Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 122 (2014) 100–106
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Molecular modeling and spectroscopic studies on the interaction of the chiral drug venlafaxine hydrochloride with bovine serum albumin Nahid Shahabadi ⇑, Saba Hadidi Inorganic Chemistry Department, Faculty of Chemistry, Razi University, Kermanshah, Iran
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Spectroscopic techniques
In present work we have studied the interaction of racemic mixture of the antidepressant drug ‘‘S,RVenlafaxine hydrochloride (VEN)’’ with BSA.
combination with molecular modeling were used. The hydrogen bonding and weak van der Waals force played a major role in the interaction. Through the site marker competitive experiment, VEN located in subdomain IIIA of BSA. Molecular docking studies showed that both S and R isomers have similar interactions with BSA.
a r t i c l e
i n f o
Article history: Received 2 September 2013 Received in revised form 3 November 2013 Accepted 5 November 2013 Available online 16 November 2013 Keywords: Venlafaxine hydrochloride Bovine serum albumin Protein–drug interaction Multi-spectroscopy methods Molecular docking Binding mechanism
a b s t r a c t This study was designed to examine the interaction of racemic antidepressant drug ‘‘S,R-venlafaxine hydrochloride (VEN)’’ with bovine serum albumin (BSA) under physiological conditions. The mechanism of interaction was studied by spectroscopic techniques combination with molecular modeling. Stern– Volmer analysis of fluorescence quenching data shows the presence of the static quenching mechanism. The thermodynamic parameters indicated that the hydrogen bonding and weak van der Waals interactions are the predominant intermolecular forces stabilizing the complex. The number of binding sites (n) was calculated. Through the site marker competitive experiment, VEN was confirmed to be located in subdomain IIIA of BSA. The binding distance (r = 4.93 nm) between the donor BSA and acceptor VEN was obtained according to Förster’s non-radiative energy transfer theory. According to UV–vis spectra and CD data binding of VEN leaded to conformational changes of BSA. Molecular docking simulations of S and R-VEN revealed that both isomers have similar interaction and the same binding sites, from this point of view S and R isomers are equal. Ó 2013 Elsevier B.V. All rights reserved.
Introduction Serum albumins are the most abundant proteins in the circulatory system of a wide variety of organisms. Bovine serum albumin (BSA) being the major macromolecule in blood plasma of animals accounting to about 60% of the total protein corresponding to a ⇑ Corresponding author. Tel./fax: +98 831 8360795. E-mail address: [email protected] (N. Shahabadi). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.11.016
concentration of 42 g dm3 [1]. It consists of a single chain of 582 amino acids, globular nonglycoprotein cross-linked with 17 cysteine residues (eight disulfide bonds and one free thiol). It is divided into three linearly arranged, structurally distinct and evolutionarily related domains (I–III); each domain is composed of two subdomains (A and B) [2]. BSA has two tryptophans, embedded in two different domains: Trp-134, located on the surface of domain I and Trp-213, located within the hydrophobic pocket of domain II. The binding cavities associated with subdomains IIA
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and IIIA are also referred to as site I and site II according to the terminology proposed by Sudlow et al. [3]. The most exceptional property of a serum albumin is that it serves as a depot protein and a transport protein for numerous endogenous and exogenous compounds [4]. The exogenous substances that bound to protein with a high affinity are drugs. In this work, BSA is selected as our protein model because of its medically important, abundance, low cost, ease of purification, unusual ligand-binding properties, stability [5,6], and the results of all the studies are consistent with the fact that human and bovine serum albumins are homologous proteins [6–8]. Venlafaxine ‘‘1-[2-(dimethylamino)-1-(4-methoxyphenyl)ethyl] cyclohexanol hydrochloride’’ commercially known as ‘‘Effexor’’ is a representative of a new class of antidepressants. For VEN there is one chiral center. VEN is administered as a racemate, composed of equal amounts the S-(+) and R-() enantiomeric forms (Fig. 1) and is usually categorized as a serotonin–nor epinephrine reuptake inhibitor (SNRI) but it has been referred to as a serotonin– norepinephrine–dopamine reuptake inhibitor. Its higher solubility in water results in burst effect with sudden peak levels of drug in blood. In humans, 90% of the total VEN decrease is accounted for by enantioselective O-desmethylation through cytochrome P450 (CYP) CYP2D6 leading to the active metabolite O-desmethylvenlafaxine (ODV) and 10% for no-enantioselective N-desmethylation by CYP3A4, leading to N-desmethylvenlafaxine (NDV) [9]. A key process in the development of new drugs is elucidation of the nature of the interaction between the drug molecule and the target protein. Since albumin serves as a transport carrier for drugs, it is important to study the interactions of these drugs with this protein. The effectiveness of these compounds as pharmaceutical agents depends on their binding ability. Fluorescence and UV–vis absorption spectroscopy are effective techniques to study the small molecules–proteins interactions, because of their sensitivity, reproducibility and convenience. These approaches can reveal the binding affinity of small molecules with proteins and help to understand their binding mechanisms. Circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy are reliable methods for analyzing the contents of secondary conformation forms of proteins, which can explain the conformational changes of proteins induced by ligands [10]. In present work, we use several spectral methods including fluorescence, UV–vis absorption and CD spectroscopy combination with molecular docking to obtain information on the quenching mechanism, thermodynamic parameters, the special binding site and the effect of VEN on the secondary structure of BSA, which could reveal important information for the study of the structure–activity relationship of the drug and BSA. Experimental details Apparatus Fluorescence measurements were performed with a JASCO spectrofluorimeter Model FP-6200 equipped with a thermostat bath, using a 1.0 cm quartz cell. UV–vis absorption spectra were
measured on an Agilent UV–vis spectrophotometer Model 8453 using a 1.0 cm cell. CD measurements were recorded on a JASCO spectropolarimeter Model J-810, using a 0.1 cm quartz cell. pH measurements were carried out with a digital pH-meter with a combined glass-calomel electrode. Reagents Bovine serum albumin (BSA) was purchased from Sigma– Aldrich. All BSA solutions were prepared in the 0.1 M phosphate buffer (pH 7.4), BSA solutions stored in the dark at 4 °C. A stock solution of VEN was prepared by dissolving its crystals in 50 mM of the phosphate buffer at pH 7.4. Doubly distilled water was used to prepare the buffers. Procedure Fluorescence spectra Fluorescence measurements were performed by keeping the concentration of BSA constant (5 106 M) while varying the rVEN concentration from 0.0 to 4.7 105 M at different temperatures (278, 288, 310). Displacement experiments The displacement experiments were performed using the site probes including ibuprofen and warfarin by keeping the concentration of protein and probe constant (each of 5 106 M). The fluorescence quenching titration was used to determine the binding constants of VEN–BSA systems in presence of above site probes for sites I and II. UV–vis absorption studies The absorbance measurements were performed by keeping the BSA concentration constant (1 105 M) while varying the racVEN concentration from 0 to 1.9 105 M (ri = [VEN]/[BSA] = 0.0, 0.7, 1.1 and 1.9). The samples were incubated at room temperature for 10 min and the spectra were recorded in the range of 200– 400 nm. Circular dichroism (CD) measurements Circular dichroism (CD) measurements were conducted by keeping the concentration of BSA constant (3 106 M) while varying the VEN concentration from 0 to 1.8 106 M (ri = [VEN]/[BSA] = 0.0, 0.4, 0.5 and 0.6). Molecular docking MGL tools 1.5.4 with AutoGrid 4 and AutoDock 4 were used to set up and perform blind docking calculations between R and SVEN with BSA. The structure of free BSA (PDB id: 3V03, chain A) obtained by X-ray crystallography was used as a template. Receptor (BSA) and ligands (R and S-VEN) files were prepared using AutoDock Tools. The BSA was enclosed in a box with number of grid points in x y z directions, 126 126 126 and a grid spacing of 0.703 Å. Lamarckian genetic algorithms, as implemented in AutoDock, were employed to perform docking calculations. All other parameters were default settings. For each of the docking cases, the lowest energy docked conformation, according to the Autodock scoring function, was selected as the binding mode. The output from AutoDock was rendered with PyMol. In AutoDock, the overall docking energy of a given ligand molecule in its active site is expressed as follows:
X Aij Bij DG ¼ DGvdw 6 r 12 r ij ij ij þ DGelec Fig. 1. Molecular structure of R and S-venlafaxine hydrochloride.
