.1%. The concentration of ct-DNA was kept a constant (7.3  105 mol/L) while varying the concentration of gefitinib. The corresponding buffer (0.025 mol/L) was used as reference solution. 2.6. Molecular docking

F

Cl Fig. 1. Molecular structure of gefitinib [(N-(3-Chloro-4-fluoro-phenyl)-7-methoxy6-(3-morpholin-4-ylpropoxy) quinazolin-4-amine)].

The starting geometry of gefitinib was constructed using Chem3D Ultra (version 8.0, Cambridgesoft Com., USA). The geometry of gefitinib was optimized successively through semi-empirical theory at PM3 level and density functional theory (DFT) at B3lyp/6-

J.-H. Shi et al. / Journal of Photochemistry and Photobiology B: Biology 147 (2015) 47–55 Table 1 Six DNA sequences were used for molecular docking. DNA PDB ID

DNA sequences

Centers of grid boxes

2ELG 1K2K 463D 1VTJ 104D 121D

(CGCGCG)2 (CGTACG)2 (CGCGAATTCGCG)2 (CGCGATATCGCG)2 (CGCGTATACGCG)2 (CGCAAATTTGCG)2

5.178, 0.126, 5.367 0.256, 1.998, 7.42 9.948, 12.636, 0.448 10.27, 21.001, 77.358 6.774, 0.418, 16.126 11.448, 23.065, 72.049

31+g(d,p) level until all eigenvalue of the Hessian matrix were positive using the Gaussian 03 software [22,23]. The optimized geometry of gefitinib with the lowest energy was used in the following molecular docking. Crystal structures of six DNA fragments with the B-form were extracted from the Protein Data Bank (http://pubchem.ncbi.nlm. nih.gov/summary/summary.cgi?cid=216239&loc=ec_rcs). The molecular dockings of gefitinib with B-DNAs were accomplished by AutoDock 4.2 software from the Scripps Research Institute (TSRI) (http://autodock.scripps.edu/). Firstly, the polar hydrogen atoms were added into B-DNA molecules. Then, the partial atomic charges of the B-DNA and gefitinib molecules were calculated using Gasteiger-marsili [24] and Kollman methods [25], respectively. In the process of molecular docking, the grid maps of dimensions (60 Å  60 Å  60 Å) with a grid-point spacing of 0.375 Å and the centers of grid boxes were set as shown in Table 1. The number of genetic algorithm runs and the number of evaluations was set to 100 and 2.5 million, respectively. All other parameters were default settings. Cluster analysis was performed on the results of docking by using a root mean square (RMS) tolerance of 2.0 Å, and this was dependent on the binding free energy. Finally, the dominating configuration of the binding complex of gefitinib and B-DNA fragments with minimum binding energy can be obtained.

and ct-DNA were shown in Fig. 2. The experimental results revealed that there was a characteristic absorption peak at near 260 nm for ct-DNA solution in the absence and presence of gefitinib which belongs to p ? p⁄ transition of base pairs of ct-DNA and the intensity of the absorption band decreased with the gradual addition of gefitinib while the peak position did not obviously change (Fig. 2A). Meanwhile, the intensities of the absorption bands for gefitinib centered at 226, 248 and 331 nm, respectively, which belong to the p ? p⁄ transition of gefitinib, decreased regularly (hypochromism) with the increase of ct-DNA concentration while the position of absorption band almost did not change (Fig. 2B). Generally, in the case of the intercalation, the Pi antibonding orbital (p⁄) of intercalated molecule can couple with Pi bonding orbital (p) of ct-DNA base pairs, resulting in the decrease of the p ? p⁄ transition energy and the bathochromism (red shift) (>10 nm) of absorption band. Additionally, the coupling p⁄ orbital is partially filled by electrons, resulting in the decrease of electronic transition probability and the emergence of hypochromism (up to 40%) [26–28]. In the case of electrostatic binding, hyperchromic effect for the absorption bands of the DNA and the bound molecules 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 [11]. However, in the case of the groove binding mode, 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 [29]. Based on the above viewpoints, it can be concluded that there is the binding interaction of ct-DNA with gefitinib and the main binding mode may be groove binding interaction. 3.2. Binding constant and binding forces In order to further study the binding interaction of gefitinib with DNA, the binding constant (Kb) and the thermodynamic parameters in the binding process of ct-DNA with gefitinib were determined. For the binding interaction of gefitinib with ct-DNA, there is always an equilibrium between gefitinib and ct-DNA as following

