- Email: [email protected]
Study on the interaction of ertugliflozin with human serum albumin in vitro by multispectroscopic methods, molecular docking, and molecular dynamics simulation
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 219 (2019) 83–90
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Study on the interaction of ertugliflozin with human serum albumin in vitro by multispectroscopic methods, molecular docking, and molecular dynamics simulation Wenjing Wang a, Na Gan a, Qiaomei Sun a, Di Wu b,⁎⁎, Ruixue Gan a, Man Zhang a, Peixiao Tang a, Hui Li a,⁎ a b
School of Chemical Engineering, Sichuan University, Chengdu 610065, China Key Laboratory of Meat Processing of Sichuan, College of Pharmacy and Biological Engineering, Chengdu University, Chengdu 610106, China
a r t i c l e
i n f o
Article history: Received 24 January 2019 Received in revised form 15 April 2019 Accepted 17 April 2019 Available online 18 April 2019 Keywords: Human serum albumin (HSA) Ertugliflozin Fluorescence spectroscopy Molecular docking Molecular dynamics (MD) simulation analyses
a b s t r a c t Ertugliflozin is a potent and selective inhibitor of sodium-dependent glucose cotransporters 2 (SGLT2) and used as a monotherapy to improve glycemic control in adult patients with type 2 diabetes. In this study, ertugliflozin binding to human serum albumin (HSA) was investigated by multispectroscopic and computer simulations. The fluorescence spectra demonstrated that the quenching mechanism of ertugliflozin and HSA was static quenching. Thermodynamic parameters indicated that hydrogen bonding and van der Waals forces played a key role in the binding. Fluorescence competition experiments and molecular docking revealed that ertugliflozin bound to HSA sites II. In three-dimensional fluorescence, circular dichroism spectroscopy, and molecular dynamics simulation, ertugliflozin did not affect the basic skeleton structure of HSA but slightly increased the α-helical structure content and changed the microenvironment around amino acid residues. Results provide valuable information on the basis of the interaction of ertugliflozin with HSA. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Ertugliflozin, (1S,2S,3S,4R,5S)-5-{4-chloro-3-[(4-ethoxyphenyl) methyl]phenyl}-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]octane2,3,4-triol (Fig. 1), belongs to a new class of potent and selective sodium-dependent glucose cotransporter 2 (SGLT2) inhibitors and is currently being developed for treatment of type 2 diabetes mellitus [1]. Ertugliflozin was approved by the FDA as a monotherapy on December 22, 2017 and can be combined with sitagliptin or metformin hydrochloride. Ertugliflozin is an oral preparation that enters the bloodstream through the digestive system after oral administration and is transported to the target by plasma proteins. Ertugliflozin has oral bioavailability of approximately 65% and plasma protein binding rate of 94%–96% [2]. Thus, studying the binding of ertugliflozin to plasma protein has important implications for improving its bioavailability and understanding physiological characteristics in human body. Human serum albumin (HSA) is the most abundant protein in plasma and is capable of binding to a large variety of ligands (fatty acids, hormones, amino acids, and drugs). As an important transporter,
⁎ Correspondence to: H. Li, School of Chemical Engineering, Sichuan University, Chengdu, Sichuan, China. ⁎⁎ Correspondence to: D. Wu, Key Laboratory of Meat Processing of Sichuan, College of Pharmacy and Biological Engineering, Chengdu University, Chengdu, Sichuan, China. E-mail addresses: [email protected] (D. Wu), [email protected] (H. Li).
https://doi.org/10.1016/j.saa.2019.04.047 1386-1425/© 2019 Elsevier B.V. All rights reserved.
this binding property of HSA can significantly affect its distribution and efficacy in the body and help or prevent many drugs from reaching their target sites [3]. The interaction between the ligand and HSA is important to understand the pharmacokinetic behavior of the drug in the human body; in this regard, drug–HSA interactions have gained increasing attention, and most studies have focused on the binding sites and modes of drug–HSA [4]. The binding properties of some other diabetes drugs to HSA have been studied, such as metformin [5]. However, the binding properties between ertugliflozin and HSA have not been reported. Studying the binding properties of ertugliflozin and HSA can provide some theoretical insights into the transport in vivo and binding mode of ertugliflozin. Therefore, the present study focused on the interaction between ertugliflozin and HSA. This comprehensive in vitro study evaluates the interaction between ertugliflozin and HSA by using multispectroscopic, circular dichroism (CD), molecular docking, and molecular dynamics (MD) simulation analyses. These methods conform to one another. Fluorescence spectroscopy was used to determine whether ertugliflozin binds to HSA and identify the binding mechanism. The thermodynamic parameters of the ertugliflozin–HSA system and the binding types involved were obtained based on the fluorescence experiments. In addition, fluorescence competitive studies were performed to determine binding sites and molecular docking simulations were conducted for validation. The effects of ertugliflozin on HSA conformation and amino acid residue microenvironment were investigated by CD and 3D fluorescence
84
W. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 219 (2019) 83–90
At room temperature, the 3D fluorescence spectra of the solution of HSA (2 μM) and ertugliflozin–HSA ([ertugliflozin]/[HSA] = 30:1) were measured. The excitation wavelength was set to 200–400 nm, and the emission wavelength range was 200–500 nm. The wavelength increments were both 5 nm. The possible binding positions were determined by fluorescence competitive studies. DNSA and DP are known to bind to Site I and Sites II of HSA [6,7], respectively. Different concentrations (0, 140, 240, 320 and 400 μM) of ertugliflozin solution were gradually added into the test solutions, in which the final concentrations of HSA (2 μM) and DNSA/DP (20 μM) were unchanged. The excitation wavelengths for DNSA and DP were 326 and 375 nm, respectively, and the fluorescence spectra of DNSA/DP-HSA were recorded at 298 K. Theoretically, fluorescence quenching has various causes, including the inner-filter effect, which refers to the absorption of radiation that enters (excitation) or emits from (emission) fluorophores. The innerfilter effect should be corrected by the following equation to increase the reliability of fluorescence data [8]: Aex þAem 2
F corr ¼ F obs e
;
ð1Þ
where Fcorr is the corrected fluorescence intensities, and Fobs is the fluorescence intensities obtained in the experiment. The system absorption at the excitation (Aex) and emission (Aem) wavelengths were measured using a TU-1901 UV–Vis spectrophotometer (Persee, Beijing, China). 2.3. Fluorescence lifetime Fluorescence lifetime was measured by a Jobin Yvon Fluorolog-3 spectrofluorometer (Horiba, LesUlis, FRA) at λex = 280 nm and λem = 337 nm. The HSA concentration was 2 μM, and ertugliflozin were 80 and 140 μM. Fig. 1. Molecular structure of ertugliflozin.
spectroscopy. These experimental results were verified by molecular docking and molecular dynamic simulation. This study will help us to understand the binding mechanism between ertugliflozin and HSA through experiment and simulation in vitro.
2.4. CD spectroscopy
2. Experimental
A Chirascan-plus circular dichroism spectrometer (Applied Photophysics, Surrey, UK) was used to record the CD spectra. The mixed solution contained HSA (2 μM) and ertugliflozin (0, 80, and 140 μM). The spectra of 190–280 nm was acquired with a step size of 0.2 nm and a bandwidth of 1 nm. Each sample was scanned three times to calculate the mean for plotting and data analysis.
2.1. Materials
2.5. Molecular docking studies
HSA was purchased from Sigma Chemical Company (St. Louis, USA) and prepared at 20 μM in phosphate buffer saline (PBS, containing 0.1 M NaCl). Ertugliflozin was obtained from Shanghai Rongtai Pharmatech Co., Ltd. (Shanghai, China). The 5-dimethylaminonaphthalene-1sulfonamide (DNSA) was acquired from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Dansyl-L-proline (DP) was supplied by Heowns Biochemical Technology Co., Ltd. (Tianjin, China). Ertugliflozin, DNSA, and DP were dissolved in ethanol at 10 mM. All of the other reagents were of analytical grade without further purification. PBS (pH 7.4) was used to dilute the solution.
Simulation of the binding sites of ertugliflozin at HSA was conducted by YASARA package (v18.3.23) using VINA docking method [9]. The HSA crystal structure was obtained from the Protein Data Bank (PDB ID: 1H9Z). The 3D ligand structures of ertugliflozin were downloaded from PubChem (PubChem CID: 44814423). HSA was pretreated by removing water molecules, adding missing hydrogen atoms and distributing the Gasteiger charges. Other parameters were set to their default values. The ligand was bound to site I and site II by local docking. The docking model with the highest binding energy was selected as the optimal configuration for analysis.
2.2. Fluorescence spectroscopy
2.6. MD simulation
A Cary Eclipse fluorophotometer (Varian, California, USA) was used to record the fluorescence spectra. The slit widths of the fluorescence excitation and emission were set to 5 and 10 nm, respectively, and the excitation wavelength was 280 nm. The fluorescence emission spectra of 290–500 nm were measured, and the measured temperatures were 298, 304, and 310 K. The HSA solution was diluted to 2 μM, and the final concentrations of ertugliflozin were from 0 μM to 140 μM. All solutions were incubated for half an hour before measuring to achieve a steady state.