X qi qj ij
eðrij Þrij
!
X C ij Dij þ DGhbond 10 þ Ehbond r 12 rij ij ij
þ DGtor Ntor þ DGsol
!
X ðr 2 =2r2 Þ Si V j e ij ij
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where DGvdw, DGhbond, DGelec, DGtor, and DGsol are free energy coefficients of van der Waals, hydrogen bond, electrostatic interactions, torsional term, and desolvation energy of protein–ligand complex, respectively. rij, Aij, Bij, Cij, and Dij represent the interatomic distance, the depths of energy well, and the equilibrium separations between the two atoms, respectively. The first three terms are in vacuo force field energies for intermolecular interactions. The fourth term accounts for the internal steric energy of the ligand molecule. The energies used and reported by AutoDock should be distinguished since there are docked energies, which include the intermolecular and intramolecular interaction energies, and are used during dockings to predict free energies; including the intermolecular energy and torsional free energy, and are only reported at the end of a docking [11]. Results and discussion Fluorescence quenching mechanism of BSA by VEN Fluorescence spectroscopy has been usually used in the study of molecular interactions between ligands and proteins, mainly due to the high sensitivity of this methodological approach and the variety of parameters related with the interaction molecular that can be obtained, so in this work fluorescence measurements were carried out to investigate the binding mechanism of VEN with BSA. The fluorescence spectra of BSA with varying concentrations of VEN are shown in Fig. 2. The fluorescence of BSA regularly decreased with the increasing concentration of VEN, but no significant shift of the emission maximum wavelength was observed, indicating that VEN interacted with BSA and quenched its intrinsic fluorescence. Fluorescence quenching is the decrease of the quantum yield of fluorescence from a fluorophore induced by a variety of molecular interactions with quencher molecule. Under the conditions of fixed pH, temperature and ionic strength, fluorescence quenching may result from ground complex formation, energy transfer and dynamic quenching processes [12]. Dynamic quenching refers to a process that the fluorophore and the quencher come into contact during the lifetime of the excited state, whereas static quenching refers to fluorophore–quencher complex formation. One way to distinguish static quenching from dynamic quenching is to examine their differing dependence on temperature [13]. Dynamic quenching depends upon diffusion: higher temperatures result in larger diffusion coefficients. As a result, the bimolecular quenching constants are expected to increase with temperature rising but the reverse effect would be observed for static quenching.
Fig. 2. Fluorescence spectra of BSA in the absence and the presence of VEN, [BSA] = 5 106 M and [VEN] = 0.0–4.7 105 M.
In order to confirm the type of BSA fluorescence quenching between VEN and BSA, the fluorescence quenching data were analyzed using the Stern–Volmer equation [14]:
F0 ¼ 1 þ K sv ½Q ¼ 1 þ K q s0 ½Q F where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively. Ksv is the Stern–Volmer quenching constant, which was determined by linear regression of a plot of F0/F against [Q]. Kq is the quenching rate constant of biomolecule, s0 is the average lifetime of the fluorophore without quencher, the value of s0 of the biopolymer is 108 s and [Q] is the concentration of quencher. The results in Table 1 indicates that the probable quenching mechanism of BSA by VEN involves static quenching, because Ksv is decreases with increasing temperature [15]. As described above, static quenching refers to fluorophore–quencher complex formation. This phenomenon can be described by UV–vis absorption spectra. Fig. 3 shows the absorption spectra of VEN in the presence and absence of BSA. The intensity of the spectral band of VEN was significantly enhanced in the presence of BSA (1:1 ratio), and a 4 nm red shift was noted. All the evidences indicated that the possible quenching mechanism of VEN–BSA interaction was initiated by static quenching and the VEN–BSA complex was formed. Binding constants and binding site For static quenching, the relationship between fluorescence quenching intensity and the concentration of quenchers can be described by the double logarithm regression curve [16]:
log
F0 F ¼ log K b þ nlog½Q F
where Kb is the binding constant and n is the number of binding sites per BSA. The values of Kb and n were calculated from the values of intercept and slope of the plots, respectively, and the results were listed in Table 1. Table 1 shows that Kb decreases slightly with the temperatures rising, but n is approximately equal to 1, indicating that VEN and BSA formed the mol ratio 1:1 complex and the complex would be partly decomposed with the temperature rising. Location of binding site The structure of BSA consists of three homologous domains (I– III), and each domain is composed of two subdomains (A and B). There are two major specific ligands-binding sites in BSA and the principal regions usually located in hydrophobic cavities in subdomains IIA and IIIA, which are called site I and site II, respectively [17]. To identify the VEN binding site on BSA, the site marker competitive experiment was carried out. We used ibuprofen and warfarin which specifically bound to a known site on BSA as site marker fluorescence probes to monitor sites II and I of BSA, respectively [18]. For this, varied amounts of the VEN were added to a solution containing fixed amounts of BSA and site probe (5 106 M) and fluorescence intensities were noted down upon excitation at 290 nm (Figs. 4 and 5). The binding constant values were evaluated. The corresponding values of binding constant (Kb) at 278 K were obtained to be 7.37 102 L mol1 for ibuprofen–VEN–BSA system and 1.33 104 L mol1 for warfarin–VEN– BSA system. Compared with the binding constant, 1.37 104 L mol1of VEN–BSA system, the value of Kb decreased remarkably upon the addition of ibuprofen, whereas warfarin had a little effect on the binding of VEN to BSA. The results meant that the binding site for VEN and ibuprofen was the same in BSA; that is to say, the VEN was most likely bound to the hydrophobic pocket located in subdomain IIIA (site II).