3. Results and discussion 3.1. UV–vis absorption spectra UV–vis absorption spectroscopy is usually used to determine the binding strength and mode of DNA with small molecules [10]. The UV absorption spectra of mixture solutions of gefitinib

A

Gefitinib ðGÞ þ DNA G—DNA

B

0.40

0.8 0.7

0.25

7 0.20 0.15

0.5

7

0.4 0.3

0.10

0.2

0.05

0.1

0.00

1

0.6

1 Absorbance

0.30

ð1Þ

As is well known, the equilibrium constant is given by

0.35

Absorbance

49

0.0

225 250 275 300 325 350 375 400

Wavelength (nm)

225 250 275 300 325 350 375 400

Wavelength (nm)

Fig. 2. (A) UV–vis absorption spectra of ct-DNA (CDNA = 5.0  105 mol/L) in the absence and presence of gefitinib. The concentration of gefitinib from 1 to 7 were 0, 0.5  105, 1.0  105, 2.0  105, 3.0  105, 4.0  105 and 5.0  105 mol/L, respectively. The corresponding solution of gefitinib was used as the reference solution. (B) UV–vis absorption spectra of gefitinib (Cgefitinib = 2.5  105 mol/L) in the absence and presence of ct-DNA. The concentration of DNA from 1 to 7 were 0, 0.5  105, 1.0  105, 1.5  105, 2.0  105, 2.5  105 and 3.0  105 mol/L, respectively. The corresponding solution of DNA was used as the reference solution.

J.-H. Shi et al. / Journal of Photochemistry and Photobiology B: Biology 147 (2015) 47–55

C G—DNA C G—DNA ¼ C G  C DNA ðC G;0  C G—DNA Þ  ðC DNA;0  C G—DNA Þ

Ka ¼

10.0

ð2Þ

where CG–DNA is the concentration of binding complex of gefitinib with ct-DNA. CG and CDNA are the concentrations of the unbinded gefitinib and ct-DNA, respectively. CG,0 and CDNA,0 are the initial concentrations of gefitinib and ct-DNA, respectively. Then, Eq. (2) can be translated into Eq. (3).

  1 C G—DNA þ C G;0 C DNA;0 ¼ 0 ðC G—DNA Þ2  C G;0 þ C DNA;0 þ Ka

9.5

lnKb

50

9.0

ð3Þ

8.5

r=0.9389

The solution of Eq. (3) is given by

C G—DNA ¼

8.0

  rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi  2 1 C G;0 þ C DNA;0 þ K a  C G;0 þ C DNA;0 þ K1a  4C G;0 C DNA;0

0.00324

2

0.00336

Fig. 4. Van’t Hoff plot for the gefitinib–ct-DNA complex.

In addition, the absorption of ct-DNA can be ignored at 331 nm. The initial absorbance of gefitinib (A0) can be described by

A0 ¼ eG ½G0

ð5Þ

When the ct-DNA was added into the solution of gefitinib, the absorbance (A) is contributed by the unbinded gefitinib and ct-DNA, that is

A ¼ AG þ AG—DNA ¼ eG ½G þ eG—DNA ½G—NDNA ¼ eG ð½G0  ½G—DNAÞ þ eG—DNA ½G—NDNA ¼ A0  ðeG  eG—DNA Þ½G—DNA

ð6Þ

DA ¼ A0  A ¼ ðeG  eG—DNA Þ½G—DNA ¼ De½G—DNA

ð7Þ

where eG and eG–DNA are the molar extinction coefficient of gefitinib and gefitinib–ct-DNA complex, respectively. Therefore, Eq. (4) can be translated into Eq. (8).

