binding interaction between sorafenib and calf thymus dna: spectroscopic methodology, viscosity...

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2014) xxx–xxx

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Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

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Binding interaction between sorafenib and calf thymus DNA:Spectroscopic methodology, viscosity measurement and moleculardocking

http://dx.doi.org/10.1016/j.saa.2014.09.0561386-1425/� 2014 Elsevier B.V. All rights reserved.

⇑ Corresponding author at: College of Pharmaceutical Science, Zhejiang Univer-sity of Technology, Hangzhou 310032, China. Tel./fax: +86 571 8832 0064.

E-mail address: [email protected] (J.-H. Shi).

Please cite this article in press as: J.-H. Shi et al., Binding interaction between sorafenib and calf thymus DNA: Spectroscopic methodology, viscositsurement and molecular docking, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), http://dx.doi.org/1j.saa.2014.09.056

Jie-Hua Shi a,b,⇑, Jun Chen a, Jing Wang a, Ying-Yao Zhu a

a College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032, Chinab 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

� Sorafenib binds to DNA via minorgroove binding and forms 1:1complex with it.� The main interaction forces were van

der Waals and hydrogen bondinginteractions.� There was slight change of the

secondary structure of DNA due tobinding sorafenib.� The flexibility of sorafenib plays an

important role in increasing thesorafenib–DNA stability.

g r a p h i c a l a b s t r a c t

It was confirmed that sorafenib interacts with ct-DNA via minor groove binding mode through spectro-scopic methods (such as UV–vis absorption spectroscopy, and fluorescence emission spectroscopy) andmolecular doching.

CF3Cl O

O

OHMolecular docking

H H

0.00

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0.30

Abs

orba

nce

wavelength (nm)

1 ( Csorafenib =0)

7 (C sorafenib = 5.52×10 -5 M)

250 300 350 400

400 450 500 550 600 6500

50

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Fluo

resc

ence

inte

nsity

(a.u

.)

Wavelength (nm)

1 ( Csorafenib =0)

6 (Csorafenib=3×10 -5 M)

λ ex = 365 nm

Note: all solutions conclude fixed concentration of DNA and Hoechst 33258

Sorafenib

DNA

Sorafenib - DNA complex

UV-vis absorption spectroscopy

Fluorescence emission spectroscopy

a r t i c l e i n f o

Article history:Received 18 June 2014Received in revised form 27 August 2014Accepted 18 September 2014Available online xxxx

Keywords:SorafenibCalf thymus-DNAUV–vis spectroscopyCircular dichroismFluorescence spectroscopyMolecular docking

a b s t r a c t

The binding interaction of sorafenib with calf thymus DNA (ct-DNA) was studied using UV–vis absorptionspectroscopy, fluorescence emission spectroscopy, circular dichroism (CD), viscosity measurement andmolecular docking methods. The experimental results revealed that there was obvious binding interac-tion between sorafenib and ct-DNA. The binding constant (Kb) of sorafenib with ct-DNA was5.6 � 103 M–1 at 298 K. The enthalpy and entropy changes (DH0 and DS0) in the binding process of sorafe-nib with ct-DNA were –27.66 KJ mol–1 and –21.02 J mol–1 K–1, respectively, indicating that the main bind-ing interaction forces were van der Waals force and hydrogen bonding. The docking results suggestedthat sorafenib preferred to bind on the minor groove of A-T rich DNA and the binding site of sorafenibwas 4 base pairs long. The conformation change of sorafenib in the sorafenib–DNA complex was obvi-ously observed and the change was close relation with the structure of DNA, implying that the flexibilityof sorafenib molecule played an important role in the formation of the stable sorafenib–ct-DNA complex.

� 2014 Elsevier B.V. All rights reserved.

