Spectroscopic and molecular docking studies of binding interaction of gefitinib, lapatinib and sunitinib with bovine serum albumin (BSA)

Journal of Photochemistry & Photobiology, B: Biology 153 (2015) 380–390

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Spectroscopic and molecular docking studies of binding interaction of gefitinib, lapatinib and sunitinib with bovine serum albumin (BSA) Guo-Feng Shen a, Ting-Ting Liu a, Qi Wang a, Min Jiang a, Jie-Hua Shi a,b,⁎ 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 25 July 2015 Received in revised form 26 October 2015 Accepted 27 October 2015 Available online 7 November 2015 Keywords: Gefitinib Lapatinib Sunitinib Bovine serum albumin Binding interaction

a b s t r a c t The binding interactions of three kinds of tyrosine kinase inhibitors (TKIs), such as gefitinib, lapatinib and sunitinib, with bovine serum albumin (BSA) were studied using ultraviolet spectrophotometry, fluorescence spectroscopy, circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR) and molecular docking methods. The experimental results showed that the intrinsic fluorescence quenching of BSA induced by the three TKIs resulted from the formation of stable TKIs–BSA complexes through the binding interaction of TKIs with BSA. The stoichiometry of three stable TKIs–BSA complexes was 1:1 and the binding constants (Kb) of the three TKIs–BSA complexes were in the order of 104 M−1 at 310 K, indicating that there was a strong binding interaction of the three TKIs with BSA. Based on the analysis of the signs and magnitudes of the free energy change (ΔG 0), enthalpic change (ΔH 0) and entropic change (ΔS 0) in the binding process, it can be deduced that the binding process of the three TKIs with BSA was spontaneous and enthalpy-driven process, and the main interaction forces between the three TKIs and BSA were van der Waals force and hydrogen bonding interaction. Moreover, from the results of CD, FT-IR and molecular docking, it can be concluded that there was a significant difference between the three TKIs in the binding site on BSA, lapatinib was located on site II (m) of BSA while gefitinib and sunitinib were bound on site I of BSA, and there were some changes in the BSA conformation when binding three TKIs to BSA but BSA still retains its secondary structure α-helicity. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Serum albumin is the most abundant plasma protein in mammals with a high affinity for a wide range of metabolites and drugs, which plays key physiological roles in transportation, distribution and metabolism of many endogenous and exogenous ligands in vivo [1]. It is generally suggested that the strong binding of drug with serum albumin can decrease the concentration of free drug in plasma, while weak binding can lead to a short lifetime or poor distribution of the drug. Therefore, it is imperative to investigate the binding interaction of drug molecules with serum albumin, which has been an interesting research field in life sciences, chemistry and clinical medicine [2–7]. Recently, the crystal structure of bovine serum albumin (BSA) has been determined and the amino acid sequence shows that BSA is comprised of 583 residues and consists of three homologous αhelical domains (I, II, III). Each domain comprises sub-domain A and sub-domain B. Subdomains IIA, IIIA and IB among them are known as Sudlow's site I, II and III, respectively. BSA has become one of the most extensively studied proteins in evaluating the ⁎ Corresponding author at: College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032, China. E-mail address: [email protected] (J.-H. Shi).

http://dx.doi.org/10.1016/j.jphotobiol.2015.10.023 1011-1344/© 2015 Elsevier B.V. All rights reserved.

interaction of drug with protein due to a similar sequence and configuration with human serum albumin (HSA), clear structure and low cost [8]. Gefitinib, lapatinib and sunitinib, which belong to tyrosine kinase inhibitors (TKIs), are the new targeted anti-tumor drugs in which the mechanism of action is to inhibit cellular pathways used for the cancer cell to grow [9]. Up to now, many studies, both in vivo and in vitro, have mainly focused on its pharmacological effects and clinical application. It has also been demonstrated that these TKIs have a wide range of antitumor activity [10–15]. Previously, we have reported the characterization of interactions between sorafenib and ct-DNA [16], between sorafenib and BSA [17] and between gefitinib and ct-DNA [18] through spectroscopic methods and molecular docking. In view of the above background, the aim of the present work is to delineate the mechanism by which gefitinib, lapatinib and sunitinib interact with BSA, so as to obtain the critical information about the interaction of the three TKIs with BSA such as the quenching mechanism of fluorescence, the specific binding site, the effect of the three TKIs on the micro-environmental and conformational changes of BSA and the interaction forces. To study the binding interactions of the three TKIs with BSA, ultraviolet spectrophotometry, fluorescence spectroscopy, synchronous fluorescence, circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR) and molecular docking methods were

G.-F. Shen et al. / Journal of Photochemistry & Photobiology, B: Biology 153 (2015) 380–390

used in this work. The study of the interaction of the three TKIs with BSA has great significance in helping to clarify the store and transport process of the three TKIs in vivo and the mechanism of action and pharmacokinetics.

