Molecular modeling and multispectroscopic studies of the interaction of hepatitis B drug, adefovir dipivoxil with human serum albumin

Author’s Accepted Manuscript Molecular modeling and multispectroscopic studies of the interaction of hepatitis B drug, adefovir dipivoxil with human serum albumin Nahid Shahabadi, Monireh Falsafi, Saba Hadidi www.elsevier.com/locate/jlumin

PII: DOI: Reference:

S0022-2313(15)00378-6 http://dx.doi.org/10.1016/j.jlumin.2015.07.006 LUMIN13455

To appear in: Journal of Luminescence Received date: 9 January 2015 Revised date: 3 July 2015 Accepted date: 5 July 2015 Cite this article as: Nahid Shahabadi, Monireh Falsafi and Saba Hadidi, Molecular modeling and multispectroscopic studies of the interaction of hepatitis B drug, adefovir dipivoxil with human serum albumin, Journal of Luminescence, http://dx.doi.org/10.1016/j.jlumin.2015.07.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Molecular modeling and multispectroscopic studies of the interaction of hepatitis B drug, adefovir dipivoxil with human serum albumin

Nahid Shahabadi1,2*, Monireh Falsafi1, Saba Hadidi1,2 1

Department of Chemistry, Faculty of Science, Razi University, Kermanshah, Iran.

2

Medical Biology Research Center (MBRC) Kermanshah University of Medical Sciences,

Kermanshah, Iran.

* Corresponding author: Tel / Fax: +98-83-34274559 E-mail: [email protected]

1

Abstract The interaction of hepatitis B drug, adefovir dipivoxil with human serum albumin (HSA) was studied by using UV–vis, fluorometric, circular dichroism (CD) and molecular docking techniques. The results indicated that the binding of the drug to HSA caused fluorescence quenching through static quenching mechanism with binding constant of 1.3 × 103 M-1. The thermodynamic parameters indicated that the hydrophobic force contacts are the major forces in the stability of protein-drug complex (ΔH > 0 and ΔS > 0). The displacement experiments using the site probes viz., warfarin and ibuprofen showed that adefovir dipivoxil could bind to the site III of HSA. The results of CD and UV–vis spectroscopy indicated that the binding of the drug induced some conformational changes in HSA. Furthermore, the study of molecular docking also confirmed binding of adefovir dipivoxil to the site III of HSA by hydrophobic interaction.

Keywords: Adefovir dipivoxil, Human serum albumin (HSA), Fluorescence quenching, Molecular docking, Hydrophobic interaction

1. Introduction Adefovir dipivoxil was approved by the Food and Drug Administration for the treatment of chronic hepatitis B in adults with evidence of active viral replication and either evidence of persistent elevations in serum amino transferases (ALT or AST) or histologically active disease [1]. Adefovir dipivoxil is currently in phase III clinical trials for the treatment of HIV and phase II clinical trials for the treatment of Hepatitis B Virus (HBV) infections [2]. Adefovir dipivoxil, an orally bioavailable prodrug of adefovir, includes two pivaloyl oxymethyl units (Fig. 1), making it a prodrug form of adefovir [3]. Adefovir (A, 9-(2phosphono methoxy ethyl) adenine), an acyclic nucleoside phosphonate analogue is active 2

against retroviruses, hepadna viruses and herpes viruses [2]. It is known that dispense, free concentration and the metabolism of diverse drugs are strongly affected by drug–protein interactions in the blood stream [4-6]. This sort of interaction can also influence the drug stability and toxicity during the chemotherapeutic process [6]. Human serum albumin (HSA) as the most abundant carrier protein in blood plasma plays an important role in the transport and disposition of many endogenous and exogenous substances such as metabolites, drugs, and other biologically active compounds present in blood [7,8]. It often increases the solubility of hydrophobic drugs in plasma and modulates their delivery to cell in vivo and in vitro [9]. Human serum albumin (∼600 μM), is a 66.5 kDa monomer and consists of a single polypeptide chain of 585 amino acid residues with 17 tyrosyl residues and only one tryptophan (Trp) located at position 214 along the chain. It contains three homologous helical domains (I–III): I (residues 1–195), II (196–383), III (384–585) [10] which each of them divided into A and B subdomains. Subdomain A contains six helices and subdomain B with four helices. This protein is divided into nine loops by 17 disulphide bonds, and its secondary structure is dominated by α-helix [11]. The protein binds a wide variety of endogenous ligands including non-esterified fatty acids, bilirubin, hemin and thyroxine and lipophilic compounds in multiple sites. Many commonly used drugs with acidic or electronegative features (e.g. warfarin, diazepam, ibuprofen) also bind to HSA, usually at one of two primary sites (1 and 2), located in subdomains IIA and IIIA, respectively [12,13]. Plasma protein binding of drugs assumes great importance because of it influences their pharmacokinetic and pharmacodynamic properties. It may also cause interference with the binding of other endogenous and/or exogenous ligands as a result of overlap of binding sites and/or conformational changes. Therefore, detailed investigation of drug–protein interaction assumes importance for thorough understanding of the pharmacokinetic behavior of a drug and for the design of analogues with effective pharmacological properties [14-16].