Introduction

Deoxyribonucleic acid (DNA) plays key physiological roles inthe life process because it carries important genetic information

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2 J.-H. Shi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2014) xxx–xxx

and guides the biological synthesis of proteins and enzymesthrough the process of duplication, transportation and translationof genetic information. Small molecules can bind to DNA viacovalent or non-covalent interactions, resulting in alteration orinhibition of DNA function [1,2]. Therefore, the investigation onthe binding interaction of DNA with small molecules is helpful tounderstand the structural features of DNA, origin of some diseasesand action mechanism of some drugs, and design improved drugsthat target cellular DNA. The researches on the binding interactionbetween DNA and small molecules have been considered as one ofthe key topics in the field of life sciences, chemistry and medicine.Currently, the methods used to investigate the binding interactionof DNA with small molecules mainly contain the experimentalmethods [3–7] and the molecular docking methods [8–14].

Sorafenib (Fig. 1), which is an inhibitor of several tyrosine pro-tein kinases and Raf kinases, is used for the treatment of primarykidney cancer and advanced primary liver cancer [15–19]. It hasalso been demonstrated that sorafenib has the preclinical andclinical activity against several tumor types such as renal cell car-cinoma, and non small cell lung cancer [18,20–23]. In addition, Todet al studied that the binding interaction between sorafenib andplasma using the quenching fluorescence method and the influ-ence of albuminemia and bilirubinemia on sorafenib dispositionin cancer patients, suggesting that sorafenib is highly bound toplasma (>99.5%) and the major influence of albuminemia onsorafenib clearance [24]. However, to our best knowledge, thestudy on the intermolecular interactions of sorafenib with DNAusing UV–vis absorption spectroscopy, fluorescence emission spec-troscopy, circular dichroism (CD) and molecular modeling methodshas not been reported.

In this work, the binding interaction between sorafenib and calfthymus DNA (ct-DNA) was studied using UV–vis absorptionspectroscopy, fluorescence emission spectroscopy, circular dichro-ism (CD), viscosity measurement and molecular docking in orderto obtain the detailed information about the binding interactionof sorafenib with ct-DNA such as specific binding site, bindingmodes, binding constant, effect of sorafenib on conformation ofct-DNA, interaction forces, among others. The study of the bindinginteraction of ct-DNA with sorafenib molecule has great signifi-cance in helping to elucidate the mechanism of action andpharmacokinetics.

Materials and methods

Chemical and reagents

The highly polymerized ct-DNA and Hoechst 33258 werepurchased from Sigma Chemical Co., Ltd. and were used withoutfurther purification. Sorafenib tosylate was provided from NanjingAnge Pharmacetutical Co., Ltd. Tris(hydroxymethyl) aminometh-ane (Tris) (>99%) was purchased from Shanghai Bobo biotechnol-ogy Co., Ltd. Other chemicals were of analytical reagent gradeand were used without further purification.

CF3

Cl

N N

O

H H

6

1

23

4

5

78

9 10

1112

15

Fig. 1. The structur

Please cite this article in press as: J.-H. Shi et al., Binding interaction between ssurement and molecular docking, Spectrochimica Acta Part A: Molecuj.saa.2014.09.056

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 solu-tion of ct-DNA was prepared by dissolving of ct-DNA in 0.050 M ofthe Tris–HCl buffer (pH = 7.4). The stock solution of ct-DNA wasstored at 4 �C in the dark for 5 days only and was stirred at fre-quent intervals to ensure the formation of a homogenous solution.The stock solution of ct-DNA gave a ratio of UV absorbance at260 nm and 280 nm of above 1.8, indicating that DNA was suffi-ciently free from protein [3,25]. The final concentration of DNAin the stock solution was determined by UV absorption spectros-copy using the molar absorption coefficient of 6600 M–1 cm–1.