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of phenylbutazone (3 × 10−3 mol/L) and ibuprofen (3 × 10−3 mol/L) were prepared in ethanol, respectively. These stock solutions were kept in the dark at below 4 °C. 2.3. UV Spectroscopy Measurements

2. Materials and Methods 2.1. Chemical and Reagents Gefitinib, lapatinib and sunitinib (Fig. 1) (99% purity) were obtained from Guangzhou Eastbang Pharmaceuticals Co., Ltd. Bovine serum albumin (BSA) was purchased from Shanghai Boao Biotechnology Co., Ltd., which was used without further purification. Tris(hydroxymethyl)aminomethane (Tris) (99% purity) was purchased from Shanghai Bobo biotechnology Co., Ltd. Phenylbutazone was purchased from Hubei Hengshuo Chemical Co., Ltd. Ibuprofen was obtained from College of Pharmaceutical Sciences in Zhejiang University of Technology. All other reagents were of analytical grade. The redistilled water was used without further purification.

UV spectra of all BSA solutions in the absence and presence of the three TKIs were recorded on a UV-1601 spectrophotometer (Shimadzu, Kyoto, Japan) equipped with 1 cm quartz cells from 205 to 350 nm at room temperature. The corresponding solution of the TKIs was used as the reference solution. 2.4. Fluorescence Measurements The fluorescence measurements were performed using F96s Spectrofluorimeter (Shanghai Lengguang Technology Co., Ltd., China) with 1.0 cm quartz cell under physiological conditions. The excitation wavelength was set at 289 nm, and the fluorescence emission spectra of all BSA solutions were recorded from 250 to 520 nm using 10/10 nm slit widths at different temperatures (298, 303 and 310 K).

2.2. Sample Preparation 2.5. Synchronous Fluorescence Measurements Tris–HCl buffer solution (pH = 7.40) consisted of Tris (0.050 mol/L) and was adjusted to pH = 7.40 by 36% HCl solution. BSA was dissolved in the Tris–HCl buffer solution (pH = 7.40) with 0.050 mol/L NaCl (to keep the ion strength), in which the concentration of BSA was 1.5 × 10−6 mol/L. The stock solutions of the three TKIs were prepared in ethanol (6 × 10−4 mol/L), respectively. Similarly, the stock solutions

The synchronous fluorescence measurements of all BSA solutions were recorded on Fluoromax-4 Spectrometer with 5/5 slit widths (HORIBA Jobin Yvon, Ltd., Japan) at Δλ = 15 nm and Δλ = 60 nm (Δλ = λem − λex) using a 1.0 cm quartz cell at room temperature. 2.6. Circular Dichroism Measurements The circular dichroism measurements were recorded on a JASCOJ815 Spectrophotometer (Japan Spectroscopic Company, Tokyo, Japan) with 1 cm quartz cell at room temperature. The CD spectra were collected with an interval of 1 nm and with a scan speed of 100 nm/min from 200 to 400 nm. Each spectrum was the average of three scans and was corrected by corresponding buffer blanks. 2.7. FT-IR Measurements FT-IR spectra of all BSA solutions were measured at room temperature on Nicolet 5700 FT-IR spectrophotometer (Thermo Nicolet, America) equipped with a Ge/KBr beamsplitter and a DTGS detector. The sample solutions were placed between ZnS windows. For all spectra 100 scans recorded at 4 cm−1 resolution were averaged. The Nicolet Omnic software (version 7.2) was used for all data manipulation. Firstly, the IR spectrum was analyzed using the self-deconvolution with second derivative resolution enhancement implemented in the Nicolet Omnic software (version 7.2) to characterize each peak position for overlap peak. The data files were transferred to a computer. Then, the IR spectrum was retreated using Gaussian multi-peak fitting implemented in Origin 8.0. The error associated to the calculated percentages of the fitting peak area was lower than 0.1%. 2.8. Molecular Docking

Fig. 1. The structures of gefitinib (a), lapatinib (b) and sunitinib (c).

The starting geometries of gefitinib, lapatinib and sunitinib were constructed using Chem3D Ultra (version 8.0, Cambridgesoft Com., USA). These geometries were firstly treated by energy minimization using MM2 method implemented in Chem 3D Ultra and then optimized by density functional theory (DFT) at 6–311 g ++ (d,p) level implemented in Gaussian 03 until all eigenvalue of the Hessian matrix were positive [19,20]. The structure of BSA (PDB ID: 3V03) is download from the PDB data base (http://www.pdb.org/pdb/home/home.do), which is then optimized through energy minimization in a spherical water box with the radius of 49.1 nm with 0.15 M NaCl to maintain the charge at neutral using the CHARMM 27 force field, Langevin force

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Table 1 The coordinates of centers of grid boxes in the molecular docking process. Domainsa Ligand Gefitinib Lapatinib Sunitinib a b c d

Site Ib

Site II (m)c

Site II (l)d

34.725, 35.225, 45.308 38.454, 37.402, 41.184 39.591, 32.608, 44.539

20.906, 16.818, 42.676 19.580, 13.240, 42.673 19.946, 16.411, 45.007

36.669, 13.936, 61.667 34.686, 10.531, 61.238 40.079, 15.479, 63.442

Domain is a hydrophobic cavity, which can embed almost all of the amino acid residues. BSA is composed of domains I, II and III, and each domain has subunit domains A and B. Site I is one of the binding sites for the drug to bind BSA, which is in the subunit domain IIA of BSA. Site II (m) is one of the binding sites for the drug to bind BSA, which is in the middle subunit domain IIIA of BSA. Site II (l) is one of the binding sites for the drug to bind BSA, which is in the left subunit domain IIIA of BSA.