MD simulations were performed in YASARA v18.3.23 [10], and the force field was AMBER14. A series of preparations, including hydrogenation, optimization, and calibration, was carried out prior to the simulation. The AM1-BCC model was used to calculate the partial atomic charge of the ligand. After the minimization of the system initial energy, the pH was set to 7.4 and the operating temperature was constant at 298 K for MD simulation. The complex was placed in a water box with a size of 100.04 Å × 100.04 Å × 100.04 Å along the x-, y-, and z-axes. Periodic boundary conditions were then applied. Counter ions were
W. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 219 (2019) 83–90
85
randomly added by replacing water molecules with Na+ or Cl− to provide a charge–neutral system. The Particle-Mesh Ewald algorithm and a cut-off of 8.0 Å were applied to calculate the Coulomb forces. The simulation time steps of the intramolecular force and the intermolecular force were 1.25 fs and 2.50 fs, respectively [11]. Data were recorded every 10 ps, and data analysis could be conducted after 5.0 ns, with a total running time of 50 ns.
ertugliflozin and HSA result in an increased hydrophobicity and a reduced polarity of amino acid residues. In general, the following are the two fluorescence quenching mechanisms: dynamic quenching and static quenching, which can be distinguished by the relationship between quenching constant (Ksv) and temperature. The Stern–Volmer equation was used to calculate the relevant data as follows [17]:
3. Results and discussion
F0 ¼ 1 þ K SV ½Q ¼ 1 þ kq τ0 ½Q ; F
ð2Þ
3.1. Fluorescence quenching analysis In the excited state, HSA will produce a certain intensity of fluorescence due to the presence of three fluorophores, namely, tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) residues [12,13]. The intensity of the HSA emission spectra depends mainly on Tyr and Trp. Many small molecular ligands can alter the microenvironment around the HSA chromophore, ultimately leading to changes in the intrinsic fluorescence intensity of HSA [14]. The fluorescence spectra of HSA in the presence and absence of different concentrations of ertugliflozin at 298 K are shown in Fig. 2(A). Notably, ertugliflozin itself has no fluorescence. With the addition of ertugliflozin concentration, the fluorescence intensity of HSA gradually decreases, that is, fluorescence quenching occurs, and a blue shift exists. Usually, the blue shift of the maximum emission peak (λmax) of the protein means that the amino acid residues are in a hydrophobic environment, whereas the red shift of λmax reflects an increase in the polarity of amino acid residues [15,16]. The result testified that ertugliflozin bound to HSA and the combination of
where F0 and F are the fluorescence intensities of HSA in the absence and presence of ertugliflozin, respectively; [Q] is the quencher concentration; kq is the biomolecular quenching constant, and τ0 is the HSA lifetime in the absence of the quencher (τ0 = 5.520 × 10−9 s). Fig. 2(B) shows the Stern–Volmer curve of ertugliflozin–HSA at different concentrations. In dynamic quenching, intermolecular collision occurs between the quencher and fluorescence that ultimately results in an increase of Ksv with the increase of temperature. In addition, in static quenching, the ground state complex is formed by the weak interaction between the ertugliflozin and HSA, leading to decreased Ksv with the increase of temperature [18,19]. As shown in Fig. 2(B) and Table 1, Ksv is negatively correlated with temperature, and kq was much greater than the maximum scatter collision quenching constant (2 × 1010 L mol−1 s−1) of quenchers with biopolymer. Therefore, the fluorescence quenching of HSA caused by ertugliflozin belonged to static quenching, manifesting that HSA and ertugliflozin formed an unstable complex.
Fig. 2. (A) Fluorescence emission spectra of HSA in the presence and absence of various ertugliflozin concentrations at 298 K. CHSA = 2 μM, Certugliflozin = 0 (a), 20 (b), 40 (c), 60 (d), 80 (e), 100 (f), 120 (g), and 140 (h) μM. (B) Stern–Volmer curve of ertugliflozin–HSA system at different temperatures. (C) Plot of log(F0 − F)/F vs. log[Q] for the ertugliflozin–HSA system at three different temperatures. In (B) and (C), the concentrations of HSA and ertugliflozin were the same as the fluorescence emission spectra. (D) Fluorescence lifetime curves of ertugliflozin– HSA systems in the presence of various concentrations of ertugliflozin. CHSA = 2 μM, Certugliflozin = 80 and 140 μM.
86
W. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 219 (2019) 83–90
Table 1 Stern–Volmer quenching constants of the ertugliflozin–HSA system at different temperatures. T(K)
Ksv(×103 M−1)
kq(×1011 M−1 s−1)
Ka(×103 M−1)
n
298 304 310
4.566 ± 0.175 4.304 ± 0.152 4.127 ± 0.266
8.272 ± 0.175 7.797 ± 0.152 7.476 ± 0.266
1.914 ± 0.464 1.225 ± 0.189 0.575 ± 0.205
0.894 0.849 0.770
exposed to water. However, the average fluorophore lifetime (τav) can be expressed in terms of fractional contribution (fi) of each decay time to the steady state fluorescence intensity as [23]: X τav ¼
X
f i τi ¼ X
α i τ2i
i
i
αi τi
:
ð5Þ
i
For static quenching, assuming that the ligand has n binding sites with the protein, the fluorescence intensity of the ligand–protein system, the ligand concentration, the binding constant (Ka), and the number of binding sites (n) satisfy the modified Stern–Volmer equation [20]: logð F 0 − F Þ ¼ logK a þ nlog½Q : F
ð3Þ
Table 1 shows the values of Ka and n. Fig. 2(C) is obtained by plotting the curve of log(F0 − F)/F vs. log[Q] for the ertugliflozin–HSA system. Ka decreased with increasing temperature, showing that temperature reduced the binding between HSA and ertugliflozin. The n value ranging from 0.7 to 0.9 in Table 1 suggested approximately one binding site between the HSA and ertugliflozin.
Remarkably, the τav values with different concentrations of ertugliflozin are almost constant within a certain range. The result testified that the quenching mechanism between HSA and ertugliflozin was static quenching. 3.3. Thermodynamic parameters and binding modes Four atomic affinities, namely, hydrogen bonding, van der Waals forces, hydrophobic interactions, and electrostatic interactions, are observed between the HSA and ertugliflozin. Usually, the thermodynamic parameters of the ligand–protein system, such as enthalpy change (△H), entropy change (△S), and Gibbs free energy (△G), are calculated by the Gibbs–Helmholtz equation (Eq. (6)) and the Van't Hoff equation (Eq. (7)). The main binding forces of ligand–protein system were also judged by their numerical magnitude as positive or negative. △G ¼ △H−T△S;
3.2. Fluorescence lifetime analysis Static quenching and dynamic quenching differ in the changes of fluorescence lifetime, which can be used to distinguish between the two quenching mechanisms. For dynamic quenching, intermolecular collisions reduce the fluorescence lifetime of the protein, whereas in static quenching, ligands and proteins form unstable complexes that do not result in reduced fluorescence lifetime [21]. Fig. 2(D) shows the fluorescence lifetime curves of ertugliflozin–HSA systems in the absence and presence of various concentrations of ertugliflozin. As can be seen from the figure, the fluorescence lifetime of HSA did not change before and after the addition of ertugliflozin, illustrating the quenching mechanism of ertugliflozin–HSA system involving static quenching. The initial fluorescence lifetime data were processed by biexponential iterative fitting, and the results are listed in Table 2. Eq. (4) was used to calculate the mean fluorescence lifetime (bτN) [22]:
ln K a ¼ −
ð6Þ
ΔH ΔS þ : RT R
ð7Þ
ΔH and ΔS were obtained from the slope and the intercept of the Van't Hoff plot, respectively. Ross et al. [24] proved that △H b 0 and △S N 0 refer to electrostatic interactions, △H N 0 and △S N 0 manifest as typically hydrophobic interactions, and △H b 0 and △S b 0 indicate hydrogen bonding and van der Waals forces. The thermodynamic parameters of HSA and ertugliflozin are shown in Table 3. △G b 0 meant that the combination of HSA and ertugliflozin was spontaneous. △H and △S are negative, confirming that hydrogen bonds and van der Waals forces play a major role in the bonding, and △H b 0 suggested that the temperature rise was not conducive to the bonding reactions. This finding was consistent with the conclusion of Section 3.1. 3.4. Analysis of HSA conformational changes caused by ertugliflozin
bτ N ¼ α 1 τ 1 þ α 2 τ2 ;
ð4Þ
where α1 and α2 are the pre-exponential factors, and τ1 and τ2 are the decay times. Chi-square (χ2) was used to evaluate the performance of the fitting results. If χ2 b 1.3, the fitting results were within an acceptable tolerance interval. As can be seen from Table 2, with the increase of ertugliflozin, the HSA lifetime decreased slightly. This result was inconsistent with that of the fluorescence quenching analysis. In general, the conformational heterogeneity of protein structures, the interaction of fluorophores with water molecules, and the interaction of the Trp with nearby amino acid residues may alter the fluorescence lifetime of HSA. Although insulating the specific cause of increased lifetime is difficult, in this study, the most likely cause of a slight increase in fluorescence lifetime is the change in HSA secondary structure (see Section 3.4 for details) to increase the concentration of fluorophores Table 2 Fluorescence decay fitting parameters of ertugliflozin–HSA in the presence of various HSA concentrations. Sample
τ1(ns)
τ2(ns)
α1
α2
Free HSA HSA -40ertugliflozin HSA-70ertugliflozin
2.499 2.527 2.741
6.565 6.706 6.776
0.257 0.301 0.358
0.743 0.699 0.642
bτN(ns) 5.520 5.448 5.331
The effects of ertugliflozin on HSA conformation were investigated by 3D fluorescence spectroscopy and CD spectroscopy. The results of these three methods are mutually verified to improve the reliability of the experimental results. 3.4.1. CD spectroscopy CD is another indispensable mean widely used to study protein conformational changes and qualify the amount of α-helix [25,26]. The CD spectra of HSA have two negative characteristic peaks at 208 and 222 nm, which are related to the α-helix-rich secondary structure of HSA. As shown in Fig. 3, with the addition of ertugliflozin solution, the characteristic peak intensity of HSA increased, but the peak type and peak position did not change significantly. This phenomenon explains that the binding of ertugliflozin and HSA did not destroy the α-helix structure of protein. The change in the α-helix content caused by Table 3 Thermodynamic parameters of the ertugliflozin–HSA system at different temperatures.