0.12

298 K, r=0.9790 304 K, r=0.9879 310 K, r=0.9908

0.08

ΔA

0.00332 −1

1/T (K )

ð4Þ

0.10

0.00328

0.06 0.04 0.02

DA ¼ De 

  rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi  2 C G;0 þ C DNA;0 þ K1a  4C G;0 C DNA;0 C G;0 þ C DNA;0 þ K1a  2 ð8Þ 5

In this study, the value of CG,0 was 2.5  10 mol/L. The plots of DA versus CDNA,0 were made as shown in Fig. 3. The binding constants (Ka) were calculated by nonlinear fitting and the results were listed in Table 2. The results revealed that it can be found that there are better non-linear relationship between DA and CDNA,0 at the different temperatures, indicating that the stoichiometry of gefitinib–ct-DNA complex is 1:1. The estimated values of Kb were in the order of 103–104 in the range from 298 to 310 K, which fell on the range of the groove binding constant of DNA with small molecule [30,31]. It is further indicated that the binding mode of gefitinib on ct-DNA may be groove binding model. In addition, extensive research results have confirmed that small molecules usually bind to DNA in a non-covalent binding mode [3,31]. For the non-covalent interactions, the main interaction forces are generally classified as hydrogen bonding interaction, van der Waals forces, hydrophobic interaction and electrostatic interaction [32]. However, the signs and magnitudes of the thermodynamic parameters such as enthalpy (DH0), entropy (DS0) and Gibbs free energy (DG0) changes in the binding process of biomacromolecule with small molecule can be used to confirm the binding modes. The thermodynamic parameters in the binding process can be calculated by the van’t Hoff equations [33]:

ln K b ¼ 

DH 0 DS0 þ RT R

0.00 0.000008

0.000016

0.000024

0.000032

CDNA, 0(mok/L) Fig. 3. Plots of DA versus CDNA,0 for gefitinib–DNA complex in Tris–HCl buffer solution (pH = 7.4) at different temperatures. The initial concentration of gefitinib (CG,0) was 2.5  105 mol/L. r is the correlation coefficient.

Table 2 The results of the determination of the apparent binding constants (Kb) at different temperatures and thermodynamic parameters of gefitinib–ct-DNA complex. T (K) 298 304 310 a

Kb (L mol1) 4

1.29  10 6.50  103 5.03  103

DG0exp = RT ln Kb.

DGa (kJ mol1)

DH° (kJ mol1)

DS° (J mol1 K1)

23.4 22.1 21.9

60.4

124.7

DG0 ¼ RT ln K b where R is the gas constant. Therefore, the thermodynamic parameters for the gefitinib–ct-DNA complex can be calculated from the van’t Hoff plot as shown in Fig. 4 and the results were listed in Table 2. The results revealed that the values of DH0, DS0 and DG0 were negative, indicating that the binding interaction between gefitinib and ct-DNA was exothermic and spontaneous process. Ross [34] suggested that the values of DH0 and DS0 are positive, implying that main interaction force is a hydrophobic interaction. The values of DH0 and DS0 are negative reflecting that the interaction force mainly is the van der Waals force and/or hydrogen bond. DH0 is almost zero and DS0 is positive, suggesting that the main interaction force is an electrostatic force. Based on Ross’s viewpoint, it can be further hypothesized that the main interaction forces are van der Waals force and/or hydrogen bonding interaction. And,

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J.-H. Shi et al. / Journal of Photochemistry and Photobiology B: Biology 147 (2015) 47–55

the binding process of gefitinib with ct-DNA is enthalpy-driven due to |DH0| > |TDS0| and DG0 < 0. 3.3. The effects of ionic strength The electrostatic interaction is one of non-covalent binding modes of small molecule on DNA. The stability of folded DNA 0.8

conformations needs interaction with metal cations (such as Na+ and Mg2+) from solution, which the process is referred as the counterion condensation. Specific interaction of organic cationic molecule with DNA neutralizes phosphate charges and results in the release of counterion (such as Na+ and Mg2+). The counterion release provides an entropic contribution to the binding free energy. However, the electrostatic interaction strongly depends on the concentration of salt such as NaCl in the system and is generally weak under physiological conditions, which is often served as an auxiliary mode to assist groove binding and intercalation Fluorescence intensity (a.u.)