field and the conjugate gradient method (30,000 cycles) implemented in NAMD 2.9 software from the Theoretical and Computational Biophysics Group (http://www.ks.uiuc.edu/Research/namd/) [21]. And, the temperature, cutoff and time step were set at 310 K, 12.0 and 2.0 fs, respectively. The optimized geometries of gefitinib, lapatinib, sunitinib and BSA were used in the following molecular docking. The intermolecular interactions of gefitinib, lapatinib and sunitinib with BSA were simulated by molecular docking using AutoDock 4.2 software downloaded from the Scripps Research Institute (TSRI) (http:// autodock.scripps.edu/) [22]. The partial atomic charges of BSA and the three TKIs (gefitinib, lapatinib and sunitinib) were calculated using Gasteiger–Marsili [23] and Kollman methods [24], respectively. Moreover, after merging non-polar hydrogen, the rotatable bonds were assigned. The Lamarckian genetic algorithm was selected for the conformational search. The grid maps of dimensions (60 × 60 × 60) with a grid-point spacing of 0.375 Å were calculated using Auto-Grid for the three TKIs, which ensured an appropriate size of the three TKIs accessible space [25]. In this work, the centers of grid boxes were set as shown in Table 1. The running times of genetic algorithm and the evaluation times were set to 100 and 2.5 million, respectively. All other parameters were default settings. In this work, all parameters were the same for each docking. Based on RMS cluster tolerance between structures, the complexes of the three TKIs with BSA were sorted into clusters. Finally, the dominating configurations of the binding complexes of the three TKIs with BSA with minimum binding energy (ΔG 0) were obtained. 3. Results and Discussion

surrounding Trp residues [26], which is often used as a probe to investigate the binding properties of drugs with protein. The fluorescence emission spectra of BSA in the presence of increasing amounts of the three TKIs were shown in Fig. 2. The results revealed that BSA had an intrinsic fluorescence band at 338 nm when exciting at 289 nm. However, with gradual addition of the three TKIs from 0 to 1.8 × 10−6 mol/L, the fluorescence intensity of BSA was obviously decreased along with a slight blue shift (b 1 nm), indicating a change in the environment surrounding Trp residues due to binding to the three TKIs and the energy transfer between the three TKIs and fluorophore (Trp residue for BSA). Therefore, it can be inferred that there are binding interactions of the three TKIs with BSA and the binding site may be located in or near the Trp residues [27]. As is well known, the fluorescence quenching mechanism can be usually classified as either dynamic quenching, caused by collisional encounters, or static quenching, caused by ground-state complex formation between fluorophores and quenchers, respectively [28]. The dynamic quenching and the static quenching can be distinguished by their dependence on temperature and excited-state lifetime. Dynamic quenching constant is expected to increase with increasing temperature because the higher temperature is likely to result in a larger diffusion coefficient and promote the process of electron transfer. In contrast, higher temperature may result in decreasing stability of the complex and thus smaller values of the static quenching constant. To elucidate the mechanism of fluorescence quenching by which the energy was transferred from BSA to TKIs, the quenching experiments were performed at three temperatures (198, 303 and 310 K) and the fluorescence quenching constant (KSV) of BSA is calculated using the wellknown Stern–Volmer equation [28]:

3.1. Fluorescence Quenching Mechanism Tryptophan (Trp) residue in protein has the strongest fluorescence intensity and is most sensitive to changes in the microenvironment

F0− F ¼ K SV ½Q  ¼ kq τ 0 ½Q  F

ð1Þ

Fig. 2. Fluorescence spectra of BSA solution (1.5 × 10−6 mol/L) in the presence of gefitinib (a), lapatinib (b) and sunitinib (c), respectively, (T = 298 K, pH = 7.40, λex = 289 nm). The concentrations of gefitinib, lapatinib and sunitinib from 1 to 7 were 0, 0.3 × 10−6, 0.6 × 10−6, 0.9 × 10−6, 1.2 × 10−6, 1.5 × 10−6 and 1.8 × 10−6 mol/L, respectively. Inset: Stern–Volmer plots for the quenching of BSA induced by the three TKIs.

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Table 2 The quenching constants for the interaction of the three TKIs with BSA in Tris–HCl buffer solution (pH = 7.40) at different temperatures. Stern–Volmer equation Ligand Gefitinib

Lapatinib

Sunitinib

a

T (K) 298 303 310 298 303 310 298 303 310

Intercept

Slope

r2 a

−0.002 ± 0.001 −0.002 ± 0.003 −0.001 ± 0.002 −0.006 ± 0.003 −0.004 ± 0.003 −0.001 ± 0.002 −0.004 ± 0.003 −0.005 ± 0.004 −0.003 ± 0.002

0.0107 ± 0.0002 0.0090 ± 0.0003 0.0078 ± 0.0003 0.0183 ± 0.0004 0.0132 ± 0.0004 0.0086 ± 0.0003 0.0148 ± 0.0004 0.0127 ± 0.0005 0.0106 ± 0.0003