τav
χ2
T(K)
△H(kcal/ mol)
△G(kcal/ mol)
△S(cal mol−1 K−1)
6.092 6.123 6.033
1.256 1.197 1.109
298 304 310
−77.12 −77.12 −77.12
−18.72 −17.97 −16.38
−195.5 −195.5 −195.5
W. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 219 (2019) 83–90
87
changes. Fig. 4 is 3D fluorescence spectra of free HSA and ertugliflozin–HSA system. Peak A is the Rayleigh scattering peak, whereas peak 1 and peak 2 are the characteristic signals of residues and polypeptide backbone structures in HSA [31,32]. The 3D fluorescence spectral parameters of HSA alone and in the presence of ertugliflozin are listed in Table 4. Notably, the fluorescence intensity of peak A slightly increased because of ertugliflozin addition. A possible reason for this phenomenon may be that ertugliflozin addition increased the HSA diameter. This phenomenon enhanced the Rayleigh scattering peak. Both the intensities of peak 1 and peak 2 diminished, indicating that the binding of ertugliflozin to HSA affected the microenvironment around the Tyr and Trp residues [33]. Based on the above results, in the presence of ertugliflozin, although the basic skeleton structure of HSA does not change significantly, this condition caused the disturbance of the polypeptide skeleton, the slight increase of the α-helical structure and the change of the microenvironment around the amino acid residues. Fig. 3. CD spectra of HSA in the absence and presence of ertugliflozin. CHSA = 2 μM, Certugliflozin = 80 μM.
ertugliflozin addition can be calculated by the mean residue ellipticity (MRE) at 208 nm, shown as follows [14,27]: MRE208 ¼
ObservedCDðmdeg Þ ; C p nl 10
α−helixð%Þ ¼
−MRE208 −4000 100; 33000−4000
ð8Þ
ð9Þ
where Cp is the molar concentration of HSA in solution; n is the number of amino acid residues (585) in HSA, and l is the path length of the cell (0.1 cm). By calculation, the α-helix (%) of free HSA was 48.64%, which agreed with about 50% obtained in other reports [28–30], and after adding 80 μM ertugliflozin, the α-helix (%) of HSA became 52.83%. An increase of the α-helix structure revealed that ertugliflozin made the HSA conformation changed. 3.4.2. 3D fluorescence spectroscopy Another conformation investigation experiment was undertaken using 3D fluorescence spectroscopy. The 3D fluorescence spectroscopy is the most effective way to characterize protein conformational
3.5. Site-selective binding of ertugliflozin on HSA HSA consists of 585 amino acid residues, and its crystal structure contains three helical domains (I–III) with similar structure; each of which is divided into A and B subdomains [34]. HSA can be combined with a variety of endogenous ligands, such as non-esterified fatty acids, bilirubin, hemin, and thyroxine. Sudlow et al. [6,7] found that when many endogenous and exogenous compounds in the blood stream bind to HSA, they are usually located in one of the two major sites in subdomains IIA and IIIA, namely, site I and site II. Site I is a binding pocket within the core of subdomain IIA, including all six helices of subdomain IIA and a loop-helix feature of subdomain IB, and site II has six helices of subdomain IIIA. Site II is smaller than site I, but due to the rotation of subdomain IIIA, its pocket entrance is more susceptible to exposure to solvent [34]. DNSA and DP are two common probes that bind to site I and site II, respectively, and their fluorescence intensities increase when they bind to HSA. According to Sudlow et al. [6], by measuring the fluorescence of the DNSA/DP–HSA system in the absence and presence of different concentrations of ligand and then calculating the percentage of probe displacement (I), the drug binding site can be predicted as follows:
I¼
F1 100%; F2
Fig. 4. 3D Fluorescence spectra of free HSA (A) and ertugliflozin-HSA system (B). CHSA = 2 μM, Certugliflozin = 60 μM.
ð10Þ
88
W. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 219 (2019) 83–90
Table 4 3D fluorescence spectral parameters of HSA alone in the presence of ertugliflozin. System HSA
Ertugliflozin-HSA (30:1)
Peak no. Peak position [λex/λem(nm/nm)] A 1 2 A 1 2
280/280 → 350/350 280/335 225/334 280/280 → 350/350 280/329 225/322
Stokes shift Intensity △λ(nm) 0 55 109 0 49 97
189.42 → 241.37 571.10 712.88 172.65 → 275.88 500.64 267.71
where F1 is the fluorescence intensity of the probe-HSA in the absence of ligand, F2 is the fluorescence intensity of the probe-HSA in the presence of various concentrations of ligand. The results of the fluorescence competition experiment are shown in Fig. 5. Free DP has low fluorescence intensity. After adding HSA to DP, the maximum emission wavelength of DP has a significant blue shift, and the fluorescence intensity is significantly higher than without HSA. Ertugliflozin addition reduced the fluorescence intensity of the DP–HSA complex. Upon addition of 400 μM ertugliflozin, the fluorescence intensity of DP decreased to approximately 74% of the initial intensity. This result suggests that both DP and ertugliflozin competed for site II in HSA, and DP was replaced by ertugliflozin from HSA. However, ertugliflozin minimally affected the fluorescence intensity of DNSA–HSA, explaining that ertugliflozin did not bind to site I or ertugliflozin had significantly less binding ability to site I than DNSA. 3.6. Analysis of molecular docking results Molecular docking is considered an important method to explore the binding sites and binding energies of small molecule on HSA, which is often corroborated by fluorescence competitive study. The binding energy in YASARA program is defined by the following formula [35]: Binding energy ¼ Receptor energy þ Ligand energy−Complex energy:
ð11Þ
According to Eq. (11), great amount of positive energy manifests good combination. When ertugliflozin binds to site I, the maximum binding energy was 9.26 kcal/mol, and when ertugliflozin binds to site II, the maximum binding energy was 9.63 kcal/mol. Therefore, the perfect binding site of ertugliflozin on HSA was site II. This finding was consistent with the result of the fluorescence competition experiment.
Fig. 5. Fluorescence spectra of DP–HSA in the absence and presence of ertugliflozin. CHSA = 2 μM, CDNSA/DP = 20 μM, Certugliflozin = 0 (a), 140 (b), 240 (c), 320 (d), and 400 (e) μM. The inset shows the effect of ertugliflozin on the fluorescence of DNSA/DP–HSA systems.
Fig. 6. Best docked result of the ertugliflozin–HSA system.
Fig. 6 shows that ertugliflozin was mainly surrounded by ARG485, ARG348, VAL344, LEU453, LEU457, PHE488, SER489, TYR411, LEU430, ALA449, ILE388, LEU387, and PRO384 when bound to HSA site II. The docked result means that ertugliflozin and these active amino acids maintain the stability of the binding conformation mainly by the hydrophobic interaction (the red dotted line in the figure) and hydrogen bonding force (the green dotted line in the figure and the bond lengths are 3.12 and 3.06 Å, respectively). 3.7. MD simulation MD simulation can further explore the stability and dynamic behavior of the ertugliflozin–HSA complex under simulated physiological conditions. The root mean square deviation (RMSD), radius of gyration (Rg), and the percentage of secondary structure, shown in Fig. 7, were measured by YASARA. The RMSD curve fluctuated to some extent as a whole, testifying that the HSA skeleton structure has changed during the simulation. However, after 10 ns, the curve tends to be horizontal, indicating that the system reached equilibration [36]. Thus, ertugliflozin can stably bind to site II. Rg represents the HSA compactness. A higher Rg value indicates a looser structure in a specific region of the protein [37]. Fig. 7B shows the initial Rg values of the free HSA and ertugliflozin–HSA complexes are both approximately 28.20 Å. During the MD simulation, the Rg value of free HSA decreased slightly, whereas the Rg value of the ertugliflozin–HSA complex was higher than that of free HSA, revealing that the combination of ertugliflozin and HSA made HSA more unconsolidated and changed the secondary structure of HSA. By extracting the free HSA and complex conformation of 30 ns (Fig. 8), it can be clearly seen from the circled part in the figure that the protein cavity becomes larger and the structure of HSA becomes more unconsolidated after ertugliflozin bound to site II. Fig. 7C and D show that the graphs of the secondary structure content of free HSA and ertugliflozin–HSA complexes. The main secondary structure of HSA is α-helix. During the MD simulation, the content of each secondary structure remained stable with only slight changes. The mean values of α-helix and turn of free HSA were 70.53% and 20.42%, respectively. This result was slightly higher than the crystallographic result (67% αhelix [38]). Because in the process of dynamic simulation, the trajectory of HSA will constantly expand, contract and slowly move, which will
W. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 219 (2019) 83–90
89
Fig. 7. RMSD (A) and Rg (B) of ertugliflozin–HSA system. Secondary structure percentages of HSA in the presence (C) and absence (D) of ertugliflozin.
also lead to the change of helix proportion. However, the mean values of α-helix and turn of ertugliflozin–HSA complex were 71.86% and 19.46%, and the increasing trend of α-helix was in line with the CD results. The number of hydrogen bonds in the ertugliflozin-HSA system was analyzed as shown in Fig. 9. MD simulation is a dynamic process, so the number of hydrogen bonds varies between 0 and 3. After 10 ns, the system reached equilibrium, and the number of hydrogen bonds was mainly 2, indicating that hydrogen bonds played a certain role in maintaining the stability of the system, which was consistent with the two hydrogen bonds shown in the docking results. The results are consistent with the experimental results, proving that adding ertugliflozin did not significantly change the HSA skeleton
but slightly affected the secondary structure content and there were two hydrogen bonds between ertugliflozin and HSA. 4. Conclusion This study presents the first exploration of the interaction between ertugliflozin and HSA through experiments and simulations. The experimental results were consistent with the simulation results. Notably, the fluorescence quenching mechanism of ertugliflozin and HSA was static quenching, which was spontaneously bound to HSA site II by hydrogen bonding and van der Waals force, and the binding was stable. After combining with ertugliflozin, the HSA diameter increased, and the α-helix
Fig. 8. Structure of free HSA and ertugliflozin-HSA complex at 30 ns MD time.