Stock solution of sorafenib (6.28 � 10–5 mol L–1) was preparedin ethanol due to its very low solubility in water. We found thatwhen the concentration of ethanol was lower than 12% the absor-bances of ct-DNA solution and the mixture solution of sorafeniband ct-DNA were about the same, indicating that the concentrationof ethanol lower than 12% did not affect on the conformational ofDNA, which was consistent with the reference reported [26,27],and on the interaction of DNA with sorafenib. Therefore, the finalconcentration of ethanol in the test solution of sorafenib and ct-DNA was controlled at lower than 10% to avoid the conformationalchange of ct-DNA and the effect on interaction of ct-DNA withsorafenib.

UV–vis absorption spectral measurements

The UV–vis spectra for all mixture solutions of ct-DNA andsorafenib were recorded from 230 to 400 nm on UV-1601 spectro-photometer with a 1.0 cm quartz cuvette (Shimadzu corporation,Kyoto, Japan) at different temperatures to quantify the bindingconstants of sorafenib to DNA and to evaluate the effect of temper-ature on binding interaction of DNA with sorafenib. The corre-sponding solution of ct-DNA was measured as reference solution.

Fluorescence emission spectral measurements

The fluorescence emission spectra of all mixture solutions ofHoechst 33258 and ct-DNA in the absence and presence of sorafe-nib were recorded on a F96S Spectrofluorimeter with 1.0 cm quartzcell (Shanghai LengGuang Industrial Co., Ltd., Shanghai, China)from 400 to 600 nm at kex = 365 nm at ambient temperature. Thesefluorescence emission spectra were measured as the average ofthree scans and the appropriate blanks corresponding to the bufferwere subtracted to correct background.

Circular dichroism measurements

Circular dichroism spectra were recorded on JASCO J-815 Spec-trophotometer with 1.0 cm quartz cell (Japan Spectroscopic Com-pany, Tokyo, Japan) at ambient temperature, in which the scanrange was from 200 to 350 nm with an interval of 1 nm at a scanrate of 100 nm min–1. Each spectrum was the average of threescans and was corrected by corresponding buffer blanks.

O

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O

H13

14

16

1718

19

2021

22 2324

e of sorafenib.

orafenib and calf thymus DNA: Spectroscopic methodology, viscosity mea-lar and Biomolecular Spectroscopy (2014), http://dx.doi.org/10.1016/



J.-H. Shi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2014) xxx–xxx 3

Viscosity measurements

Viscosity measurements were performed using Ubbelohde vis-cometer which was thermostated at 298 K in a constant tempera-ture bath and the inner diameter of capillary was 0.57 mm. Theconcentration of ct-DNA in Tris–HCl buffer solution (pH = 7.4) inthe absence and presence of sorafenib was fixed at 1.38 � 10–5 Mand the flow time was measured using a digital stop watch. Themean values of three replicated measurements were used to eval-uate the relative specific viscosity (g/g0)1/3, where g0 and g are thespecific viscosity contributions of DNA in the absence and presenceof sorafenib, respectively.

Molecular docking

The starting geometry of sorafenib (NCBI, CID 216239) wasobtained 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-empiricaltheory at PM3 level and then was optimized by density functionaltheory (DFT) at B3lyp/6-31+g(d,p) level using Gaussian 03 softwareuntil all eigenvalue of the Hessian matrix were positive [28]. Theoptimized structure of sorafenib was used for the moleculardocking calculations.

The ct-DNA is composed of two strands that wrap around eachother to form a right-handed double helix with the B-form. Thecrystal structures of B-DNAs used in molecular docking wereextracted 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 simu-lated by molecular docking method using AutoDock 4.0 program[29]. All of the water molecules were removed from B-DNA. Thepolar hydrogen atoms were added to B-DNAs and the rotatablebonds of sorafenib were set to 6 using Autodock Tools [30,31].The partial atomic charges of DNA and sorafenib were calculatedusing Gasteiger–Marsili [32] and Kollman methods [33], respec-tively. The grid maps of dimensions 60 � 60 � 60 Å with a grid-point spacing of 0.375 Å were created for six B-DNAs to ensurean appropriate size for sorafenib-accessible space. In this work,the centers of grid boxes were set as shown in Table 1. The num-ber of genetic algorithm runs and the number of evaluationswere set to 100 and 2.5 million, respectively. Other miscella-neous parameters were assigned the default values given byAutodock program. Cluster analysis was performed on the resultsof docking by using a root mean square (RMS) tolerance 2.0 Å.Finally, we obtained the dominating configuration of the bindingcomplex of sorafenib with NDA with minimum binding freeenergy (DG).