0.9986 0.9927 0.9927 0.9971 0.9950 0.9940 0.9956 0.9925 0.9971

Ksv (×104 L/mol)

kq (×1012 L/mol s)

6.4 ± 0.1 1.9 ± 0.1 1.6 ± 0.1 11.0 ± 0.3 7.9 ± 0.2 3.2 ± 0.1 8.9 ± 0.3 7.6 ± 0.3 6.4 ± 0.2

10.7 ± 0.2 9.0 ± 0.3 7.8 ± 0.3 18.3 ± 0.4 13.2 ± 0.4 8.6 ± 0.1 14.8 ± 0.4 12.7 ± 0.5 10.6 ± 0.3

r2 is the coefficient of determination.

where F0 and F are the steady-state fluorescence intensities in the absence and presence of quencher, respectively, and [Q ] is the initial concentration of quencher. kq is the quenching rate constant of BSA. τ0 is the fluorescence lifetime of BSA in the excited state without any quencher, and its value is about 6 ns [29]. KSV is the Stern–Volmer quenching constant, which can be determined by linear regression of Stern–Volmer equation. The Stern–Volmer plots of the fluorescence of BSA quenched by the three TKIs at different temperatures were shown in Fig. 2 (inset) and the calculated results were listed in Table 2. As shown in Fig. 2 (inset) and Table 2, there were good linear relationships between (F0 − F)/F and the ([Q ]τ0) × 1015 for the three TKIs. The KSV values for the interactions of the three TKIs with BSA at different temperatures were of the order of 10− 4 L/mol and decreased with increasing temperature, indicating that the strong fluorescence quenching was arisen from TKIs binding and the probable quenching process was static quenching mechanism rather than a dynamic quenching mechanism. In addition, the k q values (~ 1012 L/mol s) were 100 times greater than the maximum diffusion collision quenching rate constant (2.0 × 1010 L/mol s) of BSA with a variety of quenchers [30,31], further indicating that the quenching of BSA induced by the three TKIs is not caused by dynamic collision but by the formation of TKIs–BSA complexes. 3.2. Binding Constant and Binding Modes of TKIs–BSA Interaction For the binding interaction of TKIs with BSA, the binding constant (Kb) and the number of binding sites (n) can be determined by the following equation under the assumption that BSA has the independent binding sites [28]:

the static quenching equation log[(F0 − F)/F ] versus log[Q ] as shown in Fig. S1 and the calculated results are listed in Table 3. The results revealed that the values of n at the temperature ranges studied were approximately equal to 1 in the studied temperature range, indicating the existence of just a single binding site in BSA for the three TKIs. The estimated values of Kb were in the order of 104 M−1 at different temperatures, indicating that there was a strong binding interaction of the three TKIs with BSA. Moreover, the Kb values decreased with the increase in temperature, resulting in a decrease in the stability of the three TKIs–BSA complexes. In the binding process of drug with proteins, a drug–protein complex is stabilized through intermolecular interaction forces including hydrogen bonding interaction, van der Waals forces, hydrophobic interaction and electrostatic interaction [32]. However, the signs and magnitudes of the thermodynamic parameters including the free energy change (ΔG 0), the enthalpic change (Δ H 0) and entropic change (Δ S 0) in the binding process of proteins with drug can be used to confirm the binding modes. Based on the thermodynamic view point [33], the three possible binding modes can be (1) the positive values of both ΔH0 and ΔS 0 for hydrophobic interaction, (2) the negative values of both Δ H 0 and Δ S 0 for van der Waals force and/or hydrogen bonding interaction, and (3) the Δ H 0 close to zero and the positive value of Δ S 0 for electrostatic interaction. Then the thermodynamic parameters in the binding process of the three TKIs with BSA can be calculated by the following equations: ln K b ¼ −

Δ H 0 ΔS 0 þ RT R

ð3Þ

ΔG0 ¼ −RT lnK b

F0− F ¼ logK b þ n log½Q : log F

ð2Þ

Therefore, the binding constant (Kb) and the number of binding sites (n) for the interaction of BSA with TKIs can be calculated by the slopes of

ð4Þ

where R is the gas constant and Kb is the binding constant at corresponding temperature. The van't Hoff plots for the binding interaction of the three TKIs with BSA were showed in Fig. S2 and the calculated results were listed in Table 3. The negative values of

Table 3 Binding and thermodynamic parameters for the binding interaction between TKIs and BSA. Ligand

T(K)

log Kb

Kb

n

r2 a

ΔG0 (kJ mol−1)

Gefitinib

298 303 310 298 303 310 298 303 310

4.92 ± 0.09 4.82 ± 0.12 4.69 ± 0.22 5.35 ± 0.08 4.98 ± 0.19 4.43 ± 0.25 5.12 ± 0.10 5.00 ± 0.31 4.80 ± 0.15

8.32 × 104 6.61 × 104 4.90 × 104 2.24 × 105 9.55 × 104 2.69 × 104 1.32 × 105 1.00 × 105 6.31 × 104

1.01 ± 0.02 1.01 ± 0.02 1.00 ± 0.04 1.05 ± 0.01 1.01 ± 0.03 0.95 ± 0.04 1.02 ± 0.02 1.02 ± 0.05 0.99 ± 0.02

0.9987 0.9981 0.9934 0.9992 0.9950 0.9904 0.9986 0.9878 0.9968

−28.1 −28.0 −27.8 −30.5 −28.9 −26.3 −29.2 −29.0 −28.5

Lapatinib

Sunitinib

a

r2 is the coefficient of determination for double-log plots.