3

Fluorescence spectroscopy is an appropriate method to determine the binding parameters between small molecules such as drugs and proteins. After the protein is treated by different concentrations of quenchers, quenching of the protein intrinsic fluorescence can be used to infer the binding mechanism and to calculate the number of binding sites, binding constant and thermodynamic parameters [17].To validate the binding events more vividly, molecular modeling of drug with HSA was also employed [8]. Previously, interactions of some hepatitis B drugs with HSA were investigated such as lamivudine and tenofovir. Study of HSA interaction of lamivudine by X-ray crystallography and fluorescence spectroscopy revealed that it binds in IIA subdomain of HSA mainly by forming hydrogen bonds and hydrophobic interaction forces. Hydroxyl group (15) of lamivudine forms hydrogen bond with Arg222 and amino group (4) of lamivudine forms hydrogen bond with carbonyl of Arg257 [18]. Also investigation on tenofovir demonstrated hydrophobic interaction plays a major role in the drug–HSA complex and values of the Stern–Volmer quenching constants indicate the presence of a static component in the quenching mechanism. Furthermore, the site marker competitive experiment indicated tenofovir located in site I of HSA [19]. Previously, we have carried out DNA interaction of the drug and however, so far, none of the investigations were done by determining the adefovir dipivoxil–HSA binding constants [20]. In this study, the properties of binding between adefovir dipivoxil and HSA were investigated using fluorescence quenching method, circular dichroism (CD) and UV-vis absorption spectroscopy. The aim of this study was to analyze the fluorescence quenching mechanism of HSA by adefovir dipivoxil, the number of the biding sites, the specific binding pocket, and the effects of adefovir dipivoxil on the conformational changes of HSA. In addition, the molecular modeling was used to improve the understanding of the interaction of this drug with HSA.

4

2. Material and methods 2.1. Material Human serum albumin (HSA) was purchased from Sigma and Ethanol (99.9%), Na2HPO4, NaH2PO4, Warfarin and Ibuprofen were purchased from Merck. Adefovir dipivoxil with chemical name of 9-[2-[[bis[(pivaloyloxy)methoxy]-phosphinyl]methoxy]ethyl] adenine was purchased from OSR Health care Pvt Ltd, India. It has a molecular formula of C20H32N5O8P and a molecular weight of 501.48.

2.2. Preparation of stock solutions Adefovir dipivoxil and HSA solutions were prepared in the buffer solution adjusted to pH 7.4 with 0.1 M Na2HPO4 and NaH2PO4. HSA stock solution (10-3 M, based on its molecular weight of 66,000) was prepared in 0.01 M phosphate buffer of pH 7.4 and was kept in the dark at 4 ºC. Triple distilled water was used throughout the experiment.

2.3. Methods 2.3.1. Fluorescence spectra All fluorescence spectra were recorded with a LS-55 Spectrofluorimeter (Perkin–Elmer corporate, UK) equipped with a Xenon ampuxl-159, quartz cells (1.0 cm) and a thermostat bath. We used λex = 280 nm for fluorescence quenching study. The maximal fluorescence emission of HSA was located at 347 nm. Fluorescence spectra were recorded at 283, 298 and 310 K in the range of 300–470 nm. For fluorophores that display structured emission, it is important to maintain adequate wavelength resolution, which is adjusted by the slit widths on the monochromator. The detailed structure is nearly lost when there solution is increased [21]. Thus we carried out fluorescence spectroscopy with the spectra band widths of

5

excitation and emission slits at 5.0 nm because it was minimum band width for our instrument.Since, the drug has very little absorption at 280 nm, the fluorescence intensities in this paper were corrected for absorption of the exciting light and reabsorption of the emitted light to decrease the inner filter effect using the following relationship [21]: Fcor= Fobs× e(Aex+Aem)/2

(1)

That Fcor and Fobs are the fluorescence intensities corrected and observed, respectively and Aex and Aem are the absorption of the system at the excitation and the emission wavelength, respectively. The fluorescence intensity utilized in this paper is the corrected intensity. Synchronous fluorescence spectra of HSA titrated with various concentrations of adefovir dipivoxil were recorded from 300 to 380 nm (Δλ = 60 nm) and from 260 to 350 nm (Δλ = 15 nm), at which the spectrum only showed the spectroscopic behavior of tryptophan (Trp) and tyrosine (Tyr) residues of HSA, respectively.