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Table 1 The quenching constants (KSV), number of binding sites (n), binding constants (Kb) and relative thermodynamic parameters for the interaction of VEN with BSA at different temperatures. T(K) 278 288 310
KSV (L mol1) 103 4.14 3.39 2.40
Kq (L mol1) 1011 4.14 3.39 2.40
n 1.11 1.03 0.72
Log Kb 4.14 3.77 2.19
Kb 4
1.37 10 5.92 103 1.54 102
DG (kJ mol1)
DH (kJ mol1)
DS (j mol1 k1)
22.68 19.77 13.36
103.69
291.41
Thermodynamic analysis for detection of binding mode There are essentially four types of non-covalent interactions that play a key role in binding ligand to proteins. These are hydrogen bonds, Van der Waals forces, electrostatic and hydrophobic bonds interactions [19]. The force acting may be predicted by knowing the value of the enthalpy change (DH) and entropy change (DS), which can be evaluated using the van’t Hoff equation [20]:
ln K b ¼ Fig. 3. Absorption spectra of VEN in the absence and the presence of BSA, [BSA] = [VEN] = 1 105 M.
DH DS þ RT R
where Kb is the binding constant at the corresponding temperature and R is the gas constant. DH and DS can be calculated from the slope and ordinate of the plot of ln Kb versus 1/T, respectively. The free energy change (DG) is calculated from the Gibbs–Helmholtz relationship:
DG ¼ DH DTS The values of DH, DG and DS are listed in Table 1. The negative sign of DG values supported the assertion that all binding processes are spontaneous. The negative value of DH (103.69 kJ mol1) indicated that the binding reaction of VEN and BSA was exothermic. This meant that the higher the temperature, the weaker the bound between VEN and BSA, which was also verified by the decreasing values of Kb with increase in temperature. The formation of hydrogen bonds would be a great negative source of DH and DS, so that the negative values of DH and DS indicated that both hydrogen bond and van der Waals forces played an important role in the binding of VEN and BSA [21]. Fluorescence resonance energy transfer (FRET) from BSA to VEN Fig. 4. Effect of site marker to VEN–BSA system; [ibuprofen] = [BSA] = 5 10 and [VEN] = 0.0–4.7 105 M.
6
M
Fluorescence resonance energy transfer (FRET) is a distance dependent interaction between the different electronic excited states of molecules in which excitation energy is transferred from one molecule (donor) to another molecule (acceptor) without emission of a photon from the former molecular system. The efficiency of FRET depends mainly on the following factors: (i) the extent of overlap between the donor emission and the acceptor absorption, (ii) the orientation of the transition dipole of donor and acceptor and (iii) the distance between the donor and acceptor. The spectral studies suggested that BSA forms complex with VEN. In order to evaluate the distance, r between fluorophore (donor) of BSA and bound drug (acceptor), the fluorescence resonance energy transfer (FRET) is used. The overlap of the UV–vis absorption spectrum of VEN with the fluorescence emission spectrum of BSA is shown in Fig. 6. The efficiency of energy transfer between the donor and acceptor, E, could be calculated by the following equation [22]:
E¼ Fig. 5. Effect of site marker to VEN–BSA system; [warfarin] = [BSA] = 5 106 M and [VEN] = 0.0–4.7 105 M.
R60 R60
þ
r6
¼1
F F0
where E denotes the efficiency of energy transfer between the donor and the acceptor, and R0 is the critical distance when the
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Fig. 6. Spectral overlap [BSA] = [VEN] = 5 106 M.
of
VEN
absorption
with
BSA
fluorescence;
efficiency of energy transfer is 50%. The value of R60 can be calculated using the equation [23]:
R60 ¼ 8:8 1025 K 2 n4 UJ where K2 is the spatial orientation factor related to the geometry of the donor and acceptor of dipoles, and K2 = 2/3 for random orientation as in fluid solution; n is the averaged refracted index of the medium in the wavelength range where spectral overlap is significant; U is the fluorescence quantum yield of the donor; and J is the spectral overlap integral between the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor. The value of J can be calculated by the equation [24]:
R J¼
FðkÞeðkÞk4 dk R FðkÞdk
where F(k) is the corrected fluorescence intensity of the donor in the wavelength range from k to k + Dk; e(k) is the molar absorption coefficient of the acceptor at wavelength k. In the present case, n = 1.36 and U = 0.15 [25]. From above equations, J = 2.35 1015 cm3 L mol1, E = 2.21%, R0 = 2.62 nm, and r = 4.93 nm were calculated. As the binding distance r = 4.93 nm is less than 8 nm, and 0.5R0 < r < 1.5R0, the energy transfer from BSA to VEN occurred with high possibility [26]. Conformation investigation UV–vis absorption studies UV–vis absorption measurement is a very simple method to explore the structural change and to know the complex formation [27]. BSA has two absorption peaks. The strong absorption peak at about 210 nm reflects the framework conformation of the protein. The weak absorption peak at about 280 nm was concerned with the polarity of the microenvironment around tyrosine and tryptophan residues of BSA [28]. The difference absorption spectra of BSA with various amounts of VEN obtained by subtracting the corresponding spectrum of free VEN from those of VEN–BSA complex are shown in Fig. 7. It is clear from the figure that with the addition of VEN, the absorbance peak around 278 nm which mainly caused by the transition p ? p of aromatic amino acid residues in BSA are raised [29] and the maximum peak position of VEN–BSA was shifted slightly towards longer wavelength region which indicates formation of a complex between BSA and VEN and the change in polarity around the tryptophan residue. The binding constants of the VEN–BSA complex were also determined using UV–vis spectroscopic method as reported [30]. The double reciprocal plot of 1/(AA0) versus 1/[VEN] is linear and the binding constant (Kb = 4.3 104) can be estimated from the ratio of the intercept to the slope, A0 is the initial absorbance of the free BSA
Fig. 7. UV–vis spectra of BSA in the absence and the presence of VEN; [BSA] = 1 105 M and [VEN] = 0–1.9 105 M.
at 280 nm and A is the recorded absorbance at different VEN concentrations. Similar binding constants were observed for tamoxifen and its metabolites protein complexes [31]. Circular dichroism studies CD spectroscopy is a sensitive technique and is commonly used to investigate secondary structure of protein in solution. To gain a better understanding in VEN-protein binding mechanism and secondary structure changes of protein, further experiments were performed on the CD spectroscopy. CD spectra of BSA exhibit two negative bands at 208 and 222 nm that is the characteristic of a-helix structure [32]. Because they are, in the far ultraviolet region, related to the polypeptide backbone structure of proteins. And they are attributed to the n ? p transition in the peptide bond of a-helical [33]. The CD spectra of BSA in the absence and presence of rac-VEN are shown in Fig. 8. As shown in Fig. 8, the CD intensity of BSA decreased (shifting to zero levels) without any significant shift of the peaks with increasing molar ratio of VEN to BSA, which suggested that the a-helical content decreased. The CD results were expressed in terms of mean residue ellipticity (MRE) in deg cm2 dmol1 according to the following equation:
MRE ¼
Observ ed CD ðmdegÞ C p nl 10
where Cp is the molar concentration of the protein, n is the number of amino acid residues (583 for BSA) and l the path-length (0.1 cm). The a-helical contents of free and combined BSA were calculated from MRE values at 208 nm using the following equation:
a helixl ð%Þ ¼
MRE208 4000 33; 000 4000
Compared with the free BSA, it was obvious that the content of
a-helix was reduced from 65.81% to 39.48% at a molar ratio of
Fig. 8. CD spectra of BSA in the absence and the presence of VEN; [BSA] = 3 106 M and [VEN] = 0–1.8 106 M.