90
W. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 219 (2019) 83–90
Fig. 9. The number of hydrogen bonds of ertugliflozin-HSA system throughout MD stimulation.
content increased slightly. In summary, this study provides valuable information on the basis of the interaction of ertugliflozin with HSA. These results may provide some new ideas for the optimization of clinical use.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21808020), the Applied Basic Research Program of Sichuan Province (Grant No. 2018JY0151). References [1] Z. Miao, G. Nucci, N. Amin, R. Sharma, V. Mascitti, M. Tugnait, A.D. Vaz, E. Callegari, A.S. Kalgutkar, Pharmacokinetics, metabolism, and excretion of the antidiabetic agent ertugliflozin (PF-04971729) in healthy male subjects, Drug Metab. Dispos. 41 (2013) 445–456. [2] A.S. Kalgutkar, M. Tugnait, T. Zhu, E. Kimoto, Z. Miao, V. Mascitti, X. Yang, B. Tan, R.L. Walsky, J. Chupka, B. Feng, R.P. Robinson, Preclinical species and human disposition of PF-04971729, a selective inhibitor of the sodium-dependent glucose cotransporter 2 and clinical candidate for the treatment of type 2 diabetes mellitus, Drug Metab. Dispos. 39 (2011) 1609–1619. [3] H. Mao, P.J. Hajduk, R. Craig, R. Bell, T. Borre, S.W. Fesik, Rational design of diflunisal analogues with reduced affinity for human serum albumin, J. Am. Chem. Soc. 123 (2001) 10429–10435. [4] F. Yang, J. Yue, L. Ma, Z. Ma, M. Li, X. Wu, H. Liang, Interactive associations of drugdrug and drug-drug-drug with IIA subdomain of human serum albumin, Mol. Pharm. 9 (2012) 3259–3265. [5] E. Rahnama, M. Mahmoodian-Moghaddam, S. Khorsand-Ahmadi, M.R. Saberi, J. Chamani, Binding site identification of metformin to human serum albumin and glycated human serum albumin by spectroscopic and molecular modeling techniques: a comparison study, J. Biomol. Struct. Dyn. 33 (2015) 513–533. [6] G. Sudlow, D.J. Birkett, D.N. Wade, Further characterization of specific drug binding sites on human serum albumin, Mol. Pharmacol. 11 (1976) 1052–1061. [7] G. Sudlow, D.J. Birkett, D.N. Wade, The characterization of two specific drug binding sites on human serum albumin, Mol. Pharmacol. 11 (1975) 824–832. [8] Q. Gu, J.E. Kenny, Improvement of inner filter effect correction based on determination of effective geometric parameters using a conventional fluorimeter, Anal. Chem. 81 (2009) 420–426. [9] O. Trott, A.J. Olson, AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading, J. Comput. Chem. 31 (2010) 455–461. [10] N. Suvannang, C. Nantasenamat, C. Isarankura-Na-Ayudhya, V. Prachayasittikul, Molecular docking of aromatase inhibitors, Molecules 16 (2011) 3597–3617. [11] H. Grubmüller, P. Tavan, Multiple time step algorithms for molecular dynamics simulations of proteins: how good are they? J. Comput. Chem. 19 (1998) 1534–1552.
[12] J.M. Khan, S.K. Chaturvedi, R.H. Khan, Elucidating the mode of action of urea on mammalian serum albumins and protective effect of sodium dodecyl sulfate, Biochem. Biophys. Res. Commun. 441 (2013) 681–688. [13] P.K. Suresh, N. Divya, S. Nidhi, R. Rajasekaran, Phenytoin-bovine serum albumin interactions - modeling plasma protein - drug binding: a multi-spectroscopy and in silico-based correlation, Spectrochim. Acta A Mol. Biomol. Spectrosc. 193 (2018) 523–527. [14] Z. Suo, Q. Sun, H. Yang, P. Tang, R. Gan, X. Xiong, H. Li, Combined spectroscopy methods and molecular simulations for the binding properties of trametinib to human serum albumin, RSC Adv. 8 (2018) 4742–4749. [15] A. Szkudlarek, A. Sulkowska, M. Maciazek-Jurczyk, M. Chudzik, J. Rownicka-Zubik, Effects of non-enzymatic glycation in human serum albumin. Spectroscopic analysis, Spectrochim. Acta A Mol. Biomol. Spectrosc. 152 (2016) 645–653. [16] A.S. Abdelhameed, P. Alam, R.H. Khan, Binding of Janus kinase inhibitor tofacitinib with human serum albumin: multi-technique approach, J. Biomol. Struct. Dyn. 34 (2016) 2037–2044. [17] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Springer Science &Business Media, 2013. [18] J.R. Lakowicz, Quenching of Fluorescence, 2006 277–330. [19] X. Ding, Z. Suo, Q. Sun, R. Gan, P. Tang, Q. Hou, D. Wu, H. Li, Study of the interaction of broad-spectrum antimicrobial drug sitafloxacin with human serum albumin using spectroscopic methods, molecular docking, and molecular dynamics simulation, J. Pharm. Biomed. Anal. 160 (2018) 397–403. [20] E. Lissi, C. Calderon, A. Campos, Evaluation of the number of binding sites in proteins from their intrinsic fluorescence: limitations and pitfalls, Photochem. Photobiol. 89 (2013) 1413–1416. [21] L.A. Macmanusspencer, M.L. Tse, P.C. Hebert, H.N. Bischel, R.G. Luthy, Binding of perfluorocarboxylates to serum albumin: a comparison of analytical methods, Anal. Chem. 82 (2010) 974–981. [22] S. Patel, K.K. Sharma, A. Datta, Competitive binding of Chlorin p6 and Dansyl-lProline to Sudlow's site II of human serum albumin, Spectrochim. Acta A Mol. Biomol. Spectrosc. 138 (2015) 925–931. [23] M.M. Islam, V.K. Sonu, P.M. Gashnga, N.S. Moyon, S. Mitra, Caffeine and sulfadiazine interact differently with human serum albumin: a combined fluorescence and molecular docking study, Spectrochim. Acta A Mol. Biomol. Spectrosc. 152 (2016) 23–33. [24] P.D. Ross, S. Subramanian, Thermodynamics of protein association reactions: forces contributing to stability, Biochemistry 20 (1981) 3096–3102. [25] U. Katrahalli, S. Jaldappagari, S.S. Kalanur, Study of the interaction between fluoxetine hydrochloride and bovine serum albumin in the imitated physiological conditions by multi-spectroscopic methods, J. Lumin. 130 (2010) 211–216. [26] X. Ma, J. Yan, Q. Wang, D. Wu, H. Li, Spectroscopy study and co-administration effect on the interaction of mycophenolic acid and human serum albumin, Int. J. Biol. Macromol. 77 (2015) 280–286. [27] N.J. Greenfield, G.D. Fasman, Computed circular dichroism spectra for the evaluation of protein conformation, Biochemistry 8 (1969) 4108–4116. [28] N. Ohta, D. Chen, S. Ito, T. Futo, T. Yotsuyanagi, K. Ikeda, Effect of transdiamminedichloroplatinum (II) on human serum albumin: conformational changes through partial disulfide bond cleavage, Int. J. Pharm. 118 (1995) 85–93. [29] Y. Yue, Q. Dong, Y. Zhang, X. Li, X. Yan, Y. Sun, J. Liu, Synthesis of imidazole derivatives and the spectral characterization of the binding properties towards human serum albumin, Spectrochim. Acta A Mol. Biomol. Spectrosc. 153 (2016) 688–703. [30] X. Zhou, X. Li, X. Chen, Binding mechanism of Orange G to human serum albumin: saturation transfer difference-NMR, spectroscopic and computational techniques, Dyes Pigments 98 (2013) 212–220. [31] Z. Chen, H. Xu, Y. Zhu, J. Liu, K. Wang, P. Wang, S. Shang, X. Yi, Z. Wang, W. Shao, S. Zhang, Understanding the fate of an anesthetic, nalorphine upon interaction with human serum albumin: a photophysical and mass-spectroscopy approach, RSC Adv. 4 (2014) 25410–25419. [32] M. Saeidifar, H. Mansouri-Torshizi, A. Akbar Saboury, Biophysical study on the interaction between two palladium(II) complexes and human serum albumin by multispectroscopic methods, J. Lumin. 167 (2015) 391–398. [33] P. Zhang, Z. Li, X. Wang, Z. Shen, Y. Wang, J. Yan, Z. Zhou, W. Zhu, Study of the enantioselective interaction of diclofop and human serum albumin by spectroscopic and molecular modeling approaches in vitro, Chirality 25 (2013) 719–725. [34] J. Ghuman, P.A. Zunszain, I. Petitpas, A.A. Bhattacharya, M. Otagiri, S. Curry, Structural basis of the drug-binding specificity of human serum albumin, J. Mol. Biol. 353 (2005) 38–52. [35] YASARA, Yet another scientific artificial reality application: molecular graphics, modeling and simulation program, http://www.yasara.org/. [36] B. Hemmateenejad, M. Shamsipur, F. Samari, T. Khayamian, M. Ebrahimi, Z. Rezaei, Combined fluorescence spectroscopy and molecular modeling studies on the interaction between harmalol and human serum albumin, J. Pharm. Biomed. Anal. 67-68 (2012) 201–208. [37] S. Fujiwara, T. Amisaki, Molecular dynamics study of conformational changes in human serum albumin by binding of fatty acids, Proteins 64 (2006) 730–739. [38] X.M. He, D.C. Carter, Atomic structure and chemistry of human serum albumin, Nature 358 (1992) 209.