ΔH0 (kJ mol−1)

ΔS0 (J mol−1 K−1)

−34

−20

−136

−354

−48

−62

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Fig. 3. Synchronous fluorescence spectroscopy of BSA (1.5 × 10−6 mol/L) under physiological condition (pH = 7.4) in the presence of gefitinib at room temperature when Δλ setting at 60 nm and 15 nm, respectively. The concentrations of gefitinib from 1 to 6 were 0, 0.3 × 10−6, 0.6 × 10−6, 0.9 × 10−6, 1.2 × 10−6 and 1.5 × 10−6 mol/L.

both Δ H 0 and Δ S 0 for the binding interaction of the three TKIs with BSA were observed from Table 3, indicating that the interaction mode for the three TKIs binding on BSA is mainly van der Waals force and/or hydrogen bonding interaction. Moreover, it could be suggested that the binding of the three TKIs with BSA was a spontaneous and enthalpy-driven process due to | ΔH°| N | TΔS°| and ΔG° b 0.

It was obvious that there were a decrease in absorption intensity and a red-shift in the maximum wavelength at near 210 nm with the addition of gefitinib, lapatinib or sunitinib, indicating a change in the framework conformation of BSA and the content of α-helix due to the formation of TKIs–BSA complexes. This result further confirmed that the quenching was mainly arisen by the formation of TKI–BSA complex.

3.3. Conformational Change of BSA Induced by TKIs

3.3.3. Circular Dichroism Spectroscopy Circular dichroism spectroscopy is a powerful method for identifying the conformational changes of protein whose secondary structure may vary due to the interaction of protein with the drug. To prove the possible effect of the three TKIs binding action on the secondary structure of BSA, the CD spectra of BSA in the absence and presence of the three TKIs were measured and shown in Fig. 5 and Fig. S5. As shown in Fig. 5 and Fig. S5, BSA and its complex bound with the three TKIs exhibited two negative absorption bands at near 208 nm and 222 nm, respectively, which are contributed to π → π* and n → π* transfer for the

3.3.1. Synchronous Fluorescence Spectra The synchronous fluorescence can offer characteristic information of the Tyr and Trp residues when the scanning interval Δλ (Δλ = λem − λex) is set at 15 and 60 nm, respectively, which has been used to study the micro-environment of amino acid residues by the determination of the shift of the maximum emission wavelength. Generally, the shift of the maximum emission wavelength reveals the alteration of the polarity microenvironment around Tyr or Trp residues [34]. Generally, the red shift of the maximum emission wavelength implies the decrease of hydrophobicity surrounding Tyr or Trp residue and the increase of the stretching extent of the peptide chain while the blue shift of the maximum emission wavelength indicates the increase in the hydrophobicity surrounding the Tyr or Trp residue and the increase in the folding state for the macromolecule. The results revealed that when values of Δλ were set at 15 and 60 nm, respectively, the intensity of synchronous fluorescence decreased instantaneously upon addition of the three TKIs in agreement with the steady-state fluorescence result (Fig. 3 and Fig. S3). However, the maximum wavelength of the emission peaks of Trp residues (Δλ setting at 60 nm) was a slight blue shift (b1 nm) when adding the three TKIs from 0 to 1.5 × 10−6 mol/L while the maximum emission wavelength of Tyr almost did not change, indicating the slight increase in the hydrophobicity surrounding Trp residues and in the folding state. 3.3.2. UV Spectroscopy UV absorption measurement is a very simple but effective method for exploring the structural change and understanding the complex formation. The UV spectra of all BSA solutions in the presence of TKIs were shown in Fig. 4 and Fig. S4. The results revealed that there were two absorption bands for all BSA solutions in the presence of the three TKIs. The absorption band at near 210 nm, which is connected with α-helix, reflected the framework conformation of BSA. And a weak absorption band at near 280 nm belonged to the π–π* transition of the aromatic amino acids such as Trp, Tyr and Phe residues.

Fig. 4. UV spectra of BSA (1.5 × 10−6 mol/L) in the presence of gefitinib under physiological condition (pH = 7.4) at room temperature. The concentrations of gefitinib from 1 to 7 were 0, 3.0, 6.0, 9.0, 12.0, 15.0 and 18.0 × 10−6 mol/L, respectively.