2.3.2. Displacement experiments The displacement experiments (n = 3 replicates) were performed at 298 K using the site probes viz., warfarin and ibuprofen by keeping the concentration of protein and also probe constant (each of 10-5M). The fluorescence quenching titration was used to determine the binding constants of drug–HSA systems in the presence of above site probes for it's I and II.

2.3.3. UV-spectrophotometry Absorbance spectra were recorded in the range 200–400 nm using an HP spectrophotometer (Agilent 8453), equipped with a thermostat bath (Huber polysat cc1). Absorption titration experiments were carried out by keeping the concentration of HSA constant (3 × 10-5 M) while varying the adefovir dipivoxil concentration from 0 to 6.0× 10-5M (ri = [drug] / [HSA] = 0.0, 0.33, 0.67, 1.3, 1.7, 2.0). Aliquots of the HSA solution were treated with the drug at

6

several input molar ratios (ri). Absorbance values were recorded after each successive addition of adefovir dipivoxil solution and equilibration (ca.3 min). Control experiments were carried out by making identical titration of the drug in the absence of HSA. The spectra of interaction were obtained by subtracting the spectra of adefovir (control system: 0 to 6.0 × 105

M adefovir in absence of HSA) from the corresponding total spectra (0 to 6.0 × 10-5M

adefovir in the presence of HSA).

2.3.4. Energy transfer between protein and adefovir dipivoxil The absorption spectra of adefovir dipivoxil (10-5 M) were recorded at 298 K in the range 300–470 nm (n = 3 replicates). The emission spectrum of HSA (10-5M) was also recorded at 298 K in the same range (n = 3 replicates) and the emission spectrum of buffer was subtracted from it in range 300-310 nm. Then, the overlap of the UV absorption spectrum of adefovir dipivoxil with the fluorescence emission spectrum of HSA was used to calculate the energy transfer.

2.3.5. Circular dichroism studies Circular

dichroism

(CD)

measurements

were

recorded

on

a

JASCO

(J-810)

spectropolarimeter (200–250 nm) and cell length path was (0.1 cm) by keeping the concentration of HSA constant (2.2 × 10-5 M) while varying the adefovir dipivoxil concentration from 0 to 3.2× 10-5 M (ri = [adefovir] / [HSA] = 0.0, 0.25, 0.5, 0.75, 1.0, 1.5).

2.3.6. Molecular docking MGL tools 1.5.4 with AutoGrid4 and AutoDock4 were used to set up and exert blind docking calculations between the adefovir dipivoxil and HSA.The known crystal structure of HSA

7

(PDB ID: 1BM) was obtained from the Brookhaven Protein Data Bank. Drug structure obtained from the Drug Bank (Drug ID: DB00718) was used for the docking studies. Receptor (HSA) and ligand (drug) files were provided using AutoDock Tools. First of all the heteroatoms including water molecules were deleted. The HSA was enclosed in a box with number of grid points in x×y×z directions, 106 × 126 × 94 and a grid spacing of 0.897 A˚. Lamarckian genetic algorithms, as accomplished 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. Visualization of the docked pose has been carried out by using PyMol molecular graphics program.

3. Results and discussion 3.1. Fluorescence quenching mechanism of HSA by adefovirdipivoxil The intrinsic fluorescence of HSA is a sensitive tool to investigate the conformation of serum albumin when its environment or structure gets change. The fluorescence of HSA is due to the tryptophan, tyrosine and phenylalanine residues. But, the intrinsic fluorescence of HSA is essentially due to tryptophan alone, because phenylalanine has a very low quantum yield and the fluorescence of tyrosine is totally quenched if it is ionized or is near an amino group, a carboxyl group, or a tryptophan residue [22,23].The wavelength 295 nm causes the excitation of tryptophan residue in HSA while wavelength 280 nm excites tyrosyl residues in addition to the tryptophan ones [23]. The excitation of HSA was of done at 280 nm wavelength.Some small molecules can change the microenvironment of tryptophan residues, which can generate changes of intrinsic fluorescence intensity of HSA. The fluorescence quenching mechanisms are mostly classified as dynamic quenching, static quenching, and dynamic and static participate in it simultaneously, respectively. Dynamic 8