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Table 2 Docking results of BSA and S-VEN by using the AutoDock program generated different ligand conformers using Lamarkian Genetic Algorithm. These values show the minimum energy and should be bold in the table. Rank
Run
DG (kcal/mol)
Einter-mol (kcal/mol
Evdw (kcal/mol)
Eelec (kcal/mol)
Etotal (kcal/mol)
Etorsional (kcal/mol)
Ka (M1)
1 2 3 4 5 6 7 8 9 10
16 78 25 55 48 96 90 34 92 36
5.19 5.22 5.31 5.45 5.47 5.55 5.7 6.05 6.26 6.32
6.98 7.01 7.1 7.24 7.26 7.33 7.49 7.84 8.05 8.11
6.29 6.5 5.97 6.78 6.38 7.15 6.97 7.11 7.42 5.98
0.69 0.51 1.13 0.46 0.88 0.19 0.52 0.73 0.63 2.13
0.7 0.46 0.61 0.55 0.6 0.6 0.39 0.45 0.27 0.47
1.79 1.79 1.79 1.79 1.79 1.79 1.79 1.79 1.79 1.79
1.2 104 1.3 104 1.5 104 1.93 104 2 104 2.3 104 3.03 104 5.7 104 8.3 104 9.3 104
Table 3 Docking results of BSA and R-VEN by using the AutoDock program generated different ligand conformers using Lamarkian Genetic Algorithm. These values show the minimum energy and should be bold in the table. Rank
Run
DG (kcal/mol)
Einter-mol (kcal/mol)
Evdw (kcal/mol)
Eelec (kcal/mol)
Etotal (kcal/mol)
Etorsional (kcal/mol)
Ka (M1)
1 2 3 4 5 6 7 8 9 10
71 43 36 94 21 65 77 55 64 53
4.68 4.83 4.99 5.12 5.24 5.32 5.45 5.5 5.63 6.04
6.47 6.62 6.78 6.91 7.03 7.11 7.24 7.29 7.42 7.83
5.2 5.93 4 6.53 6.02 4.31 6.44 6.58 6.33 6.87
1.27 0.68 2.77 0.38 1.01 2.79 0.8 0.71 1.09 0.97
0.68 0.52 0.57 0.66 0.57 0.62 0.47 0.19 0.56 0.47
1.79 1.79 1.79 1.79 1.79 1.79 1.79 1.79 1.79 1.79
4.8 103 6.3 103 8.4 103 1.06 104 1.3 104 1.5 104 1.9 104 2.1 104 2.6 104 5.6 104
Table 4 Molecular docking analysis of the S and R-VEN binding on BSA. Docking parameter
S-VEN
R-VEN
Binding energy Location of binding site Distance between the VEN and Trp-134 Distance between the VEN and Trp-213 Residues surrounded VEN
5.45 kcal mol1 Subdomain IIIA (site II) 4.82 nm 4.13 nm Thr-411, Arg-412, Gln-416, Tyr-496, Val-497, Pro-498, Lys-499, Lys-533 OH group of VEN with the adjacent O atom of Gln-416 (2.01 Å)
5.12 kcal mol1 Subdomain IIIA (site II) 4.78 nm 4.11 nm Thr-411, Arg-412, Gln-416, Tyr-496, Val-497, Pro-498, Lys-499, Lys-533 OH group of VEN with the adjacent O atom of Gln-416 (1.69 Å)
Hydrogen bonding
VEN to BSA of 0.6:1. This result indicated that VEN bound with the amino acid residues of main polypeptide chain of the protein and destroyed their hydrogen bonding networks, resulting in some unfolding of the polypeptides of BSA [34]. Furthermore, the CD spectra of BSA in the absence and presence of VEN were observed to be similar in shape, implying the structure of the protein was also predominantly a-helix even after binding to VEN [35].
Molecular docking studies In order to understand the efficacy of a drug as a therapeutic agent, it is necessary to explore the binding site of that drug in proteins. S and R-VEN molecules were docked to BSA to determine the preferred binding sites on the protein. Figs. S1 and S2 shown the best energy ranked results, S and R-VEN molecules were most likely bound to the hydrophobic pocket located in subdomain IIIA (site II). For both isomers, that is, S and R-VEN we have considered one hydrogen bond between the OH group of VEN and the adjacent O atom of Gln-416, which stabilizes VEN–BSA complexes (Figs. S1(c) and S2(c)). Binding free energy (DG), total energy of the complex, (Etotal), electrostatic interactions (Eelec) and van der Waals energies (Evdw)
between both isomers of VEN and BSA were calculated on the basis of force field energy calculations and listed in Tables 2 and 3. From the docking simulation the observed free energy change of binding (DG) for the complex S-VEN–BSA and R-VEN–BSA are calculated to be 5.45 and 5.12 kcal mol1 respectively, are slightly lower than the experimental free energy of binding (6.32 kcal mol1) obtained from UV–vis spectroscopy at 298 K. The relationship between binding constant, Kbinding and the binding free energy change of binding, DGbinding is as follows:
DGbinding ¼ RTLnK binding the binding constant for VEN obtained by UV–vis studies (4.3 104 L mol1) matches roughly to the binding constant calculated by docked S and R-VEN–BSA model (1.93 104 and 1.06 104 L mol1 respectively). Finally molecular modeling presented in Table 4 and Fig. S3 shown no clear differences in the binding parameters and binding sites of S and R-VEN with BSA and indicated both isomers have similar interaction but it should be noted that the binding energy of S-VEN and BSA (5.45 kcal mol1) slightly larger than corresponding value (5.12 kcal mol1) for R-VEN and BSA.
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Conclusions In the present work, the binding of a racemic mixture of antidepressant drug ‘‘S,R-venlafaxine hydrochloride (VEN)’’ to BSA was carried out by employing spectroscopic techniques and molecular modeling under physiological conditions. These results are listed: The results of the Stern–Volmer quenching constant KSV is inversely correlated with temperature, which indicates that, the probable quenching mechanism of the VEN–BSA binding reaction is initiated by complex formation. The values of n revealed the presence of a single class of binding site on BSA for VEN. According to competitive binding experiments, the binding site is located in the hydrophobic pocket of subdomain IIIA. Negative DH and DS values indicated that the van der Waals forces and hydrogen bond formation are the predominant intermolecular forces stabilizing the complex. The distance between donor and acceptor was obtained based on fluorescence resonance energy transfer. As the binding distance r = 4.93 nm is less than 8 nm, the energy transfer from BSA to VEN occurred with high possibility. The results of UV–vis spectra and CD data indicate that the conformation of BSA molecules is changed significantly in the presence of VEN. The experimental results were in agreement with the results obtained via a molecular docking study. According to molecular docking studies both isomers have similar interaction with BSA and same binding site, from this point of view S and R isomers are equal. The biological significance of this work is evident since albumin serves as a carrier protein for multiple drugs. Since, the interaction of BSA with racemic mixture of the drug ‘‘Venlafaxine hydrochloride’’ has not been studied so far, the present study assumes importance in pharmacology and clinical medicine.
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Acknowledgments We acknowledge the financial support from Razi University Research Center and thank Miss Maryam Maghsudi for her kind assistance. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2013.11.016.