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Study on the interaction of ertugliflozin with human serum albumin in vitro by multispectroscopic methods, molecular docking, and molecular dynamics simulation Wenjing Wang a, Na Gan a, Qiaomei Sun a, Di Wu b,⁎⁎, Ruixue Gan a, Man Zhang a, Peixiao Tang a, Hui Li a,⁎ a b
School of Chemical Engineering, Sichuan University, Chengdu 610065, China Key Laboratory of Meat Processing of Sichuan, College of Pharmacy and Biological Engineering, Chengdu University, Chengdu 610106, China
a r t i c l e
i n f o
Article history: Received 24 January 2019 Received in revised form 15 April 2019 Accepted 17 April 2019 Available online 18 April 2019 Keywords: Human serum albumin (HSA) Ertugliflozin Fluorescence spectroscopy Molecular docking Molecular dynamics (MD) simulation analyses
a b s t r a c t Ertugliflozin is a potent and selective inhibitor of sodium-dependent glucose cotransporters 2 (SGLT2) and used as a monotherapy to improve glycemic control in adult patients with type 2 diabetes. In this study, ertugliflozin binding to human serum albumin (HSA) was investigated by multispectroscopic and computer simulations. The fluorescence spectra demonstrated that the quenching mechanism of ertugliflozin and HSA was static quenching. Thermodynamic parameters indicated that hydrogen bonding and van der Waals forces played a key role in the binding. Fluorescence competition experiments and molecular docking revealed that ertugliflozin bound to HSA sites II. In three-dimensional fluorescence, circular dichroism spectroscopy, and molecular dynamics simulation, ertugliflozin did not affect the basic skeleton structure of HSA but slightly increased the α-helical structure content and changed the microenvironment around amino acid residues. Results provide valuable information on the basis of the interaction of ertugliflozin with HSA. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Ertugliflozin, (1S,2S,3S,4R,5S)-5-{4-chloro-3-[(4-ethoxyphenyl) methyl]phenyl}-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]octane2,3,4-triol (Fig. 1), belongs to a new class of potent and selective sodium-dependent glucose cotransporter 2 (SGLT2) inhibitors and is currently being developed for treatment of type 2 diabetes mellitus [1]. Ertugliflozin was approved by the FDA as a monotherapy on December 22, 2017 and can be combined with sitagliptin or metformin hydrochloride. Ertugliflozin is an oral preparation that enters the bloodstream through the digestive system after oral administration and is transported to the target by plasma proteins. Ertugliflozin has oral bioavailability of approximately 65% and plasma protein binding rate of 94%–96% [2]. Thus, studying the binding of ertugliflozin to plasma protein has important implications for improving its bioavailability and understanding physiological characteristics in human body. Human serum albumin (HSA) is the most abundant protein in plasma and is capable of binding to a large variety of ligands (fatty acids, hormones, amino acids, and drugs). As an important transporter,
⁎ Correspondence to: H. Li, School of Chemical Engineering, Sichuan University, Chengdu, Sichuan, China. ⁎⁎ Correspondence to: D. Wu, Key Laboratory of Meat Processing of Sichuan, College of Pharmacy and Biological Engineering, Chengdu University, Chengdu, Sichuan, China. E-mail addresses: [email protected] (D. Wu), [email protected] (H. Li).
https://doi.org/10.1016/j.saa.2019.04.047 1386-1425/© 2019 Elsevier B.V. All rights reserved.
this binding property of HSA can significantly affect its distribution and efficacy in the body and help or prevent many drugs from reaching their target sites [3]. The interaction between the ligand and HSA is important to understand the pharmacokinetic behavior of the drug in the human body; in this regard, drug–HSA interactions have gained increasing attention, and most studies have focused on the binding sites and modes of drug–HSA [4]. The binding properties of some other diabetes drugs to HSA have been studied, such as metformin [5]. However, the binding properties between ertugliflozin and HSA have not been reported. Studying the binding properties of ertugliflozin and HSA can provide some theoretical insights into the transport in vivo and binding mode of ertugliflozin. Therefore, the present study focused on the interaction between ertugliflozin and HSA. This comprehensive in vitro study evaluates the interaction between ertugliflozin and HSA by using multispectroscopic, circular dichroism (CD), molecular docking, and molecular dynamics (MD) simulation analyses. These methods conform to one another. Fluorescence spectroscopy was used to determine whether ertugliflozin binds to HSA and identify the binding mechanism. The thermodynamic parameters of the ertugliflozin–HSA system and the binding types involved were obtained based on the fluorescence experiments. In addition, fluorescence competitive studies were performed to determine binding sites and molecular docking simulations were conducted for validation. The effects of ertugliflozin on HSA conformation and amino acid residue microenvironment were investigated by CD and 3D fluorescence
84
W. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 219 (2019) 83–90
At room temperature, the 3D fluorescence spectra of the solution of HSA (2 μM) and ertugliflozin–HSA ([ertugliflozin]/[HSA] = 30:1) were measured. The excitation wavelength was set to 200–400 nm, and the emission wavelength range was 200–500 nm. The wavelength increments were both 5 nm. The possible binding positions were determined by fluorescence competitive studies. DNSA and DP are known to bind to Site I and Sites II of HSA [6,7], respectively. Different concentrations (0, 140, 240, 320 and 400 μM) of ertugliflozin solution were gradually added into the test solutions, in which the final concentrations of HSA (2 μM) and DNSA/DP (20 μM) were unchanged. The excitation wavelengths for DNSA and DP were 326 and 375 nm, respectively, and the fluorescence spectra of DNSA/DP-HSA were recorded at 298 K. Theoretically, fluorescence quenching has various causes, including the inner-filter effect, which refers to the absorption of radiation that enters (excitation) or emits from (emission) fluorophores. The innerfilter effect should be corrected by the following equation to increase the reliability of fluorescence data [8]: Aex þAem 2
F corr ¼ F obs e
;
ð1Þ
where Fcorr is the corrected fluorescence intensities, and Fobs is the fluorescence intensities obtained in the experiment. The system absorption at the excitation (Aex) and emission (Aem) wavelengths were measured using a TU-1901 UV–Vis spectrophotometer (Persee, Beijing, China). 2.3. Fluorescence lifetime Fluorescence lifetime was measured by a Jobin Yvon Fluorolog-3 spectrofluorometer (Horiba, LesUlis, FRA) at λex = 280 nm and λem = 337 nm. The HSA concentration was 2 μM, and ertugliflozin were 80 and 140 μM. Fig. 1. Molecular structure of ertugliflozin.
spectroscopy. These experimental results were verified by molecular docking and molecular dynamic simulation. This study will help us to understand the binding mechanism between ertugliflozin and HSA through experiment and simulation in vitro.
2.4. CD spectroscopy
2. Experimental
A Chirascan-plus circular dichroism spectrometer (Applied Photophysics, Surrey, UK) was used to record the CD spectra. The mixed solution contained HSA (2 μM) and ertugliflozin (0, 80, and 140 μM). The spectra of 190–280 nm was acquired with a step size of 0.2 nm and a bandwidth of 1 nm. Each sample was scanned three times to calculate the mean for plotting and data analysis.
2.1. Materials
2.5. Molecular docking studies
HSA was purchased from Sigma Chemical Company (St. Louis, USA) and prepared at 20 μM in phosphate buffer saline (PBS, containing 0.1 M NaCl). Ertugliflozin was obtained from Shanghai Rongtai Pharmatech Co., Ltd. (Shanghai, China). The 5-dimethylaminonaphthalene-1sulfonamide (DNSA) was acquired from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Dansyl-L-proline (DP) was supplied by Heowns Biochemical Technology Co., Ltd. (Tianjin, China). Ertugliflozin, DNSA, and DP were dissolved in ethanol at 10 mM. All of the other reagents were of analytical grade without further purification. PBS (pH 7.4) was used to dilute the solution.
Simulation of the binding sites of ertugliflozin at HSA was conducted by YASARA package (v18.3.23) using VINA docking method [9]. The HSA crystal structure was obtained from the Protein Data Bank (PDB ID: 1H9Z). The 3D ligand structures of ertugliflozin were downloaded from PubChem (PubChem CID: 44814423). HSA was pretreated by removing water molecules, adding missing hydrogen atoms and distributing the Gasteiger charges. Other parameters were set to their default values. The ligand was bound to site I and site II by local docking. The docking model with the highest binding energy was selected as the optimal configuration for analysis.
2.2. Fluorescence spectroscopy
2.6. MD simulation
A Cary Eclipse fluorophotometer (Varian, California, USA) was used to record the fluorescence spectra. The slit widths of the fluorescence excitation and emission were set to 5 and 10 nm, respectively, and the excitation wavelength was 280 nm. The fluorescence emission spectra of 290–500 nm were measured, and the measured temperatures were 298, 304, and 310 K. The HSA solution was diluted to 2 μM, and the final concentrations of ertugliflozin were from 0 μM to 140 μM. All solutions were incubated for half an hour before measuring to achieve a steady state.