quenching is mainly caused by collisional encounters between the fluorophoreand the quencher while static quenching is mainly resulted from the formation of stable compound between fluorophore and quencher. No matter which quenching mechanism can evince information about structural changes of certain amino acid residues. Static and dynamic can be distinguished by their difference constants dependence on temperature and viscosity, or presumably by the change of their fluorescence lifetimes [24]. Figure 2 shows the fluorescence emission spectra of HSA in the presence of various concentrations of adefovir dipivoxil following excitation at 280 nm. HSA exhibited strong emission with a peak at 347 nm. It was observed that the fluorescence intensity of HSA decreased significantly with increasing of concentration of adefovir dipivoxil. When HSA was titrated with different amounts of the drug, a remarkable intrinsic fluorescence decrease of HSA was observed. If it is assumed that the fluorescence quenching mechanism of HSA by adefovir dipivoxil is dynamic quenching process, fluorescence quenching can be described by Stern–Volmer equation [25]:

F0  1  K q 0 Q  1  K sv Q F

(2)

where F and F0 are the relative fluorescence intensities in the presence and absence of quencher (drug), respectively; [Q] is the concentration of quencher, Ksv is the Stern–Volmer quenching constant, which measures the efficiency of quenching. Kq is the quenching rate constant of the biomolecule (HSA), τ0 is the average lifetime of the biomolecule (HSA) in the absence of quencher evaluated at about 10 ns [26]. According to the literatures [26,27], for dynamic quenching, the maximum scatter collision quenching rate constant of various quenchers with biopolymers is 2.0 × 1010 L mol−1 s−1. The quenching rate constants of HSA induced by adefovir dipivoxil are greater than 2.0 × 1010, 9

this indicated that the quenching process might not be dynamic quenching process but it was from the formation of complex between HSA and drug. Fig. 3 shows the Stern–Volmer plots of F0/F versus quencher concentrations at different temperatures (283, 298, and 310 K) at λex = 280 nm. The Stern–Volmer curves were linear, and the slopes decreased with the temperature arising (Table 1). As we known, Ksv values decrease with an increase in temperature for static quenching, though the reverse effect would be observed for dynamic quenching [28, 29]. The Ksv values,in our study, decreased with the increase in temperature, which indicated that the quenching process was static quenching process.

3.2. Binding constant and binding sites When small molecules such asdrugs are bound independently to a set of equivalent sites on a macromolecule, relationship between free and bound molecules can be described by following equation [30]:

Log(F0−F)/F=log Kb + n log[L]

(3)

here Kb is the binding constant, [L] is the concentration of the free drug and n is the number of binding sites. F0 and F have the same meanings with Eq. (3). As it is difficult to obtain the values of [L], for increasing accuracy of the calculation, we applied a modified equation as follows[31]:

(4) Log

F0  F F F  n lg K b  nLog{[ Dt ]  n 0 [ H t ]} F F0

where [Ht] and [Dt] denote the total concentration of the HSA and drug in the reaction system. The supposed value of n in the bracket is equal to 1, the curve of log(F0-F)/F versus 10

log{[Dt]-[Ht](F0-F)/F0} is depicted and fitted linearly, then the value of n can be obtained from the slope of the plot. If the n value obtained is not equal to 1, then it is substituted into the bracket and the curve of log(F0-F)/F versus log{[Dt]-n[H](F0-F)/F0} is drawn again. The above process was repeated again and again till n was procured only a single value or a circulating value. Based on the values of n at different temperatures obtained, the binding constant Kb can be also obtained. The result was shown in Table 1. The results illustrate that the binding constant between the drug and HSA decreases with respect to increasing the temperature.The values of n approximately equal to 1 indicated the existence of just a single binding site in HSA for adefovir dipivoxil.

3.3. Thermodynamic parameters and nature of the binding forces The interaction forces between drug molecule and HSA include hydrogen bond, van der Waals force, electrostatic force and hydrophobic force [32]. The thermodynamic parameters can be calculated from van’t Hoff equation:

ln Kb= H/RT+ S/R

G = H T S

(5)

(6)

where Kb is the binding constant at the corresponding temperature and R is the gas constant. ∆H, ∆G and ∆S are standard enthalpy change, standard free energy change and standard entropy change, respectively. The experiments were carried out at 283, 298, and 310 K, and the thermodynamic parameters were calculated and summarized in Table 1. The negative value of ∆G revealed that the interaction process was spontaneous. The positive ΔH and ΔS values indicated that hydrophobic forces play main roles in the binding of adefovir dipivoxil to protein [33].