[31] [32] [33] [34] [35]
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Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Molecular modeling and spectroscopic studies on the interaction of the chiral drug venlafaxine hydrochloride with bovine serum albumin Nahid Shahabadi ⇑, Saba Hadidi Inorganic Chemistry Department, Faculty of Chemistry, Razi University, Kermanshah, Iran
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Spectroscopic techniques
In present work we have studied the interaction of racemic mixture of the antidepressant drug ‘‘S,RVenlafaxine hydrochloride (VEN)’’ with BSA.
combination with molecular modeling were used. The hydrogen bonding and weak van der Waals force played a major role in the interaction. Through the site marker competitive experiment, VEN located in subdomain IIIA of BSA. Molecular docking studies showed that both S and R isomers have similar interactions with BSA.
a r t i c l e
i n f o
Article history: Received 2 September 2013 Received in revised form 3 November 2013 Accepted 5 November 2013 Available online 16 November 2013 Keywords: Venlafaxine hydrochloride Bovine serum albumin Protein–drug interaction Multi-spectroscopy methods Molecular docking Binding mechanism
a b s t r a c t This study was designed to examine the interaction of racemic antidepressant drug ‘‘S,R-venlafaxine hydrochloride (VEN)’’ with bovine serum albumin (BSA) under physiological conditions. The mechanism of interaction was studied by spectroscopic techniques combination with molecular modeling. Stern– Volmer analysis of fluorescence quenching data shows the presence of the static quenching mechanism. The thermodynamic parameters indicated that the hydrogen bonding and weak van der Waals interactions are the predominant intermolecular forces stabilizing the complex. The number of binding sites (n) was calculated. Through the site marker competitive experiment, VEN was confirmed to be located in subdomain IIIA of BSA. The binding distance (r = 4.93 nm) between the donor BSA and acceptor VEN was obtained according to Förster’s non-radiative energy transfer theory. According to UV–vis spectra and CD data binding of VEN leaded to conformational changes of BSA. Molecular docking simulations of S and R-VEN revealed that both isomers have similar interaction and the same binding sites, from this point of view S and R isomers are equal. Ó 2013 Elsevier B.V. All rights reserved.
Introduction Serum albumins are the most abundant proteins in the circulatory system of a wide variety of organisms. Bovine serum albumin (BSA) being the major macromolecule in blood plasma of animals accounting to about 60% of the total protein corresponding to a ⇑ Corresponding author. Tel./fax: +98 831 8360795. E-mail address: [email protected] (N. Shahabadi). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.11.016
concentration of 42 g dm3 [1]. It consists of a single chain of 582 amino acids, globular nonglycoprotein cross-linked with 17 cysteine residues (eight disulfide bonds and one free thiol). It is divided into three linearly arranged, structurally distinct and evolutionarily related domains (I–III); each domain is composed of two subdomains (A and B) [2]. BSA has two tryptophans, embedded in two different domains: Trp-134, located on the surface of domain I and Trp-213, located within the hydrophobic pocket of domain II. The binding cavities associated with subdomains IIA
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and IIIA are also referred to as site I and site II according to the terminology proposed by Sudlow et al. [3]. The most exceptional property of a serum albumin is that it serves as a depot protein and a transport protein for numerous endogenous and exogenous compounds [4]. The exogenous substances that bound to protein with a high affinity are drugs. In this work, BSA is selected as our protein model because of its medically important, abundance, low cost, ease of purification, unusual ligand-binding properties, stability [5,6], and the results of all the studies are consistent with the fact that human and bovine serum albumins are homologous proteins [6–8]. Venlafaxine ‘‘1-[2-(dimethylamino)-1-(4-methoxyphenyl)ethyl] cyclohexanol hydrochloride’’ commercially known as ‘‘Effexor’’ is a representative of a new class of antidepressants. For VEN there is one chiral center. VEN is administered as a racemate, composed of equal amounts the S-(+) and R-() enantiomeric forms (Fig. 1) and is usually categorized as a serotonin–nor epinephrine reuptake inhibitor (SNRI) but it has been referred to as a serotonin– norepinephrine–dopamine reuptake inhibitor. Its higher solubility in water results in burst effect with sudden peak levels of drug in blood. In humans, 90% of the total VEN decrease is accounted for by enantioselective O-desmethylation through cytochrome P450 (CYP) CYP2D6 leading to the active metabolite O-desmethylvenlafaxine (ODV) and 10% for no-enantioselective N-desmethylation by CYP3A4, leading to N-desmethylvenlafaxine (NDV) [9]. A key process in the development of new drugs is elucidation of the nature of the interaction between the drug molecule and the target protein. Since albumin serves as a transport carrier for drugs, it is important to study the interactions of these drugs with this protein. The effectiveness of these compounds as pharmaceutical agents depends on their binding ability. Fluorescence and UV–vis absorption spectroscopy are effective techniques to study the small molecules–proteins interactions, because of their sensitivity, reproducibility and convenience. These approaches can reveal the binding affinity of small molecules with proteins and help to understand their binding mechanisms. Circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy are reliable methods for analyzing the contents of secondary conformation forms of proteins, which can explain the conformational changes of proteins induced by ligands [10]. In present work, we use several spectral methods including fluorescence, UV–vis absorption and CD spectroscopy combination with molecular docking to obtain information on the quenching mechanism, thermodynamic parameters, the special binding site and the effect of VEN on the secondary structure of BSA, which could reveal important information for the study of the structure–activity relationship of the drug and BSA. Experimental details Apparatus Fluorescence measurements were performed with a JASCO spectrofluorimeter Model FP-6200 equipped with a thermostat bath, using a 1.0 cm quartz cell. UV–vis absorption spectra were
measured on an Agilent UV–vis spectrophotometer Model 8453 using a 1.0 cm cell. CD measurements were recorded on a JASCO spectropolarimeter Model J-810, using a 0.1 cm quartz cell. pH measurements were carried out with a digital pH-meter with a combined glass-calomel electrode. Reagents Bovine serum albumin (BSA) was purchased from Sigma– Aldrich. All BSA solutions were prepared in the 0.1 M phosphate buffer (pH 7.4), BSA solutions stored in the dark at 4 °C. A stock solution of VEN was prepared by dissolving its crystals in 50 mM of the phosphate buffer at pH 7.4. Doubly distilled water was used to prepare the buffers. Procedure Fluorescence spectra Fluorescence measurements were performed by keeping the concentration of BSA constant (5 106 M) while varying the rVEN concentration from 0.0 to 4.7 105 M at different temperatures (278, 288, 310). Displacement experiments The displacement experiments were performed using the site probes including ibuprofen and warfarin by keeping the concentration of protein and probe constant (each of 5 106 M). The fluorescence quenching titration was used to determine the binding constants of VEN–BSA systems in presence of above site probes for sites I and II. UV–vis absorption studies The absorbance measurements were performed by keeping the BSA concentration constant (1 105 M) while varying the racVEN concentration from 0 to 1.9 105 M (ri = [VEN]/[BSA] = 0.0, 0.7, 1.1 and 1.9). The samples were incubated at room temperature for 10 min and the spectra were recorded in the range of 200– 400 nm. Circular dichroism (CD) measurements Circular dichroism (CD) measurements were conducted by keeping the concentration of BSA constant (3 106 M) while varying the VEN concentration from 0 to 1.8 106 M (ri = [VEN]/[BSA] = 0.0, 0.4, 0.5 and 0.6). Molecular docking MGL tools 1.5.4 with AutoGrid 4 and AutoDock 4 were used to set up and perform blind docking calculations between R and SVEN with BSA. The structure of free BSA (PDB id: 3V03, chain A) obtained by X-ray crystallography was used as a template. Receptor (BSA) and ligands (R and S-VEN) files were prepared using AutoDock Tools. The BSA was enclosed in a box with number of grid points in x y z directions, 126 126 126 and a grid spacing of 0.703 Å. Lamarckian genetic algorithms, as implemented in AutoDock, were employed to perform docking calculations. All other parameters were default settings. For each of the docking cases, the lowest energy docked conformation, according to the Autodock scoring function, was selected as the binding mode. The output from AutoDock was rendered with PyMol. In AutoDock, the overall docking energy of a given ligand molecule in its active site is expressed as follows:
X Aij Bij DG ¼ DGvdw 6 r 12 r ij ij ij þ DGelec Fig. 1. Molecular structure of R and S-venlafaxine hydrochloride.