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Fig. 5. CD spectra of BSA (7 × 10−7 mol/L) in the presence of gefitinib. The concentration of gefitinib was 9 × 10−7 mol/L.

peptide bond of α-helix and are the characteristic bands of the structure of BSA [35]. However, compared with the free BSA, the intensities of these bands for the three TKIs–BSA complexes had a

slight change while the peak position almost did not change, suggesting that the structures of the three TKIs–BSA complexes was predominantly α-helical. And, the contents of α-helical in free

Fig. 6. Second derivative resolution enhancement and curve-fitted amide I region (1700–1600 cm−1) for BSA (0.1 mM) in Tris–HCl buffer solution (pH = 7.40) in the absence (a) and presence of gefitinib (b), lapatinib (c) and sunitinib (d), respectively, at room temperature. The concentration of TKIs was 0.15 mM.

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Table 4 Fraction of secondary structure elements of BSA in the absence and presence of the three TKIs. Target

α-Helix (%)

β-Sheet (%)

β-Turn (%)

β-Antiparallel (%)

Random coil (%)

Free BSA Gefitinib–BSA Lapatinib–BSA Sunitinib–BSA

52.5 44.7 48.6 44.0

7.8 12.4 5.9 10.2

14.8 13.2 4.9 14.3

6.8 11.0 24.5 14.2

18.1 18.6 16.1 17.2

Table 5 Binding constant of the three TKIs with BSA in the presence of site markers at room temperature. Ligand

Site marker

log Kb

r2 a

Gefitinib

Blank Ibuprofen Phenylbutazone Blank Ibuprofen Phenylbutazone Blank Ibuprofen Phenylbutazone

4.92 ± 0.09 4.90 ± 0.17 4.58 ± 0.11 5.35 ± 0.08 2.06 ± 0.05 5.28 ± 0.08 5.12 ± 0.10 5.10 ± 0.10 4.28 ± 0.14

0.9989 0.9960 0.9981 0.9992 0.9986 0.9991 0.9986 0.9987 0.9969

Lapatinib

Sunitinib

BSA and the three TKIs–BSA complexes can be calculated by the following equations [17]:

MRE ¼

ObservedCD ðm degÞ 10C p nl

α−helix ð%Þ ¼

−ðMREÞ208 −4000 CD ðm degÞ  100 33000−4000

ð5Þ

ð6Þ

where MRE is mean residue ellipticity. Cp is the molar concentration of BSA. n is the number of amino acid residues (n = 583, for BSA). l is the path length. MRE208 is the observed MRE value at 208 nm. 33,000 is the MRE value of a pure α-helical at 208 nm. 4000 is the MRE of the β-form and random coil conformation cross at 208 nm. The results revealed that, compared with the free BSA (53.3%), the contents of αhelical for the three TKIs–BSA complexes, which were 52.2%, 52.2% and 51.5% for gefitinib–BSA, lapatinib–BSA and sunitinib–BSA, had a slight decrease. This indicated that the three TKIs bound to BSA may induce some changes in the conformation of the protein. Simultaneously, this result was basically consistent with the results of synchronous fluorescence and UV spectroscopy. In addition, the CD spectrum on the near UV region from 240 to 350 nm is usually representative for the tertiary structure of the protein. From Fig. 5, some weaker character bands in the range from 240 to 340 nm were also observed. Among these bands, the character bands at near 291 and 300 nm belong to the fine bands of tryptophan (Trp) residues, the character bands at near 274 and 281 nm contribute to the fine bands of tyrosine (Tyr) residues, and the bands at near 254, 258 and 267 nm belong to the fine bands of phenylalanine (Phe) residues [36,37]. When adding the three TKIs to the BSA solution, respectively, the slight changes in the position and intensity of these character bands were observed, indicating that the tertiary structure of BSA also slightly changed due to binding of TKIs on BSA.

a

r2 is the coefficient of determination.

3.3.4. FT-IR Measurements Infrared spectroscopy and its derivative methods can be used to characterize the conformational change of protein, which can offer spectral shifting and intensity variations of protein amide I band (mainly C_O stretch) [38]. The amide I band (1700–1600 cm−1) is sensitive to the weak change of secondary structures of protein, which generally used to characterize secondary structures of protein. However, the content of secondary structures can be determined quantitatively by calculation of peak area of amide I band. In order to monitor the intensity variations of amide I band and to further determine the protein secondary structures of BSA in the presence of gefitinib, lapatinib and sunitinib, the FT-IR spectra of amide I band of BSA in the absence and presence of the three TKIs (mainly C_O stretching vibration) were monitored. As shown in Fig. 6, an intense band in the amide I region centered at 1647 cm−1 could be observed in the original FTIR spectra of native BSA without TKIs, indicating that BSA was in a α-helix rich conformation. However, in the presence of gefitinib, lapatinib and sunitinib, there was a slight change in both shape and peak position as compared with that for the native BSA. It might be due to the binding of TKIs towards the C_O, C\\N and N\\H groups of protein. The interactions between TKIs and BSA probably induced the rearrangement of the polypeptide carbonyl hydrogen-bonding pattern. The secondary structures of protein are stabilized by hydrogen bonds between amide C_O and N\\H groups in protein, and the position and the area of the components could reflect the patterns and the strength of the hydrogen bonds [39,40]. Thus, a quantitative analysis of the protein secondary structure for free BSA and the three TKIs–BSA complexes was further carried out through the infrared selfdeconvolution with second derivative resolution enhancement and curve-fitting procedures and the results were shown in Fig. 6 and Table 4. The calculated results showed that free BSA was 52.5% α-helix

Fig. 7. Double-log plots against log[Q] derived from the quenching of BSA by gefitinib (a), lapatinib (b) and sunitinib (c), respectively, in Tris buffer (pH = 7.4) at room temperature in the presence of site markers.