11

3.4. Site-selective binding of adefovir dipivoxil on HSA In the structure of HSA, there are two primary sites called site I (classical example is warfarin) and site II (classical example is ibuprofen) for binding a wide variety of drugs. The inside wall of the pocket of subdomain IIA corresponding to the so-called site I is formed by hydrophobic side chains. Site II corresponds to the pocket with hydrophobic side chains in subdomain IIIA. This is almost the same size as site I. The third site in D-shaped cavity of subdomain IB named site III is typical for binding hemin[34]. To identify the adefovir dipivoxil binding site on HSA, the site marker competitive experiment was carried out by warfarin and ibuprofen [35]. Then the binding site information of adefovir dipivoxil on HSA could be obtained from the changes in the fluorescence. During the site marker competitive experiment, drug was gradually added to the solution of HSA and site markers held in equimolar concentrations (10-5M). With the addition of warfarin, the maximum emission wavelength of HSA had a slight red shift (345 nm to 353 nm) and the fluorescence intensity was obviously lower than that of without warfarin. After adding adefovir dipivoxil regularly, the fluorescence intensity of HSA gradually decreased but this decreasing is low. On the other hand, there was not change with the addition of ibuprofen, indicated that ibuprofen did not prevent the binding of adefovir dipivoxil in its usual binding location. In addition, the binding constants of competitive experiments were calculated and were 2.2×103 M-1 for warfarin and 2.2×103 M-1 for ibuprofen. Thus, in the presence of warfarin and ibuprofen, the binding constants were greater than binding constant in the presence of adefovirdipivoxil. It demonstrated that the binding of adefovirdipivoxil to HSA did not locate within site I or II of HSA and located in site III (subdomain IB) [35,36].

3.5. Synchronous Fluorescence of HSA with adefovir dipivoxil

12

Since synchronous fluorescence spectroscopy can provide fingerprints of complex samples, it was used to characterize complex mixtures. It gives information about the molecular environment in the vicinity of the chromospheres, and has several advantages, such as sensitivity, spectral simplification and avoiding different perturbing effects. When Δλ between the excitation wavelength and the emission wavelength were set at 15 or 60 nm, the synchronous fluorescence could get characteristic information of the tyrosine residues or tryptophan residues in HSA, respectively. We could explore the conformational changes of HSA, by investigating the synchronous fluorescence spectra of tyrosine residues and tryptophan residues [34]. The effect of adefovir dipivoxil on the synchronous fluorescence spectrum of HSA is shown in Figs. 4 (a,b). Fig. 4a shows that the maximum emission wavelength of tyrosine residues does not show a significant shift. In contrast, an obvious red shift (from 342 to 344 nm) of tryptophan residues (Fig. 4b) was observed, which indicated that the polarity around the tryptophan residues was increased and the hydrophobicity was decreased, while the microenvironment around the tyrosine residues had no discernable change during the binding process. Moreover, the fluorescence intensity decreased regularly with the addition of adefovir dipivoxil, which further demonstrated the occurrence of fluorescence quenching in the binding process. Also quenching of the HSA synchronous fluorescence by adefovir dipivoxil was higher when Δλ was 60 nm (16.4 %) than 15 nm (6.4 %), indicating that a significant contribution of the tryptophan residue in the fluorescence of HSA, adefovir dipivoxil was closer to the tryptophan residue compared to the tyrosine residue [37].

3.6. UV-vis absorption spectroscopy The UV-vis absorption measurement is a simple, but effective method in recognizing complex formation. A complex formed between adefovir dipivoxil and HSA was evident 13

from the data of the UV-vis absorption spectra (Fig. 5). The UV absorption intensity of HSA increased with the variation of drug concentration. Further, slightly red shift of maximum peak position was also noticed possibly due to complex formation between the drug and HSA [38,39].