X qi qj ij
eðrij Þrij
!
X C ij Dij þ DGhbond 10 þ Ehbond r 12 rij ij ij
þ DGtor Ntor þ DGsol
!
X ðr 2 =2r2 Þ Si V j e ij ij
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where DGvdw, DGhbond, DGelec, DGtor, and DGsol are free energy coefficients of van der Waals, hydrogen bond, electrostatic interactions, torsional term, and desolvation energy of protein–ligand complex, respectively. rij, Aij, Bij, Cij, and Dij represent the interatomic distance, the depths of energy well, and the equilibrium separations between the two atoms, respectively. The first three terms are in vacuo force field energies for intermolecular interactions. The fourth term accounts for the internal steric energy of the ligand molecule. The energies used and reported by AutoDock should be distinguished since there are docked energies, which include the intermolecular and intramolecular interaction energies, and are used during dockings to predict free energies; including the intermolecular energy and torsional free energy, and are only reported at the end of a docking [11]. Results and discussion Fluorescence quenching mechanism of BSA by VEN Fluorescence spectroscopy has been usually used in the study of molecular interactions between ligands and proteins, mainly due to the high sensitivity of this methodological approach and the variety of parameters related with the interaction molecular that can be obtained, so in this work fluorescence measurements were carried out to investigate the binding mechanism of VEN with BSA. The fluorescence spectra of BSA with varying concentrations of VEN are shown in Fig. 2. The fluorescence of BSA regularly decreased with the increasing concentration of VEN, but no significant shift of the emission maximum wavelength was observed, indicating that VEN interacted with BSA and quenched its intrinsic fluorescence. Fluorescence quenching is the decrease of the quantum yield of fluorescence from a fluorophore induced by a variety of molecular interactions with quencher molecule. Under the conditions of fixed pH, temperature and ionic strength, fluorescence quenching may result from ground complex formation, energy transfer and dynamic quenching processes [12]. Dynamic quenching refers to a process that the fluorophore and the quencher come into contact during the lifetime of the excited state, whereas static quenching refers to fluorophore–quencher complex formation. One way to distinguish static quenching from dynamic quenching is to examine their differing dependence on temperature [13]. Dynamic quenching depends upon diffusion: higher temperatures result in larger diffusion coefficients. As a result, the bimolecular quenching constants are expected to increase with temperature rising but the reverse effect would be observed for static quenching.
Fig. 2. Fluorescence spectra of BSA in the absence and the presence of VEN, [BSA] = 5 106 M and [VEN] = 0.0–4.7 105 M.
In order to confirm the type of BSA fluorescence quenching between VEN and BSA, the fluorescence quenching data were analyzed using the Stern–Volmer equation [14]:
F0 ¼ 1 þ K sv ½Q ¼ 1 þ K q s0 ½Q F where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively. Ksv is the Stern–Volmer quenching constant, which was determined by linear regression of a plot of F0/F against [Q]. Kq is the quenching rate constant of biomolecule, s0 is the average lifetime of the fluorophore without quencher, the value of s0 of the biopolymer is 108 s and [Q] is the concentration of quencher. The results in Table 1 indicates that the probable quenching mechanism of BSA by VEN involves static quenching, because Ksv is decreases with increasing temperature [15]. As described above, static quenching refers to fluorophore–quencher complex formation. This phenomenon can be described by UV–vis absorption spectra. Fig. 3 shows the absorption spectra of VEN in the presence and absence of BSA. The intensity of the spectral band of VEN was significantly enhanced in the presence of BSA (1:1 ratio), and a 4 nm red shift was noted. All the evidences indicated that the possible quenching mechanism of VEN–BSA interaction was initiated by static quenching and the VEN–BSA complex was formed. Binding constants and binding site For static quenching, the relationship between fluorescence quenching intensity and the concentration of quenchers can be described by the double logarithm regression curve [16]:
log
F0 F ¼ log K b þ nlog½Q F
where Kb is the binding constant and n is the number of binding sites per BSA. The values of Kb and n were calculated from the values of intercept and slope of the plots, respectively, and the results were listed in Table 1. Table 1 shows that Kb decreases slightly with the temperatures rising, but n is approximately equal to 1, indicating that VEN and BSA formed the mol ratio 1:1 complex and the complex would be partly decomposed with the temperature rising. Location of binding site The structure of BSA consists of three homologous domains (I– III), and each domain is composed of two subdomains (A and B). There are two major specific ligands-binding sites in BSA and the principal regions usually located in hydrophobic cavities in subdomains IIA and IIIA, which are called site I and site II, respectively [17]. To identify the VEN binding site on BSA, the site marker competitive experiment was carried out. We used ibuprofen and warfarin which specifically bound to a known site on BSA as site marker fluorescence probes to monitor sites II and I of BSA, respectively [18]. For this, varied amounts of the VEN were added to a solution containing fixed amounts of BSA and site probe (5 106 M) and fluorescence intensities were noted down upon excitation at 290 nm (Figs. 4 and 5). The binding constant values were evaluated. The corresponding values of binding constant (Kb) at 278 K were obtained to be 7.37 102 L mol1 for ibuprofen–VEN–BSA system and 1.33 104 L mol1 for warfarin–VEN– BSA system. Compared with the binding constant, 1.37 104 L mol1of VEN–BSA system, the value of Kb decreased remarkably upon the addition of ibuprofen, whereas warfarin had a little effect on the binding of VEN to BSA. The results meant that the binding site for VEN and ibuprofen was the same in BSA; that is to say, the VEN was most likely bound to the hydrophobic pocket located in subdomain IIIA (site II).