0.7 0.6

6

400

0.4

1 0.3 0.2 0.1 0.0 225

250

275

300

325

350

375

400

Fluorescence intensity (a.u.)

Absorbance

0.5

1 300

7

380 360 340 320 570

575

580

585

200

100 λ ex= 465 nm

Fig. 5. Effect of the concentrations of NaCl on the absorbance of gefitinib–ct-DNA complex (CDNA = 5.0  105 mol/L, Cgefitinib = 2.5  105 mol/L). The concentrations of NaCl from 1 to 6 are 0, 1.0  102, 2.0  102, 3.0  102, 4.0  102 and 5  102 mol/L, reaspectively.

0 500

550

600

650

Wavelength (nm) Fig. 7. Fluorescence emission spectra of the mixture solutions of ct-DNA (5  106 mol/L) and rhodamine B (5  106 mol/L) in the presence of gefitinib in Tris–HCl buffer (pH = 7.40). The concentrations of gefitinib from 1 to 7 were 0, 2.5  106, 5.0  106, 7.5  106, 10.0  106, 12.5  106 and 15.0  106 mol/L, respectively.

1.10

1.05

0

400

λem (nm)

Wavelength (nm)

(η/η )1/3

420

Table 4 Fluorescence emission intensions of mixture solutions of ct-DNA (1.50  106 mol/L) and EB (1.54  106 mol/L) at kem = 588 nm in the presence of gefitinib in Tris–HCl buffer (pH = 7.4).

1.00

0.95

Cgefitinib (105 mol/L) Fluorescence intensity (a.u.)

0 122.9

0.40 121.8

0.80 123.0

1.20 122.4

1.60 122.3

2.00 122.2

0.90 0.4

0.6

0.8

1.0

1.2

r =[Gefitinib]/[ct-DNA] Fig. 6. Effect of gefitinib on the viscosity of ct-DNA (2.27  105 mol/L). Table 3 The effect of gefitinib on the fluorescence intensity of fluorescent probes.

0 0.4 0.8 1.2 1.6 2.0 a

10 5

Fluorescence intensity (a.u.) Hoechest-33258a

Rhodamine Bb

EBc

320.9 380.5 440.2 450.9 543.8 617.0

396.25 393.27 398.32 398.82 393.48 396.42

72.72 73.98 73.75 71.20 73.46 75.27

Fluorescence intensity of Hoechest-33258 (2.0  105 mol/L) in Tris–HCl buffer solution (pH = 7.4) at kem = 492 nm in the absence and presence of gefitinib. b Fluorescence intensity of rhodamine B (2.0  105 mol/L) in Tris–HCl buffer solution (pH = 7.4) at kem = 576 nm in the absence and presence of gefitinib. c Fluorescence intensity of EB (3.0  105 mol/L) in Tris–HCl buffer solution (pH = 7.4) at kem = 585 nm in the absence and presence of gefitinib.

CD [medg]

Cgefitinib (105 mol/L)

DNA DNA:Gefitinib=1:0.1 DNA:Gefitinib=1:0.3 Gefitinib

15

free gefitinib

0 -5 -10

CD [medg]

0.2

-5

1

-6

3

-7 -8 -9 235

240

245

250

Wavelength (nm) -15 200 220 240 260 280 300 320 340 360 380 400

Wavelength (nm) Fig. 8. CD spectra of DNA, gefitinib and DNA–gefitinib complex. CDNA = 7.3  105 mol/L, the Cgefitinib from 1 to 3 are 0.0, 7.3  106 and 2.4  105 mol/L, respectively.