MD simulations were performed in YASARA v18.3.23 [10], and the force field was AMBER14. A series of preparations, including hydrogenation, optimization, and calibration, was carried out prior to the simulation. The AM1-BCC model was used to calculate the partial atomic charge of the ligand. After the minimization of the system initial energy, the pH was set to 7.4 and the operating temperature was constant at 298 K for MD simulation. The complex was placed in a water box with a size of 100.04 Å × 100.04 Å × 100.04 Å along the x-, y-, and z-axes. Periodic boundary conditions were then applied. Counter ions were
W. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 219 (2019) 83–90
85
randomly added by replacing water molecules with Na+ or Cl− to provide a charge–neutral system. The Particle-Mesh Ewald algorithm and a cut-off of 8.0 Å were applied to calculate the Coulomb forces. The simulation time steps of the intramolecular force and the intermolecular force were 1.25 fs and 2.50 fs, respectively [11]. Data were recorded every 10 ps, and data analysis could be conducted after 5.0 ns, with a total running time of 50 ns.
ertugliflozin and HSA result in an increased hydrophobicity and a reduced polarity of amino acid residues. In general, the following are the two fluorescence quenching mechanisms: dynamic quenching and static quenching, which can be distinguished by the relationship between quenching constant (Ksv) and temperature. The Stern–Volmer equation was used to calculate the relevant data as follows [17]:
3. Results and discussion
F0 ¼ 1 þ K SV ½Q ¼ 1 þ kq τ0 ½Q ; F
ð2Þ
3.1. Fluorescence quenching analysis In the excited state, HSA will produce a certain intensity of fluorescence due to the presence of three fluorophores, namely, tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) residues [12,13]. The intensity of the HSA emission spectra depends mainly on Tyr and Trp. Many small molecular ligands can alter the microenvironment around the HSA chromophore, ultimately leading to changes in the intrinsic fluorescence intensity of HSA [14]. The fluorescence spectra of HSA in the presence and absence of different concentrations of ertugliflozin at 298 K are shown in Fig. 2(A). Notably, ertugliflozin itself has no fluorescence. With the addition of ertugliflozin concentration, the fluorescence intensity of HSA gradually decreases, that is, fluorescence quenching occurs, and a blue shift exists. Usually, the blue shift of the maximum emission peak (λmax) of the protein means that the amino acid residues are in a hydrophobic environment, whereas the red shift of λmax reflects an increase in the polarity of amino acid residues [15,16]. The result testified that ertugliflozin bound to HSA and the combination of
where F0 and F are the fluorescence intensities of HSA in the absence and presence of ertugliflozin, respectively; [Q] is the quencher concentration; kq is the biomolecular quenching constant, and τ0 is the HSA lifetime in the absence of the quencher (τ0 = 5.520 × 10−9 s). Fig. 2(B) shows the Stern–Volmer curve of ertugliflozin–HSA at different concentrations. In dynamic quenching, intermolecular collision occurs between the quencher and fluorescence that ultimately results in an increase of Ksv with the increase of temperature. In addition, in static quenching, the ground state complex is formed by the weak interaction between the ertugliflozin and HSA, leading to decreased Ksv with the increase of temperature [18,19]. As shown in Fig. 2(B) and Table 1, Ksv is negatively correlated with temperature, and kq was much greater than the maximum scatter collision quenching constant (2 × 1010 L mol−1 s−1) of quenchers with biopolymer. Therefore, the fluorescence quenching of HSA caused by ertugliflozin belonged to static quenching, manifesting that HSA and ertugliflozin formed an unstable complex.
Fig. 2. (A) Fluorescence emission spectra of HSA in the presence and absence of various ertugliflozin concentrations at 298 K. CHSA = 2 μM, Certugliflozin = 0 (a), 20 (b), 40 (c), 60 (d), 80 (e), 100 (f), 120 (g), and 140 (h) μM. (B) Stern–Volmer curve of ertugliflozin–HSA system at different temperatures. (C) Plot of log(F0 − F)/F vs. log[Q] for the ertugliflozin–HSA system at three different temperatures. In (B) and (C), the concentrations of HSA and ertugliflozin were the same as the fluorescence emission spectra. (D) Fluorescence lifetime curves of ertugliflozin– HSA systems in the presence of various concentrations of ertugliflozin. CHSA = 2 μM, Certugliflozin = 80 and 140 μM.
86
W. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 219 (2019) 83–90
Table 1 Stern–Volmer quenching constants of the ertugliflozin–HSA system at different temperatures. T(K)
Ksv(×103 M−1)
kq(×1011 M−1 s−1)
Ka(×103 M−1)
n
298 304 310
4.566 ± 0.175 4.304 ± 0.152 4.127 ± 0.266
8.272 ± 0.175 7.797 ± 0.152 7.476 ± 0.266
1.914 ± 0.464 1.225 ± 0.189 0.575 ± 0.205
0.894 0.849 0.770
exposed to water. However, the average fluorophore lifetime (τav) can be expressed in terms of fractional contribution (fi) of each decay time to the steady state fluorescence intensity as [23]: X τav ¼
X
f i τi ¼ X
α i τ2i
i
i
αi τi
:
ð5Þ
i
For static quenching, assuming that the ligand has n binding sites with the protein, the fluorescence intensity of the ligand–protein system, the ligand concentration, the binding constant (Ka), and the number of binding sites (n) satisfy the modified Stern–Volmer equation [20]: logð F 0 − F Þ ¼ logK a þ nlog½Q : F
ð3Þ
Table 1 shows the values of Ka and n. Fig. 2(C) is obtained by plotting the curve of log(F0 − F)/F vs. log[Q] for the ertugliflozin–HSA system. Ka decreased with increasing temperature, showing that temperature reduced the binding between HSA and ertugliflozin. The n value ranging from 0.7 to 0.9 in Table 1 suggested approximately one binding site between the HSA and ertugliflozin.
Remarkably, the τav values with different concentrations of ertugliflozin are almost constant within a certain range. The result testified that the quenching mechanism between HSA and ertugliflozin was static quenching. 3.3. Thermodynamic parameters and binding modes Four atomic affinities, namely, hydrogen bonding, van der Waals forces, hydrophobic interactions, and electrostatic interactions, are observed between the HSA and ertugliflozin. Usually, the thermodynamic parameters of the ligand–protein system, such as enthalpy change (△H), entropy change (△S), and Gibbs free energy (△G), are calculated by the Gibbs–Helmholtz equation (Eq. (6)) and the Van't Hoff equation (Eq. (7)). The main binding forces of ligand–protein system were also judged by their numerical magnitude as positive or negative. △G ¼ △H−T△S;
3.2. Fluorescence lifetime analysis Static quenching and dynamic quenching differ in the changes of fluorescence lifetime, which can be used to distinguish between the two quenching mechanisms. For dynamic quenching, intermolecular collisions reduce the fluorescence lifetime of the protein, whereas in static quenching, ligands and proteins form unstable complexes that do not result in reduced fluorescence lifetime [21]. Fig. 2(D) shows the fluorescence lifetime curves of ertugliflozin–HSA systems in the absence and presence of various concentrations of ertugliflozin. As can be seen from the figure, the fluorescence lifetime of HSA did not change before and after the addition of ertugliflozin, illustrating the quenching mechanism of ertugliflozin–HSA system involving static quenching. The initial fluorescence lifetime data were processed by biexponential iterative fitting, and the results are listed in Table 2. Eq. (4) was used to calculate the mean fluorescence lifetime (bτN) [22]:
ln K a ¼ −
ð6Þ
ΔH ΔS þ : RT R
ð7Þ
ΔH and ΔS were obtained from the slope and the intercept of the Van't Hoff plot, respectively. Ross et al. [24] proved that △H b 0 and △S N 0 refer to electrostatic interactions, △H N 0 and △S N 0 manifest as typically hydrophobic interactions, and △H b 0 and △S b 0 indicate hydrogen bonding and van der Waals forces. The thermodynamic parameters of HSA and ertugliflozin are shown in Table 3. △G b 0 meant that the combination of HSA and ertugliflozin was spontaneous. △H and △S are negative, confirming that hydrogen bonds and van der Waals forces play a major role in the bonding, and △H b 0 suggested that the temperature rise was not conducive to the bonding reactions. This finding was consistent with the conclusion of Section 3.1. 3.4. Analysis of HSA conformational changes caused by ertugliflozin
bτ N ¼ α 1 τ 1 þ α 2 τ2 ;
ð4Þ
where α1 and α2 are the pre-exponential factors, and τ1 and τ2 are the decay times. Chi-square (χ2) was used to evaluate the performance of the fitting results. If χ2 b 1.3, the fitting results were within an acceptable tolerance interval. As can be seen from Table 2, with the increase of ertugliflozin, the HSA lifetime decreased slightly. This result was inconsistent with that of the fluorescence quenching analysis. In general, the conformational heterogeneity of protein structures, the interaction of fluorophores with water molecules, and the interaction of the Trp with nearby amino acid residues may alter the fluorescence lifetime of HSA. Although insulating the specific cause of increased lifetime is difficult, in this study, the most likely cause of a slight increase in fluorescence lifetime is the change in HSA secondary structure (see Section 3.4 for details) to increase the concentration of fluorophores Table 2 Fluorescence decay fitting parameters of ertugliflozin–HSA in the presence of various HSA concentrations. Sample
τ1(ns)
τ2(ns)
α1
α2
Free HSA HSA -40ertugliflozin HSA-70ertugliflozin
2.499 2.527 2.741
6.565 6.706 6.776
0.257 0.301 0.358
0.743 0.699 0.642
bτN(ns) 5.520 5.448 5.331
The effects of ertugliflozin on HSA conformation were investigated by 3D fluorescence spectroscopy and CD spectroscopy. The results of these three methods are mutually verified to improve the reliability of the experimental results. 3.4.1. CD spectroscopy CD is another indispensable mean widely used to study protein conformational changes and qualify the amount of α-helix [25,26]. The CD spectra of HSA have two negative characteristic peaks at 208 and 222 nm, which are related to the α-helix-rich secondary structure of HSA. As shown in Fig. 3, with the addition of ertugliflozin solution, the characteristic peak intensity of HSA increased, but the peak type and peak position did not change significantly. This phenomenon explains that the binding of ertugliflozin and HSA did not destroy the α-helix structure of protein. The change in the α-helix content caused by Table 3 Thermodynamic parameters of the ertugliflozin–HSA system at different temperatures.