Table 1Six DNA sequences used for molecular docking.a

DNA ID PDB ID Sequences

1 1D32 D(CGCG)2

2 1ZNA D(CGCG)2

3 1K2J D(CGTACG)2

4 1BNA D(CGCGAATTCGCG)2

5 1DNE D(CGCGATATCGCG)2

6 102D D(CGCAAATTTGCG)2

a These data were extracted from Protein Data Bank (http://www.rcsb.org/pdb/home/


Results and discussion

UV–vis absorption spectroscopy

UV–vis absorption spectroscopy, one of the simplest testingtechniques, has been widely used in the study of the interactionof DNA with small molecules by monitoring changes of UV–visabsorption bands of DNA or small molecules including the hypo-chromic effect, hyperchromic effect, bathochromic effect (red shift)and hypsochromic effect (blue shift). Generally, small moleculebinding on DNA through intercalative interaction results inhypochromism and bathochromism of absorption bands. Becauseintercalative interaction is a kind of stacking interaction betweenbase pair of DNA and chromophore of small molecules, the porbital of base pairs of DNA can couple with the p* orbital of smallmolecules and form p–p* conjugation, resulting in reducing thedifference of the p–p* transition energy, which causes a red shiftof absorption band, and the coupling p* orbital is partially filledby electron resulting in decreasing the transition probability. Thelower the probability is, the smaller the molar absorption coeffi-cient. So, when small molecule intercalated into DNA, the distancebetween the chromophore of small molecule and the base pair ofDNA will decrease, resulting in hypochromism of absorption band[2,34]. In the case of electrostatic attraction between the DNA andsmall molecules, hyperchromic effect can be observed, whichreflects the corresponding changes of the conformation and struc-ture of DNA when the electrostatic interaction of DNA with smallmolecules has occurred [2]. However, in the case of the groovebinding mode between DNA and small molecules, the hypochromiceffect can be observed while the position of the absorption bandalmost does not change, which can be associated with the overlap-ping of the electronic states of the chromophore of the complexwith the nitrogenous bases in the grooves of DNA [35–37].

The UV spectra of sorafenib in the absence and presence of ct-DNA were shown in Fig. 2. It can be seen that there is an absorptionband at 264 nm for the sorafenib solution in the absence of ct-DNA,which belongs to B-band of an aromatic moiety in sorafenib mole-cule. However, the intensity of the absorption band at 264 nmdecreases with the gradient addition of ct-DNA to sorafenib solu-tion, while the position of the absorption band almost does notchange. Based on above viewpoints, it can be concluded that thereis the binding interaction of ct-DNA with sorafenib and the mainbinding mode may be groove binding interaction.

Binding constant and thermodynamic parameters measurements

As is well known, the binding constant (Kb) for the 1:1sorafenib–DNA complex can be calculated according to Benesi-Hil-debrand equation [4]:

Unit cell constants Centers of grid boxes

a = 16.88 Å, b = 26.88 Å, c = 82.60 Å 26.789, 12.727, 10.387a = 90�, b = 90�, c = 90�

a = 31.27 Å, b = 64.67 Å, c = 19.50 Å 18.02, �8.307, 8.794a = 90�, b = 90�, c = 90�

No data 0.234, 2.024, 7.463

a = 24.87 Å, b = 40.39 Å, c = 66.20 Å 14.78, 20.976, 8.807a = 90�, b = 90�, c = 90�

a = 25.48 Å, b = 41.26 Å, c = 66.88 Å 15.296, 21.285, 75.966a = 90�, b = 90�, c = 90�

a = 24.78 Å, b = 41.16 Å, c = 65.51 Å 14.558, 21.582, 75.506a = 90�, b = 90�, c = 90�

home.do). 1ZNA: DNA without gap; 1D32: DNA with two gap.


http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=216239%26loc=ec_rcs

http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=216239%26loc=ec_rcs

http://www.rcsb.org/pdb/home/home.do





250 275 300 325 350 375 4000.00

0.05

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bsor

banc

e

Wavelength (nm)

1

7

Fig. 2. UV absorption spectra of sorafenib (6.28 � 10–6 M) in absence and presenceof 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.

40000 80000 120000 160000

-70

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-20

-10 298 K 303 K 310 K

A 0/[A-A

0]

1/CDNA (L mol-1)

Fig. 3. Plots of A0/(A � A0) versus 1/CDNA for sorafenib–DNA complex in Tris–HClbuffer solution (pH = 7.4) at different temperatures.

0.00324 0.00328 0.00332 0.00336

8.2

8.3

8.4

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8.6

8.7

ln (K

b)

1/T (K-1)

Fig. 4. Van’t Hoff plot for the sorafenib–DNA complex.

0.0 0.1 0.2 0.3 0.40.0

0.1

0.2

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0.5

Abs

orba

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CNaCl (mol/L)

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

A� A0¼ eD

eD�DNA � eDþ eD

eD�DNA � eD� 1

Kb � CDNAð1Þ

where A0 is the absorption of sorafenib at 264 nm in the absence ofct-DNA. A is the corresponding absorbance at different concentra-tion of ct-DNA. eD and eD–DNA are the molar extinction coefficientof free sorafenib and sorafenib–ct-DNA complex, respectively. CDNA

is the concentration of ct-DNA. It can be found from Fig. 3 that thereis a good linear relationship between A0/(A � A0) and 1/CDNA at dif-ferent temperatures, indicating that the stoichiometry of bindinginteractions between DNA and sorafenib is 1:1. The apparent bind-ing constants (Kb) at different temperatures are calculated from

Table 2The results for the apparent binding constants (Kb) of sorafenib–DNA complex and therm

Temperature (K) B–H equations obtained Kb (L mol–1) DG

Slope (�104) Intercept ra

298 –3.10 ± 0.08 –1.73 ± 0.58 0.9983 5.58 � 103 –2303 –3.56 ± 0.09 –1.69 ± 0.68 0.9982 4.75 � 103 –2310 –4.54 ± 0.10 –1.65 ± 0.73 0.9987 3.63 � 103 –2

a r Is the correlation coefficient.b DG0

exp, 1 = –RT ln Kb.c DG0

exp, 2 = DH0 � TDS0.


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 from298 to 310 K, which is significantly lower than that of the classicintercalation binding like EB-DNA complex (Kb = 1.4 � 106 M–1)[38]. However, it falls on the range of the groove binding constantof DNA with small molecule [7,39]. It is further indicated that thebinding mode may be the groove interaction.

In the binding process of biomacromolecule with smallmolecule, there are mainly four types of non-covalent interactionsincluding hydrogen bonding interaction, van der Waals forces,

odynamic parameters.

0exp, 1

b (kJ mol–1) DG0exp, 2

c (kJ mol–1) DH0 (kJ mol–1) DS0 (J mol–1 K–1)

1.37 –21.40 –27.66 –21.021.33 –21.291.13 –21.14




0.0 0.2 0.4 0.6 0.8 1.0 1.20.90

0.95

1.00

1.05

1.10(η

/η0)1/

3

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.