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J.-H. Shi et al. / Journal of Photochemistry and Photobiology B: Biology 147 (2015) 47–55

[3,12]. If there is a significant electrostatic binding interaction in the binding process of DNA with small molecules, the strength of total interaction will decreases with the increase of salt concentration in system. The experimental results revealed (Fig. 5) that the absorbance of gefitinib–ct-DNA complex increased with the gradient increase of NaCl, indicating that also plays a role in the gefitinib–DNA binding process. The amplitude of variation resulting from the effect of NaCl on the absorbance of gefitinib–

ct-DNA complex was very little respect to Fig. 2B, implying that there was weak electrostatic binding interaction rather than predominance interaction. 3.4. Viscosity measurements As means for further exploring the binding mode of gefitinib with ct-DNA, viscosity measurements of ct-DNA solution were

Table 5 Various energies in the binding process of gefitinib with DNAs obtained from molecular docking. The unit of all energies was kJ mol1. DNA PDB ID

a b c d e

DG a

E1b

E2c

E3d

2ELG 1K2K

27.7 33.3

39.2 44.7

6.3 1.4

32.9 43.3

463D 1VTJ

38.4 44.0

49.9 55.4

9.6 8.2

40.2 47.3

104D 121D

42.2 46.9

53.7 58.39

6.7 6.8

47.0 51.5

Hydrogen bonding DNA

Gefitinibe

Bond length (Å)

Bond angle (°)

DC9:O2 (Chain B) DC11:O2 (Chain B) DG6:H3 (Chain A) DA4:H3 (Chain A) DA18:H3 (Chain B) DC9:OP1 (Chain A) DA7:H3 (Chain A) DT19:O40 (Chain B) DA18:H3 (Chain B)

H11 H11 O70 O6 N3 H11 O7 H11 O6

1.826 1.895 2.208 1.993 2.180 1.891 2.212 2.045 1.877

177.6 141.7 126.5 127.8 131.5 145.7 132.2 173.6 164.3

DG is the binding free energy change 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. E2 is the electrostatic energy. E3 is the sum of van der Waals energy, hydrogen bonding energy and desolvation free energy. H11 is hydrogen atom linked with N11 as shown in Fig. 1. O6 and O7 are oxygen atoms linked with C6 and C7 atoms, respectively.

Fig. 9. Molecular docking results of gefitinib bound to B-form DNAs: 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 color in this figure legend, the reader is referred to the web version of this article.)

J.-H. Shi et al. / Journal of Photochemistry and Photobiology B: Biology 147 (2015) 47–55

carried out by varying the concentration of gefitinib. It is generally suggested that a classical intercalative binding mode causes a significant increase in viscosity of DNA solution because the classical intercalative interaction requires the space of adjacent base pairs to be large enough to accommodate bound small molecules and elongate the double helix. And, a partial, non-classical intercalation of molecule could bend the DNA helix, reducing its length and its viscosity. In contrast, groove binding or electrostatic interaction causes less pronounced or no changes in DNA solution viscosity [35]. The viscosities of ct-DNA solution (pH = 7.4) in the absence and presence of gefitinib were measured and the results were shown in Fig. 6. The result revealed that the viscosities of ct-DNA solution almost did not change with the increasing concentration of gefitinib, suggesting that the main binding mode of gefitinib with ct-DNA is not the intercalative binding mode. 3.5. Competitive binding experiment As is well known, the binding mode of Hoechst 33258 and rhodamine B on DNA is groove binding as the interaction mode

53

of EB on DNA is intercalation [36–38]. Hoechst 33258, rhodamine B and EB are usually used as fluorescent probes to confirm the binding mode of DNA with small molecules. However, it can be found that the fluorescence intensity of free Hoechst 33258 solution significantly increased with gradually increasing the concentration of gefitinib, indicating that the interaction of Hoechst 33258 with gefitinib exists, while the fluorescence intensities of free EB or free rhodamine B had hardly changed (Table 3) with the addition of gefitinib, suggesting that the interaction of EB with gefitinib can be ignored. So, in this work, EB and rhodamine B were used as fluorescent probes in competitive binding experiments to further confirm the binding mode of gefitinib on ct-DNA. The fluorescence emission spectra of mixture solutions of ctDNA and rhodamine B in the absence and presence of gefitinib were measured and the results were shown in Fig. 7. It can be seen from Fig. 7 that the fluorescence intensity of mixture solution of ctDNA and rhodamine B obviously decreased with gradually increasing gefitinib while there are no phenomena of red- and blue-shift for the emission band of rhodamine B. Meanwhile, the results showed that the fluorescence intensity of mixture solution of EB