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Table 1 The quenching constants (KSV), number of binding sites (n), binding constants (Kb) and relative thermodynamic parameters for the interaction of VEN with BSA at different temperatures. T(K) 278 288 310
KSV (L mol1) 103 4.14 3.39 2.40
Kq (L mol1) 1011 4.14 3.39 2.40
n 1.11 1.03 0.72
Log Kb 4.14 3.77 2.19
Kb 4
1.37 10 5.92 103 1.54 102
DG (kJ mol1)
DH (kJ mol1)
DS (j mol1 k1)
22.68 19.77 13.36
103.69
291.41
Thermodynamic analysis for detection of binding mode There are essentially four types of non-covalent interactions that play a key role in binding ligand to proteins. These are hydrogen bonds, Van der Waals forces, electrostatic and hydrophobic bonds interactions [19]. The force acting may be predicted by knowing the value of the enthalpy change (DH) and entropy change (DS), which can be evaluated using the van’t Hoff equation [20]:
ln K b ¼ Fig. 3. Absorption spectra of VEN in the absence and the presence of BSA, [BSA] = [VEN] = 1 105 M.
DH DS þ RT R
where Kb is the binding constant at the corresponding temperature and R is the gas constant. DH and DS can be calculated from the slope and ordinate of the plot of ln Kb versus 1/T, respectively. The free energy change (DG) is calculated from the Gibbs–Helmholtz relationship:
DG ¼ DH DTS The values of DH, DG and DS are listed in Table 1. The negative sign of DG values supported the assertion that all binding processes are spontaneous. The negative value of DH (103.69 kJ mol1) indicated that the binding reaction of VEN and BSA was exothermic. This meant that the higher the temperature, the weaker the bound between VEN and BSA, which was also verified by the decreasing values of Kb with increase in temperature. The formation of hydrogen bonds would be a great negative source of DH and DS, so that the negative values of DH and DS indicated that both hydrogen bond and van der Waals forces played an important role in the binding of VEN and BSA [21]. Fluorescence resonance energy transfer (FRET) from BSA to VEN Fig. 4. Effect of site marker to VEN–BSA system; [ibuprofen] = [BSA] = 5 10 and [VEN] = 0.0–4.7 105 M.
6
M
Fluorescence resonance energy transfer (FRET) is a distance dependent interaction between the different electronic excited states of molecules in which excitation energy is transferred from one molecule (donor) to another molecule (acceptor) without emission of a photon from the former molecular system. The efficiency of FRET depends mainly on the following factors: (i) the extent of overlap between the donor emission and the acceptor absorption, (ii) the orientation of the transition dipole of donor and acceptor and (iii) the distance between the donor and acceptor. The spectral studies suggested that BSA forms complex with VEN. In order to evaluate the distance, r between fluorophore (donor) of BSA and bound drug (acceptor), the fluorescence resonance energy transfer (FRET) is used. The overlap of the UV–vis absorption spectrum of VEN with the fluorescence emission spectrum of BSA is shown in Fig. 6. The efficiency of energy transfer between the donor and acceptor, E, could be calculated by the following equation [22]:
E¼ Fig. 5. Effect of site marker to VEN–BSA system; [warfarin] = [BSA] = 5 106 M and [VEN] = 0.0–4.7 105 M.
R60 R60
þ
r6
¼1
F F0
where E denotes the efficiency of energy transfer between the donor and the acceptor, and R0 is the critical distance when the
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Fig. 6. Spectral overlap [BSA] = [VEN] = 5 106 M.
of
VEN
absorption
with
BSA
fluorescence;
efficiency of energy transfer is 50%. The value of R60 can be calculated using the equation [23]:
R60 ¼ 8:8 1025 K 2 n4 UJ where K2 is the spatial orientation factor related to the geometry of the donor and acceptor of dipoles, and K2 = 2/3 for random orientation as in fluid solution; n is the averaged refracted index of the medium in the wavelength range where spectral overlap is significant; U is the fluorescence quantum yield of the donor; and J is the spectral overlap integral between the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor. The value of J can be calculated by the equation [24]:
R J¼
FðkÞeðkÞk4 dk R FðkÞdk
where F(k) is the corrected fluorescence intensity of the donor in the wavelength range from k to k + Dk; e(k) is the molar absorption coefficient of the acceptor at wavelength k. In the present case, n = 1.36 and U = 0.15 [25]. From above equations, J = 2.35 1015 cm3 L mol1, E = 2.21%, R0 = 2.62 nm, and r = 4.93 nm were calculated. As the binding distance r = 4.93 nm is less than 8 nm, and 0.5R0 < r < 1.5R0, the energy transfer from BSA to VEN occurred with high possibility [26]. Conformation investigation UV–vis absorption studies UV–vis absorption measurement is a very simple method to explore the structural change and to know the complex formation [27]. BSA has two absorption peaks. The strong absorption peak at about 210 nm reflects the framework conformation of the protein. The weak absorption peak at about 280 nm was concerned with the polarity of the microenvironment around tyrosine and tryptophan residues of BSA [28]. The difference absorption spectra of BSA with various amounts of VEN obtained by subtracting the corresponding spectrum of free VEN from those of VEN–BSA complex are shown in Fig. 7. It is clear from the figure that with the addition of VEN, the absorbance peak around 278 nm which mainly caused by the transition p ? p of aromatic amino acid residues in BSA are raised [29] and the maximum peak position of VEN–BSA was shifted slightly towards longer wavelength region which indicates formation of a complex between BSA and VEN and the change in polarity around the tryptophan residue. The binding constants of the VEN–BSA complex were also determined using UV–vis spectroscopic method as reported [30]. The double reciprocal plot of 1/(AA0) versus 1/[VEN] is linear and the binding constant (Kb = 4.3 104) can be estimated from the ratio of the intercept to the slope, A0 is the initial absorbance of the free BSA
Fig. 7. UV–vis spectra of BSA in the absence and the presence of VEN; [BSA] = 1 105 M and [VEN] = 0–1.9 105 M.
at 280 nm and A is the recorded absorbance at different VEN concentrations. Similar binding constants were observed for tamoxifen and its metabolites protein complexes [31]. Circular dichroism studies CD spectroscopy is a sensitive technique and is commonly used to investigate secondary structure of protein in solution. To gain a better understanding in VEN-protein binding mechanism and secondary structure changes of protein, further experiments were performed on the CD spectroscopy. CD spectra of BSA exhibit two negative bands at 208 and 222 nm that is the characteristic of a-helix structure [32]. Because they are, in the far ultraviolet region, related to the polypeptide backbone structure of proteins. And they are attributed to the n ? p transition in the peptide bond of a-helical [33]. The CD spectra of BSA in the absence and presence of rac-VEN are shown in Fig. 8. As shown in Fig. 8, the CD intensity of BSA decreased (shifting to zero levels) without any significant shift of the peaks with increasing molar ratio of VEN to BSA, which suggested that the a-helical content decreased. The CD results were expressed in terms of mean residue ellipticity (MRE) in deg cm2 dmol1 according to the following equation:
MRE ¼
Observ ed CD ðmdegÞ C p nl 10
where Cp is the molar concentration of the protein, n is the number of amino acid residues (583 for BSA) and l the path-length (0.1 cm). The a-helical contents of free and combined BSA were calculated from MRE values at 208 nm using the following equation:
a helixl ð%Þ ¼
MRE208 4000 33; 000 4000
Compared with the free BSA, it was obvious that the content of
a-helix was reduced from 65.81% to 39.48% at a molar ratio of
Fig. 8. CD spectra of BSA in the absence and the presence of VEN; [BSA] = 3 106 M and [VEN] = 0–1.8 106 M.