τav
χ2
T(K)
△H(kcal/ mol)
△G(kcal/ mol)
△S(cal mol−1 K−1)
6.092 6.123 6.033
1.256 1.197 1.109
298 304 310
−77.12 −77.12 −77.12
−18.72 −17.97 −16.38
−195.5 −195.5 −195.5
W. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 219 (2019) 83–90
87
changes. Fig. 4 is 3D fluorescence spectra of free HSA and ertugliflozin–HSA system. Peak A is the Rayleigh scattering peak, whereas peak 1 and peak 2 are the characteristic signals of residues and polypeptide backbone structures in HSA [31,32]. The 3D fluorescence spectral parameters of HSA alone and in the presence of ertugliflozin are listed in Table 4. Notably, the fluorescence intensity of peak A slightly increased because of ertugliflozin addition. A possible reason for this phenomenon may be that ertugliflozin addition increased the HSA diameter. This phenomenon enhanced the Rayleigh scattering peak. Both the intensities of peak 1 and peak 2 diminished, indicating that the binding of ertugliflozin to HSA affected the microenvironment around the Tyr and Trp residues [33]. Based on the above results, in the presence of ertugliflozin, although the basic skeleton structure of HSA does not change significantly, this condition caused the disturbance of the polypeptide skeleton, the slight increase of the α-helical structure and the change of the microenvironment around the amino acid residues. Fig. 3. CD spectra of HSA in the absence and presence of ertugliflozin. CHSA = 2 μM, Certugliflozin = 80 μM.
ertugliflozin addition can be calculated by the mean residue ellipticity (MRE) at 208 nm, shown as follows [14,27]: MRE208 ¼
ObservedCDðmdeg Þ ; C p nl 10
α−helixð%Þ ¼
−MRE208 −4000 100; 33000−4000
ð8Þ
ð9Þ
where Cp is the molar concentration of HSA in solution; n is the number of amino acid residues (585) in HSA, and l is the path length of the cell (0.1 cm). By calculation, the α-helix (%) of free HSA was 48.64%, which agreed with about 50% obtained in other reports [28–30], and after adding 80 μM ertugliflozin, the α-helix (%) of HSA became 52.83%. An increase of the α-helix structure revealed that ertugliflozin made the HSA conformation changed. 3.4.2. 3D fluorescence spectroscopy Another conformation investigation experiment was undertaken using 3D fluorescence spectroscopy. The 3D fluorescence spectroscopy is the most effective way to characterize protein conformational
3.5. Site-selective binding of ertugliflozin on HSA HSA consists of 585 amino acid residues, and its crystal structure contains three helical domains (I–III) with similar structure; each of which is divided into A and B subdomains [34]. HSA can be combined with a variety of endogenous ligands, such as non-esterified fatty acids, bilirubin, hemin, and thyroxine. Sudlow et al. [6,7] found that when many endogenous and exogenous compounds in the blood stream bind to HSA, they are usually located in one of the two major sites in subdomains IIA and IIIA, namely, site I and site II. Site I is a binding pocket within the core of subdomain IIA, including all six helices of subdomain IIA and a loop-helix feature of subdomain IB, and site II has six helices of subdomain IIIA. Site II is smaller than site I, but due to the rotation of subdomain IIIA, its pocket entrance is more susceptible to exposure to solvent [34]. DNSA and DP are two common probes that bind to site I and site II, respectively, and their fluorescence intensities increase when they bind to HSA. According to Sudlow et al. [6], by measuring the fluorescence of the DNSA/DP–HSA system in the absence and presence of different concentrations of ligand and then calculating the percentage of probe displacement (I), the drug binding site can be predicted as follows:
I¼
F1 100%; F2
Fig. 4. 3D Fluorescence spectra of free HSA (A) and ertugliflozin-HSA system (B). CHSA = 2 μM, Certugliflozin = 60 μM.
ð10Þ
88
W. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 219 (2019) 83–90
Table 4 3D fluorescence spectral parameters of HSA alone in the presence of ertugliflozin. System HSA
Ertugliflozin-HSA (30:1)
Peak no. Peak position [λex/λem(nm/nm)] A 1 2 A 1 2
280/280 → 350/350 280/335 225/334 280/280 → 350/350 280/329 225/322
Stokes shift Intensity △λ(nm) 0 55 109 0 49 97
189.42 → 241.37 571.10 712.88 172.65 → 275.88 500.64 267.71
where F1 is the fluorescence intensity of the probe-HSA in the absence of ligand, F2 is the fluorescence intensity of the probe-HSA in the presence of various concentrations of ligand. The results of the fluorescence competition experiment are shown in Fig. 5. Free DP has low fluorescence intensity. After adding HSA to DP, the maximum emission wavelength of DP has a significant blue shift, and the fluorescence intensity is significantly higher than without HSA. Ertugliflozin addition reduced the fluorescence intensity of the DP–HSA complex. Upon addition of 400 μM ertugliflozin, the fluorescence intensity of DP decreased to approximately 74% of the initial intensity. This result suggests that both DP and ertugliflozin competed for site II in HSA, and DP was replaced by ertugliflozin from HSA. However, ertugliflozin minimally affected the fluorescence intensity of DNSA–HSA, explaining that ertugliflozin did not bind to site I or ertugliflozin had significantly less binding ability to site I than DNSA. 3.6. Analysis of molecular docking results Molecular docking is considered an important method to explore the binding sites and binding energies of small molecule on HSA, which is often corroborated by fluorescence competitive study. The binding energy in YASARA program is defined by the following formula [35]: Binding energy ¼ Receptor energy þ Ligand energy−Complex energy:
ð11Þ
According to Eq. (11), great amount of positive energy manifests good combination. When ertugliflozin binds to site I, the maximum binding energy was 9.26 kcal/mol, and when ertugliflozin binds to site II, the maximum binding energy was 9.63 kcal/mol. Therefore, the perfect binding site of ertugliflozin on HSA was site II. This finding was consistent with the result of the fluorescence competition experiment.
Fig. 5. Fluorescence spectra of DP–HSA in the absence and presence of ertugliflozin. CHSA = 2 μM, CDNSA/DP = 20 μM, Certugliflozin = 0 (a), 140 (b), 240 (c), 320 (d), and 400 (e) μM. The inset shows the effect of ertugliflozin on the fluorescence of DNSA/DP–HSA systems.
Fig. 6. Best docked result of the ertugliflozin–HSA system.
Fig. 6 shows that ertugliflozin was mainly surrounded by ARG485, ARG348, VAL344, LEU453, LEU457, PHE488, SER489, TYR411, LEU430, ALA449, ILE388, LEU387, and PRO384 when bound to HSA site II. The docked result means that ertugliflozin and these active amino acids maintain the stability of the binding conformation mainly by the hydrophobic interaction (the red dotted line in the figure) and hydrogen bonding force (the green dotted line in the figure and the bond lengths are 3.12 and 3.06 Å, respectively). 3.7. MD simulation MD simulation can further explore the stability and dynamic behavior of the ertugliflozin–HSA complex under simulated physiological conditions. The root mean square deviation (RMSD), radius of gyration (Rg), and the percentage of secondary structure, shown in Fig. 7, were measured by YASARA. The RMSD curve fluctuated to some extent as a whole, testifying that the HSA skeleton structure has changed during the simulation. However, after 10 ns, the curve tends to be horizontal, indicating that the system reached equilibration [36]. Thus, ertugliflozin can stably bind to site II. Rg represents the HSA compactness. A higher Rg value indicates a looser structure in a specific region of the protein [37]. Fig. 7B shows the initial Rg values of the free HSA and ertugliflozin–HSA complexes are both approximately 28.20 Å. During the MD simulation, the Rg value of free HSA decreased slightly, whereas the Rg value of the ertugliflozin–HSA complex was higher than that of free HSA, revealing that the combination of ertugliflozin and HSA made HSA more unconsolidated and changed the secondary structure of HSA. By extracting the free HSA and complex conformation of 30 ns (Fig. 8), it can be clearly seen from the circled part in the figure that the protein cavity becomes larger and the structure of HSA becomes more unconsolidated after ertugliflozin bound to site II. Fig. 7C and D show that the graphs of the secondary structure content of free HSA and ertugliflozin–HSA complexes. The main secondary structure of HSA is α-helix. During the MD simulation, the content of each secondary structure remained stable with only slight changes. The mean values of α-helix and turn of free HSA were 70.53% and 20.42%, respectively. This result was slightly higher than the crystallographic result (67% αhelix [38]). Because in the process of dynamic simulation, the trajectory of HSA will constantly expand, contract and slowly move, which will
W. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 219 (2019) 83–90
89
Fig. 7. RMSD (A) and Rg (B) of ertugliflozin–HSA system. Secondary structure percentages of HSA in the presence (C) and absence (D) of ertugliflozin.
also lead to the change of helix proportion. However, the mean values of α-helix and turn of ertugliflozin–HSA complex were 71.86% and 19.46%, and the increasing trend of α-helix was in line with the CD results. The number of hydrogen bonds in the ertugliflozin-HSA system was analyzed as shown in Fig. 9. MD simulation is a dynamic process, so the number of hydrogen bonds varies between 0 and 3. After 10 ns, the system reached equilibrium, and the number of hydrogen bonds was mainly 2, indicating that hydrogen bonds played a certain role in maintaining the stability of the system, which was consistent with the two hydrogen bonds shown in the docking results. The results are consistent with the experimental results, proving that adding ertugliflozin did not significantly change the HSA skeleton
but slightly affected the secondary structure content and there were two hydrogen bonds between ertugliflozin and HSA. 4. Conclusion This study presents the first exploration of the interaction between ertugliflozin and HSA through experiments and simulations. The experimental results were consistent with the simulation results. Notably, the fluorescence quenching mechanism of ertugliflozin and HSA was static quenching, which was spontaneously bound to HSA site II by hydrogen bonding and van der Waals force, and the binding was stable. After combining with ertugliflozin, the HSA diameter increased, and the α-helix
Fig. 8. Structure of free HSA and ertugliflozin-HSA complex at 30 ns MD time.