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(1647 cm−1), 7.8% β-sheet (1627 cm−1 and 1618 cm−1), 14.8% turn structure (1669 cm−1), 6.8% β-antiparallel (1681 cm−1), and 18.1% random coil (1635 cm−1). In the presence of the three TKIs, there was a certain decrease in the amount of α-helix from 52.5% (free BSA) to 44.7% (gefitinib), 48.6% (lapatinib) and 44.0% (sunitinib). And, the changes in contents of β-sheet, β-turns, β-antiparallel and random coil were observed, indicating that some α-helix converted into other secondary structures due to TKIs binding on BSA, which was also reflected from CD spectra analysis described.

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Table 6 Energy transfer parameters of the three TKIs–BSA complexes. Ligand

J (×10−15 cm3 L mol−1)

E (%)

R0 (nm)

r0 (nm)

Gefitinib Lapatinib Sunitinib

3.17 1.02 2.46

9.56 14.8 12.2

2.02 1.68 1.94

2.94 2.24 2.69

3.4. Identification of the Binding Site of TKIs on BSA It is well known that there are two major specific drug-binding sites, that are two hydrophobic cavities in subdomains IIA and IIIA, which are defined as site I and site II, respectively. From X-ray crystallographic studies, phenylbutazone has been demonstrated to bind to site I while ibuprofen is considered as site II [41]. In order to investigate the binding site for the three TKIs on BSA, phenylbutazone (as site I marker) and ibuprofen (as site II marker) were used as the site markers in this work. In the competitive experiments of site marker, the three TKIs were gradually added to the co-solution of BSA (1.5 × 10− 6 mol/L) and markers (3 × 10−6 mol/L), respectively, and the double-log plots of the three TKIs quenching effect on BSA fluorescence in the absence and presence of site markers were shown in Fig. 7. The apparent binding constants (Kb) of the three complexes were calculated in the absence and presence of site markers according to Eq. (2) and the results were listed in Table 5. The results revealed that the Kb value of BSA with lapatinib in the presence of the ibuprofen site marker was obviously lower than that in the absence of the site marker, while the Kb value of BSA with lapatinib in the presence of the phenylbutazone site marker showed only a little difference, indicating that there was significant competition binding between lapatinib and ibuprofen. It can be concluded that the binding site of lapatinib may be mainly located within site II of BSA. Similarly, according to the data from Table 5, it can be suggested that the binding site of gefitinib and sunitinib may be mainly located within site I of BSA.

3.5. Energy Transfer from BSA to Three TKIs Förster resonance energy transfer (FRET) is a mechanism describing energy transfer from one donor molecule to another acceptor molecule through nonradiative dipole–dipole coupling, which is widely used to estimate the spatial distances between the donor (protein) and the acceptor (the interacted ligand) and to investigate the structure, conformation, spatial distribution and assembly of complex proteins. Based on Förster's non-radiative energy transfer theory, the energy transfer

Table 7 Energies of the binding complexes obtained from molecular docking.

Gefitinib

Lapatinib

Sunitinib

Fig. 8. Spectral overlaps between the fluorescence emission spectrum (a) of BSA and the absorption spectra (b) of gefitinib (A), lapatinib (B) and sunitinib (C), respectively, at room temperature. The concentrations of BSA and TKIs were 1.5 × 10−6 mol/L.

Binding site

ΔG0 (kJ mol−1)a

ΔE1 (kJ mol−1)b

ΔE2 (kJ mol−1)c

ΔE3 (kJ mol−1)d

Site I Site II (m) Site II (l) Site I Site II (m) Site II (l) Site I Site II (m) Site II (l)

−25.75 −23.36 −13.77 −25.04 −34.67 −15.20 −26.59 −26.08 −23.66

−35.75 −33.33 −23.74 −38.77 −48.40 −28.93 −35.34 −34.83 −32.36

−34.00 −31.44 −22.52 −37.47 −47.65 −28.93 −33.33 −31.69 −29.06

−1.76 −1.88 −1.21 −1.29 −0.75 0.00 −2.01 −3.14 −3.35

a ΔG0 is the binding energy change in the binding process, which is calculated in water solvent using a scoring function. b ΔE1 is intermolecular interaction energy, which is a sum of van der Waals energy, hydrogen bonding energy, desolvation free energy and electrostatic energy. c ΔE2 is the sum of van der Waals energy, hydrogen bonding energy and desolvation free energy. d ΔE3 is electrostatic energy.