200 220 240 260 280 300 320 340

-6

-4

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0

2

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6

8

CD

[med

g]

Wavelength (nm)

1

2

Fig. 8. The CD spectra of DNA (5 � 10–5 M) in the presence of sorafenib in Tris–HClbuffer solution. The concentrations of sorafenib from 1 to 2 were 0, 3 � 10–5 M,respectively.


hydrophobic interaction and electrostatic interaction [40].Meanwhile, the signs and magnitudes of the thermodynamicparameters (DG0, DH0 and DS0) in the binding process ofbiomacromolecule with small molecule can be used to confirmthe binding modes. These thermodynamic parameters can becalculated by the van’t Hoff equations [41]:

ln Kb ¼ �DH0

RTþ DS0

Rð2Þ

DG0 ¼ �RT ln Kb ð3Þ

where R is the gas constant. DG0, DH0 and DS0 are the changes inGibbs free energy, enthalpy and entropy in the binding process ofbiomacromolecule with small molecule, respectively. It is generallysuggested that both DH0 and DS0 are positive, indicating that themain interaction force is hydrophobic interaction. DH0 and DS0

are negative, suggesting that the main interaction force is van derWaals force and/or hydrogen bonding interaction. DH0 is almostzero and DS0 is positive, implying that the main interaction forceis electrostatic force [41]. For the effect of temperature on theDH0 and DS0 of the interaction of DNA with sorafenib is very smallin the range of temperature studied, the values of DH0 and DS0 canbe regarded as constant. The values of DH0 and DS0 for the

400 450 500 550 600 6500

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λex= 365 nm

Fig. 7. Fluorescence emission spectra of the DNA–Hoechst 33258 in the presence ofsorafenib in Tris–HCl buffer (pH = 7.4). The concentrations of ct-DNA and Hoechst33258 were 1 � 10–5 M, 5 � 10–7 M, respectively. The concentrations of sorafenibfrom 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 linearrelationship between lnKb and the reciprocal absolute temperatureas shown in Fig. 4 and the results are listed in Table 2. It can befound from Table 2 that the values of DG0, DH0 and DS0 arenegative, suggesting that the binding interaction of sorafenib withct-DNA is exothermic and spontaneous process. Moreover, the val-ues 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-drivenand the main interaction forces are hydrogen bonding interactionand van der Waals force.

Binding mode

Extensive research results revealed that small molecules usu-ally bind to DNA in non-covalent way. Moreover, non-covalentinteraction can be classified into three modes: (i) electrostaticbinding; (ii) groove binding; and (iii) intercalative binding [1,2].In order to further elucidate the binding mode of sorafenib withct-DNA, the effect of ionic strength on UV absorption spectra ofmixture solutions of sorafenib and ct-DNA, the effect of sorafenibon the viscosity of ct-DNA solution and the competitive bindingof sorafenib with Hoechst 33258 on ct-DNA were further studied.

The effects of ionic strengthThe electrostatic binding mode is one of non-covalent binding

modes of small molecule on DNA, which is often served as anauxiliary mode to assist groove binding and intercalation. Thesmall molecule that binds strongly to DNA usually includes theelectrostatic component. But, if the electrostatic binding interac-tion plays a dominant role in the binding interaction of DNA withsmall molecules, the strength of interaction will decrease with theincrease of salt concentration in system [1,25]. The experimentalresults showed that the absorbances of sorafenib–DNA solutionsalmost did not change with the increase of the concentration ofNaCl (Fig. 5), indicating that there was no significant electrostaticbinding interaction between ct-DNA and sorafenib.

Viscosity studiesViscosity measurement is often regarded as an effective and

accurate method to determine the binding mode between smallmolecules and DNA. It is generally suggested that a classicalintercalative binding mode causes a significant increase of DNAviscosity because the intercalative interaction requires the spaceof adjacent base pairs to be large enough to accommodate thebound small molecules and elongates the double helix [42,43].However, for the electrostatic or groove binding, there is littleeffect on the viscosity of DNA [42,44]. The viscosities of ct-DNA




Table 3The 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 E2

c 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) H9 2.23DG4: H22 (Chain A) O22 1.80