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Table 2 Docking results of BSA and S-VEN by using the AutoDock program generated different ligand conformers using Lamarkian Genetic Algorithm. These values show the minimum energy and should be bold in the table. Rank
Run
DG (kcal/mol)
Einter-mol (kcal/mol
Evdw (kcal/mol)
Eelec (kcal/mol)
Etotal (kcal/mol)
Etorsional (kcal/mol)
Ka (M1)
1 2 3 4 5 6 7 8 9 10
16 78 25 55 48 96 90 34 92 36
5.19 5.22 5.31 5.45 5.47 5.55 5.7 6.05 6.26 6.32
6.98 7.01 7.1 7.24 7.26 7.33 7.49 7.84 8.05 8.11
6.29 6.5 5.97 6.78 6.38 7.15 6.97 7.11 7.42 5.98
0.69 0.51 1.13 0.46 0.88 0.19 0.52 0.73 0.63 2.13
0.7 0.46 0.61 0.55 0.6 0.6 0.39 0.45 0.27 0.47
1.79 1.79 1.79 1.79 1.79 1.79 1.79 1.79 1.79 1.79
1.2 104 1.3 104 1.5 104 1.93 104 2 104 2.3 104 3.03 104 5.7 104 8.3 104 9.3 104
Table 3 Docking results of BSA and R-VEN by using the AutoDock program generated different ligand conformers using Lamarkian Genetic Algorithm. These values show the minimum energy and should be bold in the table. Rank
Run
DG (kcal/mol)
Einter-mol (kcal/mol)
Evdw (kcal/mol)
Eelec (kcal/mol)
Etotal (kcal/mol)
Etorsional (kcal/mol)
Ka (M1)
1 2 3 4 5 6 7 8 9 10
71 43 36 94 21 65 77 55 64 53
4.68 4.83 4.99 5.12 5.24 5.32 5.45 5.5 5.63 6.04
6.47 6.62 6.78 6.91 7.03 7.11 7.24 7.29 7.42 7.83
5.2 5.93 4 6.53 6.02 4.31 6.44 6.58 6.33 6.87
1.27 0.68 2.77 0.38 1.01 2.79 0.8 0.71 1.09 0.97
0.68 0.52 0.57 0.66 0.57 0.62 0.47 0.19 0.56 0.47
1.79 1.79 1.79 1.79 1.79 1.79 1.79 1.79 1.79 1.79
4.8 103 6.3 103 8.4 103 1.06 104 1.3 104 1.5 104 1.9 104 2.1 104 2.6 104 5.6 104
Table 4 Molecular docking analysis of the S and R-VEN binding on BSA. Docking parameter
S-VEN
R-VEN
Binding energy Location of binding site Distance between the VEN and Trp-134 Distance between the VEN and Trp-213 Residues surrounded VEN
5.45 kcal mol1 Subdomain IIIA (site II) 4.82 nm 4.13 nm Thr-411, Arg-412, Gln-416, Tyr-496, Val-497, Pro-498, Lys-499, Lys-533 OH group of VEN with the adjacent O atom of Gln-416 (2.01 Å)
5.12 kcal mol1 Subdomain IIIA (site II) 4.78 nm 4.11 nm Thr-411, Arg-412, Gln-416, Tyr-496, Val-497, Pro-498, Lys-499, Lys-533 OH group of VEN with the adjacent O atom of Gln-416 (1.69 Å)
Hydrogen bonding
VEN to BSA of 0.6:1. This result indicated that VEN bound with the amino acid residues of main polypeptide chain of the protein and destroyed their hydrogen bonding networks, resulting in some unfolding of the polypeptides of BSA [34]. Furthermore, the CD spectra of BSA in the absence and presence of VEN were observed to be similar in shape, implying the structure of the protein was also predominantly a-helix even after binding to VEN [35].
Molecular docking studies In order to understand the efficacy of a drug as a therapeutic agent, it is necessary to explore the binding site of that drug in proteins. S and R-VEN molecules were docked to BSA to determine the preferred binding sites on the protein. Figs. S1 and S2 shown the best energy ranked results, S and R-VEN molecules were most likely bound to the hydrophobic pocket located in subdomain IIIA (site II). For both isomers, that is, S and R-VEN we have considered one hydrogen bond between the OH group of VEN and the adjacent O atom of Gln-416, which stabilizes VEN–BSA complexes (Figs. S1(c) and S2(c)). Binding free energy (DG), total energy of the complex, (Etotal), electrostatic interactions (Eelec) and van der Waals energies (Evdw)
between both isomers of VEN and BSA were calculated on the basis of force field energy calculations and listed in Tables 2 and 3. From the docking simulation the observed free energy change of binding (DG) for the complex S-VEN–BSA and R-VEN–BSA are calculated to be 5.45 and 5.12 kcal mol1 respectively, are slightly lower than the experimental free energy of binding (6.32 kcal mol1) obtained from UV–vis spectroscopy at 298 K. The relationship between binding constant, Kbinding and the binding free energy change of binding, DGbinding is as follows:
DGbinding ¼ RTLnK binding the binding constant for VEN obtained by UV–vis studies (4.3 104 L mol1) matches roughly to the binding constant calculated by docked S and R-VEN–BSA model (1.93 104 and 1.06 104 L mol1 respectively). Finally molecular modeling presented in Table 4 and Fig. S3 shown no clear differences in the binding parameters and binding sites of S and R-VEN with BSA and indicated both isomers have similar interaction but it should be noted that the binding energy of S-VEN and BSA (5.45 kcal mol1) slightly larger than corresponding value (5.12 kcal mol1) for R-VEN and BSA.
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Conclusions In the present work, the binding of a racemic mixture of antidepressant drug ‘‘S,R-venlafaxine hydrochloride (VEN)’’ to BSA was carried out by employing spectroscopic techniques and molecular modeling under physiological conditions. These results are listed: The results of the Stern–Volmer quenching constant KSV is inversely correlated with temperature, which indicates that, the probable quenching mechanism of the VEN–BSA binding reaction is initiated by complex formation. The values of n revealed the presence of a single class of binding site on BSA for VEN. According to competitive binding experiments, the binding site is located in the hydrophobic pocket of subdomain IIIA. Negative DH and DS values indicated that the van der Waals forces and hydrogen bond formation are the predominant intermolecular forces stabilizing the complex. The distance between donor and acceptor was obtained based on fluorescence resonance energy transfer. As the binding distance r = 4.93 nm is less than 8 nm, the energy transfer from BSA to VEN occurred with high possibility. The results of UV–vis spectra and CD data indicate that the conformation of BSA molecules is changed significantly in the presence of VEN. The experimental results were in agreement with the results obtained via a molecular docking study. According to molecular docking studies both isomers have similar interaction with BSA and same binding site, from this point of view S and R isomers are equal. The biological significance of this work is evident since albumin serves as a carrier protein for multiple drugs. Since, the interaction of BSA with racemic mixture of the drug ‘‘Venlafaxine hydrochloride’’ has not been studied so far, the present study assumes importance in pharmacology and clinical medicine.
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Acknowledgments We acknowledge the financial support from Razi University Research Center and thank Miss Maryam Maghsudi for her kind assistance. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2013.11.016.
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