Fig. 10. The conformation of gefitinib in gefitinib–DNA complexes.

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and ct-DNA at kem = 588 nm did not almost change with the addition of gefitinib (Table 4), suggesting that there is not competitive binding interaction between EB and gefitinib on ct-DNA. Therefore, it can be concluded that the binding mode of gefitinib on ct-DNA is groove binding interaction. 3.6. Circular dichroism Circular dichroism (CD) is a powerful way in determining the conformation changes of ct-DNA after binding with small molecules and has widely been used to investigate the interaction between small molecules and DNA [39,40]. From Fig. 8, it can be found that there are four main characteristic peaks at 211, 220, 245 and 275 nm, respectively, for the ct-DNA solution, which is consistent with CD of double helix DNA in the B conformation, indicating that the structure of ct-DNA used in this work is a right-hand double helix with the B-conformation. However, the negative band at around 245 nm is assigned to the helix geometry of ct-DNA with B-form as the positive peak at near 275 nm belongs to the base staking of ct-DNA [25]. With the addition of gefitinib, the position of these peaks had no remarkable change, while the intention of the negative band at 245 nm slightly increased and the intention of the positive band almost did not change, suggesting that the conformation of ct-DNA slightly changes after binding gefitinib on ct-DNA but still maintains B-form. And, the slight increase of the negative band intention showed that the interaction of gefitinib with ct-DNA made the double helix structure of ct-DNA become tight [41,42]. 3.7. Molecular docking analysis Molecular docking is an extremely useful tool, which can be used to predict the preferred orientation of one molecule to a second when bound to each other to form a stable complex. Currently, it plays a more and more important role in drugs discovery and development. In this work, the molecular dockings of gefitinib with six B-DNA fragments were performed using AutoDock 4.2 to further clarify the binding mode of gefitinib with B-DNA and to obtain the information about interaction forces between gefitinib and DNA. Gefitinib, kept as flexible molecule, was docked into six types of rigid B-DNA fragments for searching the preferential binding site of gefitinib on B-DNAs and the molecular docking results were listed in Table 5. It can be found from Table 5 that the more adenine (A) and thymine (T) base pairs in DNA sequence is, the more negative the binding energy (DG) is, indicating that gefitinib prefers to bind on the minor groove of A–T rich DNA fragments as shown in Fig. 9, which is consistent with above experimental results. This is because the narrower and deeper shape of the minor groove can offer several sites of action during the binding process of gefitinib with DNAs, resulting in the close contact with the surface of gefitinib through van der Waals forces and hydrogen bonding interaction. Meanwhile, it can be found that the significant change of conformation of gefitinib exists in the gefitinib–DNA complexes to orient easily along the minor groove. The conformation change of gefitinib is close relation with the structure of minor groove of DNA fragments as shown in Fig. 10. It is implied that the flexibility of gefitinib molecule also plays an important role in the binding process of gefitinib with DNA. In addition, it can be found that there are van der Waals, hydrogen bonding and electrostatic interactions between gefitinib and DNAs and the contribution of van der Waals and hydrogen bonding interaction is much greater than that of the electrostatic interaction because the sum of van der Waals energy, hydrogen bonding energy and desolvation free energy is larger than the electrostatic energy (Table 5), which is consistent with the experimental results. It is further indicated that the main interactions in the binding

process of gefitinib with DNA are van der Waals and hydrogen bonding interaction and the electrostatic interaction is an auxiliary interaction to assist groove binding.

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