1K2J –41.34 –48.24 –46.90 –1.34 DC11: O4 (Chain B) H7 1.93DC11: O4 (Chain B) H9 1.92DA4: H3 (Chain A) O8 2.12DG6: H3 (Chain A) O22 1.91DG8: H22(Chain B) N19 2.01

1BNA –45.44 –51.09 –51.00 –0.04 DT8: O2 (Chain A) H7 2.18DT8: O2 (Chain A) H9 1.91DA6: H3 (Chain A) O22 1.94

1DNE –40.63 –48.08 –46.86 –1.21 DA17: H3 (Chain B) O22 1.95DT20: O4 (Chain B) H7 1.82DT20: O4 (Chain B) H9 1.94

102D –42.64 –50.17 –49.37 –0.75 DA6: H3 (Chain A) O22 2.03DT19: O4 (Chain B) H7 1.72DT19: O4 (Chain B) H9 1.88DT21: O4 (Chain B) H23 1.90

a DG is the binding energy in the binding process, which is calculated in water solvent using a scoring function.b 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.

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 yellowand 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.)


Please cite this article in press as: J.-H. Shi et al., Binding interaction between sorafenib and calf thymus DNA: Spectroscopic methodology, viscosity mea-surement and molecular docking, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), http://dx.doi.org/10.1016/j.saa.2014.09.056



Fig. 10. Conformations of free sorafenib (a) and the sorafenib bound with DNA(1BNA) (b) or DNA (1DNE) (c).


in the Tris–HCl buffer solution in the absence and presence ofsorafenib were measured and the results were shown in Fig. 6.As shown in Fig. 6, the viscosities of ct-DNA in the Tris–HCl buffersolution (pH = 7.4) almost do not change with the increasingconcentration of sorafenib, indicating that the binding mode ofsorafenib with ct-DNA is not the intercalative binding mode.Therefore, the binding mode of ct-DNA with sorafenib may bethe groove binding.

Competitive binding of sorafenib with Hoechst 33258To further affirm the binding mode of sorafenib on ct-DNA, a

competitive binding experiment using Hoechst 33258 as probemolecule was performed. Hoechst 33258, which can strongly bindto AT-rich regions of DNA via minor groove binding, is a classicalfluorescent probe [45]. In this work, the fluorescence emissionspectra of the fixed amount of ct-DNA and Hoechst in Tris–HCl buf-fer solution (pH = 7.4) were measured with gradually increasingamounts of sorafenib and the results were shown in Fig. 7. It canbe found that the fluorescence intensities of ct-DNA–Hoechst solu-tions decreased with the gradually increasing concentration ofsorafenib. Meanwhile, it was not observed that sorafenib reactedwith Hoechst 33258 resulting in the change of the fluorescenceintensity of Hoechst 33258. Based on above experimental results,it can be concluded that there is competitive binding between Hoe-chst 33258 and sorafenib on ct-DNA. Therefore, sorafenib binds toAT-rich regions of ct-DNA via minor groove binding interaction.

Circular dichroism studies

As is well known, the circular dichroism (CD) spectroscopy is apowerful way in determining the secondary structure changes ofDNA after binding with small molecules and has widely been usedto investigate the interaction between small molecules and DNA[46–48]. The CD spectra of ct-DNA in Tris–HCl buffer solution inthe absence and presence of sorafenib were shown in Fig. 8. Itcan be seen that there are four main characteristic peaks in therange from 200 to 350 nm for the free ct-DNA solution, whichare two negative peaks at 211 and 244 nm and two positive peaksat 220 and 275 nm, respectively. These bands are consistent withCD spectrum of double helix DNA in the B conformation [47]. How-ever, the negative peak at 244 nm belongs to helical geometry of B-DNA while the positive peak at 275 nm is assigned to stacking ofDNA bases. When sorafenib was added to the ct-DNA solution,the change of CD spectrum of ct-DNA was observed. The intensityof the positive band at near 275 nm decreased, while that of thenegative band at near 244 nm increased. This revealed that theconformation of ct-DNA had slightly changed due to the bindinginteraction between sorafenib and ct-DNA. The increase of the neg-ative peak intensity (244 nm) showed that the interaction ofsorafenib with ct-DNA made the double helix structure of ct-DNAbecome tight [11]. The decrease of the intensity of the positiveband (275 nm) was likely to be due to a transition from theextended nucleic acid right-handed double helix to more compactform (w structure) [49].