90
W. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 219 (2019) 83–90
Fig. 9. The number of hydrogen bonds of ertugliflozin-HSA system throughout MD stimulation.
content increased slightly. In summary, this study provides valuable information on the basis of the interaction of ertugliflozin with HSA. These results may provide some new ideas for the optimization of clinical use.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21808020), the Applied Basic Research Program of Sichuan Province (Grant No. 2018JY0151). References [1] Z. Miao, G. Nucci, N. Amin, R. Sharma, V. Mascitti, M. Tugnait, A.D. Vaz, E. Callegari, A.S. Kalgutkar, Pharmacokinetics, metabolism, and excretion of the antidiabetic agent ertugliflozin (PF-04971729) in healthy male subjects, Drug Metab. Dispos. 41 (2013) 445–456. [2] A.S. Kalgutkar, M. Tugnait, T. Zhu, E. Kimoto, Z. Miao, V. Mascitti, X. Yang, B. Tan, R.L. Walsky, J. Chupka, B. Feng, R.P. Robinson, Preclinical species and human disposition of PF-04971729, a selective inhibitor of the sodium-dependent glucose cotransporter 2 and clinical candidate for the treatment of type 2 diabetes mellitus, Drug Metab. Dispos. 39 (2011) 1609–1619. [3] H. Mao, P.J. Hajduk, R. Craig, R. Bell, T. Borre, S.W. Fesik, Rational design of diflunisal analogues with reduced affinity for human serum albumin, J. Am. Chem. Soc. 123 (2001) 10429–10435. [4] F. Yang, J. Yue, L. Ma, Z. Ma, M. Li, X. Wu, H. Liang, Interactive associations of drugdrug and drug-drug-drug with IIA subdomain of human serum albumin, Mol. Pharm. 9 (2012) 3259–3265. [5] E. Rahnama, M. Mahmoodian-Moghaddam, S. Khorsand-Ahmadi, M.R. Saberi, J. Chamani, Binding site identification of metformin to human serum albumin and glycated human serum albumin by spectroscopic and molecular modeling techniques: a comparison study, J. Biomol. Struct. Dyn. 33 (2015) 513–533. [6] G. Sudlow, D.J. Birkett, D.N. Wade, Further characterization of specific drug binding sites on human serum albumin, Mol. Pharmacol. 11 (1976) 1052–1061. [7] G. Sudlow, D.J. Birkett, D.N. Wade, The characterization of two specific drug binding sites on human serum albumin, Mol. Pharmacol. 11 (1975) 824–832. [8] Q. Gu, J.E. Kenny, Improvement of inner filter effect correction based on determination of effective geometric parameters using a conventional fluorimeter, Anal. Chem. 81 (2009) 420–426. [9] O. Trott, A.J. Olson, AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading, J. Comput. Chem. 31 (2010) 455–461. [10] N. Suvannang, C. Nantasenamat, C. Isarankura-Na-Ayudhya, V. Prachayasittikul, Molecular docking of aromatase inhibitors, Molecules 16 (2011) 3597–3617. [11] H. Grubmüller, P. Tavan, Multiple time step algorithms for molecular dynamics simulations of proteins: how good are they? J. Comput. Chem. 19 (1998) 1534–1552.
[12] J.M. Khan, S.K. Chaturvedi, R.H. Khan, Elucidating the mode of action of urea on mammalian serum albumins and protective effect of sodium dodecyl sulfate, Biochem. Biophys. Res. Commun. 441 (2013) 681–688. [13] P.K. Suresh, N. Divya, S. Nidhi, R. Rajasekaran, Phenytoin-bovine serum albumin interactions - modeling plasma protein - drug binding: a multi-spectroscopy and in silico-based correlation, Spectrochim. Acta A Mol. Biomol. Spectrosc. 193 (2018) 523–527. [14] Z. Suo, Q. Sun, H. Yang, P. Tang, R. Gan, X. Xiong, H. Li, Combined spectroscopy methods and molecular simulations for the binding properties of trametinib to human serum albumin, RSC Adv. 8 (2018) 4742–4749. [15] A. Szkudlarek, A. Sulkowska, M. Maciazek-Jurczyk, M. Chudzik, J. Rownicka-Zubik, Effects of non-enzymatic glycation in human serum albumin. Spectroscopic analysis, Spectrochim. Acta A Mol. Biomol. Spectrosc. 152 (2016) 645–653. [16] A.S. Abdelhameed, P. Alam, R.H. Khan, Binding of Janus kinase inhibitor tofacitinib with human serum albumin: multi-technique approach, J. Biomol. Struct. Dyn. 34 (2016) 2037–2044. [17] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Springer Science &Business Media, 2013. [18] J.R. Lakowicz, Quenching of Fluorescence, 2006 277–330. [19] X. Ding, Z. Suo, Q. Sun, R. Gan, P. Tang, Q. Hou, D. Wu, H. Li, Study of the interaction of broad-spectrum antimicrobial drug sitafloxacin with human serum albumin using spectroscopic methods, molecular docking, and molecular dynamics simulation, J. Pharm. Biomed. Anal. 160 (2018) 397–403. [20] E. Lissi, C. Calderon, A. Campos, Evaluation of the number of binding sites in proteins from their intrinsic fluorescence: limitations and pitfalls, Photochem. Photobiol. 89 (2013) 1413–1416. [21] L.A. Macmanusspencer, M.L. Tse, P.C. Hebert, H.N. Bischel, R.G. Luthy, Binding of perfluorocarboxylates to serum albumin: a comparison of analytical methods, Anal. Chem. 82 (2010) 974–981. [22] S. Patel, K.K. Sharma, A. Datta, Competitive binding of Chlorin p6 and Dansyl-lProline to Sudlow's site II of human serum albumin, Spectrochim. Acta A Mol. Biomol. Spectrosc. 138 (2015) 925–931. [23] M.M. Islam, V.K. Sonu, P.M. Gashnga, N.S. Moyon, S. Mitra, Caffeine and sulfadiazine interact differently with human serum albumin: a combined fluorescence and molecular docking study, Spectrochim. Acta A Mol. Biomol. Spectrosc. 152 (2016) 23–33. [24] P.D. Ross, S. Subramanian, Thermodynamics of protein association reactions: forces contributing to stability, Biochemistry 20 (1981) 3096–3102. [25] U. Katrahalli, S. Jaldappagari, S.S. Kalanur, Study of the interaction between fluoxetine hydrochloride and bovine serum albumin in the imitated physiological conditions by multi-spectroscopic methods, J. Lumin. 130 (2010) 211–216. [26] X. Ma, J. Yan, Q. Wang, D. Wu, H. Li, Spectroscopy study and co-administration effect on the interaction of mycophenolic acid and human serum albumin, Int. J. Biol. Macromol. 77 (2015) 280–286. [27] N.J. Greenfield, G.D. Fasman, Computed circular dichroism spectra for the evaluation of protein conformation, Biochemistry 8 (1969) 4108–4116. [28] N. Ohta, D. Chen, S. Ito, T. Futo, T. Yotsuyanagi, K. Ikeda, Effect of transdiamminedichloroplatinum (II) on human serum albumin: conformational changes through partial disulfide bond cleavage, Int. J. Pharm. 118 (1995) 85–93. [29] Y. Yue, Q. Dong, Y. Zhang, X. Li, X. Yan, Y. Sun, J. Liu, Synthesis of imidazole derivatives and the spectral characterization of the binding properties towards human serum albumin, Spectrochim. Acta A Mol. Biomol. Spectrosc. 153 (2016) 688–703. [30] X. Zhou, X. Li, X. Chen, Binding mechanism of Orange G to human serum albumin: saturation transfer difference-NMR, spectroscopic and computational techniques, Dyes Pigments 98 (2013) 212–220. [31] Z. Chen, H. Xu, Y. Zhu, J. Liu, K. Wang, P. Wang, S. Shang, X. Yi, Z. Wang, W. Shao, S. Zhang, Understanding the fate of an anesthetic, nalorphine upon interaction with human serum albumin: a photophysical and mass-spectroscopy approach, RSC Adv. 4 (2014) 25410–25419. [32] M. Saeidifar, H. Mansouri-Torshizi, A. Akbar Saboury, Biophysical study on the interaction between two palladium(II) complexes and human serum albumin by multispectroscopic methods, J. Lumin. 167 (2015) 391–398. [33] P. Zhang, Z. Li, X. Wang, Z. Shen, Y. Wang, J. Yan, Z. Zhou, W. Zhu, Study of the enantioselective interaction of diclofop and human serum albumin by spectroscopic and molecular modeling approaches in vitro, Chirality 25 (2013) 719–725. [34] J. Ghuman, P.A. Zunszain, I. Petitpas, A.A. Bhattacharya, M. Otagiri, S. Curry, Structural basis of the drug-binding specificity of human serum albumin, J. Mol. Biol. 353 (2005) 38–52. [35] YASARA, Yet another scientific artificial reality application: molecular graphics, modeling and simulation program, http://www.yasara.org/. [36] B. Hemmateenejad, M. Shamsipur, F. Samari, T. Khayamian, M. Ebrahimi, Z. Rezaei, Combined fluorescence spectroscopy and molecular modeling studies on the interaction between harmalol and human serum albumin, J. Pharm. Biomed. Anal. 67-68 (2012) 201–208. [37] S. Fujiwara, T. Amisaki, Molecular dynamics study of conformational changes in human serum albumin by binding of fatty acids, Proteins 64 (2006) 730–739. [38] X.M. He, D.C. Carter, Atomic structure and chemistry of human serum albumin, Nature 358 (1992) 209.