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Fig. 9. The conformations of gefitinib–BSA, lapatinib–BSA and sunitinib–BSA complexes with the lowest energy obtained from molecular docking. BSA is represented by the ribbon structure. Gefitinib, lapatinib and sunitinib are represented by sphere model. Inset: TKIs inserting into the hydrophobic cavity in BSA.

efficiency (E) from the donor (BSA) to the acceptor (three TKIs) can be determined by the following equation [42,43]: E ¼ 1−

F R60 ¼ F 0 R 60 þ r 60

ð7Þ

where E is the energy transfer efficiency between the donor and the acceptor. F 0 and F are the donor fluorescence intensities in the absence and presence of an acceptor, respectively. r 0 denotes the distance between the acceptor and the donor. R0 is the Förster distance of the pair of donor and acceptor, i.e. the distance at

Fig. 10. Amino acid residues surrounding gefitinib (a), lapatinib (b) and sunitinib (c) within 8 Å. Gefitinib (a), lapatinib (b) and sunitinib(c) are represented using pink. The hydrogen bonding interactions of gefitinib (a), lapatinib (b) and sunitinib (c) with amino acid residues of BSA are shown as a thin green line.

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which the energy transfer efficiency is 50%, which can be estimated by Eq.(8). R 60 ¼ 8:79  10−25  k  n−4  Φ  J ðλÞ 2

ð8Þ

where k2 is spatial orientation factor of the dipole with k2 = 2/3 for random orientation in fluid solution. n is the average refractive index of the medium in the wavelength range where spectral overlap is significant, and n = 1.336 in the present case. Φ denotes the fluorescence quantum yield of the donor in the absence of the acceptor with Φ = 0.118 for BSA [42]. J(λ) is the spectral overlap integral between the emission spectrum of the donor and the absorption spectrum of the acceptor as shown in Fig. 8 and can be calculated by Eq.(9). Z J ðλÞ ¼

0

∞

F ðλÞ  εðλÞ  λ4  dλ Z ∞ FðλÞ  dλ

ð9Þ

0

where F(λ) denotes the normalized fluorescence intensity of the donor at the wavelength of λ. ε(λ) denotes the extinction coefficient of the acceptor at λ. So, the E, J(λ), r0 and R0 values can be calculated according to Eqs. (7)–(9) and the calculated results are listed in Table 6. As the distance between the BSA (as donor) and the interacted TKIs (as acceptor) is 2–8 nm scale with the rule 0.5R0 b r0 b 1.5R0 [33], it can be deduced that the energy transfer from BSA to the three TKIs occurs with high probability and the distance obtained by Förster resonance energy transfer with higher accuracy. In addition, the value of r0 is larger than R0, further indicating that the fluorescence quenching mechanism of BSA induced by TKIs is static quenching. 3.6. Molecular Docking In order to further determine the preferred binding sites of the three TKIs on BSA and the interaction forces of the three TKIs to BSA, the binding interactions of the three TKIs with BSA were simulated by molecular docking method implemented in AutoDock 4.2. The simulated results were presented in Table 7. The results revealed that the change in the binding free energy (ΔG0) for the binding of lapatinib within the hydrophobic cavity in site II (m) of BSA was more negative than that within the hydrophobic cavities in site I and site II (l), indicating that the binding of lapatinib within site II (m) is energetically favorable. Similarly, it could be deduced that the binding of gefitinib and sunitinib within site I is energetically favorable. This result is consistent with the results observed in the site marker competitive experiments. And, the dominating configurations of binding complexes of BSA with the three TKIs with the lowest binging free energy (ΔGo) were shown in Fig. 9. As shown in Fig. 9, gefitinib, lapatinib and sunitinib molecules insert into the hydrophobic cavities of BSA, respectively. However, gefitinib, lapatinib and sunitinib molecules were surrounded by various kinds of hydrophobic, polar and charge residues and there were hydrogen bonding interactions of gefitinib, lapatinib and sunitinib with some residues (Fig. 10). Meanwhile, as shown in Table 7, the ΔE2 (the sum of van der Waals energy, hydrogen bonding energy and desolvation free energy) is obviously more negative than ΔE3 (electrostatic energy). Therefore, it can be further suggested that the main interactions between BSA and the three TKIs (gefitinib, lapatinib and sunitinib) are van der Waals and hydrogen bonding interactions in nature. This result is in full agreement with one obtained from thermodynamic parameter analysis. 4. Conclusion In summary, the binding interactions between the three TKIs (such as gefitinib, lapatinib and sunitinib) and BSA were researched by

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spectroscopic and molecular docking methods. The following conclusions can be drawn from the above results: (1) The binding interaction of the three TKIs (such as gefitinib, lapatinib and sunitinib) with BSA forms stable complexes with the binding constants (Kb) of more than 104 M−1, resulting in the fluorescence quenching of BSA. That is the fluorescence quenching of BSA induced by gefitinib, lapatinib and sunitinib is the static quenching mechanism. In addition, we found that there is a certain correlation between the times needed to achieve the peak plasma concentration of TKIs and the binding constants of TKIs with BSA. (2) There is a significant difference between the three TKIs in the binding site on BSA. Lapatinib is located on site II (m) of BSA while gefitinib and sunitinib bind on site I of BSA. (3) The binding process of the three TKIs with BSA is spontaneous and enthalpy-driven process. The main interaction forces are van der Waals force and hydrogen bonding interaction. (4) The binding of the three TKIs to BSA induces some change in the BSA conformation and BSA still retains its secondary structure αhelicity.

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