Molecular docking

Molecular docking has gained growing interests in theinvestigation of binding interaction mechanism of biological mac-romolecule with small molecules. It plays a more and more impor-tant role in drug discovery and development [8–14]. In this work,the molecular docking of sorafenib with DNA was carried out usingAutodock 4.0 in order to further clarify the binding mode of sorafe-nib on DNA and the binding structure of DNA-sorafenib complex.Sorafenib, kept as flexible molecule, was docked into the six typesof rigid DNAs to search the preferential binding site of sorafenib on


different DNAs and the docking results were listed in Table 3. Gen-erally, the more negative the binding energy is, the stronger theinteraction between small molecule and DNA, the most stablethe complex formed by small molecule and DNA is. From Table 3,it can be found that the binding free energy (DG) is obviously lowerwhen there are adenine (A) and thymine (T) base pairs in the DNAsequences, indicating that the preferential binding site of sorafenibon the A-T rich sequence of DNA. However, sorafenib prefers tobind on the minor groove of A-T rich DNAs, which is consistentwith above experimental results, and the binding site is 4 basepairs long and involves A-T residues as shown in Fig. 9. And, signif-icant change of conformation of sorafenib occurs in the bindingprocess with DNA to orient easily along the minor groove. The con-formation change of sorafenib is close relation with the structure ofDNA minor groove (Fig. 10). In addition, it can be found from thedocking results that there are hydrogen bonding interactionsbetween sorafenib and DNAs (Table 3), suggesting that duringthe binding process of sorafenib with DNA, the narrower and dee-per shape of the minor groove can offer several sites of action,resulting in the close contact with the surface of sorafenib throughvan der Waals forces and hydrogen bonding interaction. This fur-ther illustrates that the main forces of the interaction of sorafenibwith DNAs are hydrogen bonds and van der Waals in binding pro-cess of sorafenib with DNA, which is consistent with the result ofthe thermodynamic parameter analysis, and the flexibility ofsorafenib molecule plays an important role in the binding processof DNA with sorafenib.

From Table 3, it can be also seen that the electrostatic energy isvery much lower than the sum of van der Waals energy, hydrogenbonding energy and desolvation free energy in the binding process





of sorafenib with DNAs, indicating that the main interaction modebetween sorafenib and DNAs is not electrostatic binding mode,which is also consistent with the results of the effects of ionicstrength on the UV–vis absorbance of sorafenib–ct-DNA solution.

Conclusion

In this work, UV–vis absorption spectroscopy, fluorescenceemission spectroscopy, circular dichroism (CD), viscosity measure-ment and molecular docking were carried out to research the bind-ing interaction between sorafenib and ct-DNA. It can be found thatsorafenib interacts with ct-DNA via minor groove binding modewith the binding constant (Kb) of 5.4 � 103 at 298 K. In the bindingprocess of sorafenib with ct-DNA, the main interaction forces werevan-der Waals force and hydrogen bonding force. Additionally, theconformation change of sorafenib is obvious, indicating that theflexibility of sorafenib molecule plays an important role in theformation of the stable sorafenib–ct-DNA complex.

The present study reveals the details of binding affinity, mode ofbinding interaction, main interaction forces of sorafenib withct-DNA and structure of sorafenib–ct-DNA complex. Therefore, thisstudy of the interaction mechanisms of sorafenib with ct-DNAwould provide useful information in further understanding themechanism of action and pharmacokinetics.

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