3.7. Tryptophan Fluorescence Resonance Energy Transfer (FRET) from HSA to adefovir dipivoxil According to Förster’snon-radiactive energy transfer theory, the energy transfer is possible when the fluorescence emission spectrum of the donor (fluorophore) and UV absorption spectrum of the acceptor (drug) have suitable overlap, and the donor and the acceptor are within the characteristic Förster distance [40]. The fluorescence spectrum of HSA (10-5 M) and absorption spectrum of adefovir dipivoxil (10-5 M) were scanned between 300 to 450 nm then buffer emission at 300 to 310 nm was subtracted from HSA emission spectrum. The spectral overlap of the donor (W214 of HSA) and acceptor adefovir dipivoxil is shown in Fig. 6. The efficiency of energy transfer (E) is calculated using the following equation:

E = 1- F/F0 = R06 / (R06 + r6)

(7)

Where F and F0 are the fluorescence intensities of HSA in the presence and absence of adefovir dipivoxil, r is the distance between the acceptor and the donor, and R0 is the critical distance when the energy transfer efficiency is 50%. R0 can be calculated using the equation:

R0  8.8 1025 K 2 N 4J 6

(8)

Where K2 is the spatial factor of orientation (2/3), n is the refractive index of the medium (1.33) and Φ is the fluorescence quantum yield of the donor (0.118). The overlap integral of 14

the fluorescence emission spectrum of the donor and absorption spectrum of the acceptor, J is calculated from the equation:

 F ( ) ( ) d J ( )   F ( )d 4

d

a

(9)

d

Here F(λ) is the fluorescence of the donor (HSA) in the wavelength range λ and λ+dλ, and ε(λ) is the molar extinction coefficient of the acceptor (drug) at the wavelength λ. From Equations (5)-(7), J, R0 (nm), E and r (nm) were calculated [35]. J = 1.42 10-14cm3 l mol−1, E = 0.229, R0 = 3.82 nm, and r = 4.67 nm. The FRET mechanism allowed for the determination of the distance between the HSA and the bound drug. The value obtained for the distance from the ligand to the tryptophan residue of the protein, r < 7 nm, 0.5R0< r < 1.5 R0, indicated that the energy transfer from HSA to adefovir dipivoxil occurs with high possibility [41]. 3.8. Changes of the HSA secondary structure induced by adefovir dipivoxil binding from CD To obtain more information on the binding of adefovir dipivoxil to HSA, CD spectroscopy was used to study the structure of HSA and adefovir dipivoxil–HSA complex. CD is a sensitive technique to monitor the conformational change in the protein. The CD spectra of HSA and adefovir dipivoxil–HSA complex were shown in Fig. 7. The CD spectrum of HSA exhibit two negative bands in the ultraviolet region at 208 and 222 nm, which is a characteristic of α-helical structure of protein. The reasonable explanation is that the negative peak in 208–209 nm is for π→π* transfer and peak of 222–223 nm is contributed by n→π* transfer for the peptide bond of α-helical [8, 41]. The addition of adefovir dipivoxil to HSA leads to a decrease in the ellipticity without significant shift of the peaks, indicating that the binding of adefovir dipivoxil to HSA induces a decrease in the α-helical content of HSA. However, the CD spectra of HSA in the presence and absence of adefovir dipivoxil were 15

similar in shape, which means the structure of HSA was also predominated by α-helix after the binding of adefovir dipivoxil. The CD results were expressed in terms of mean residue ellipticity (MRE) in degcm2 dmol−1 according to the following equation:

MRE 

observedCD(m deg ree) C p nl  10

(10)

Where Cp is the molar concentration of the protein (HSA), n is the number of amino acid (585 for HSA) residues of the protein and l is the path length (1 cm). The α-helical contents of free and combined HSA were calculated from MRE values at 208 nm using the (Eq. (11)): (11)   Helix (%) 

MRE208  4000  100 33000  4000

The calculated secondary structure content of HSA was 54.32% α-helical. With the addition of the adefovir dipivoxil to HSA caused the reduction of the α-helical from 54.32% to 51.15%. The decrease of α-helical content for HSA and adefovir dipivoxil indicated that the binding of adefovir dipivoxil to the HSA induced some conformational changes, but the secondary structure of HSA remains predominantly α-helix [42, 43].

3.9. Molecular Docking Analysis Molecular docking technique is an attractive method to understand the ligand–protein interactions which can corroborate our experimental results. The docking programs, when used prior to experimental screening to reduce labor and cost needed for the development of effective medicinal compounds. It can also use after experimental screening for help in better understanding of bioactivity mechanisms [44].

16

Some of the ranked results are shown in Table 2 and the docked structure (Fig. 8) shows that adefovir dipivoxil is located within the binding pocket of site 3 which is surrounded by the amino acid residues such as, Arg 117, Val 120, Met 123, Tyr 161, Phe 165, Arg 166, Leu-182 (Fig. 9). The docking result shows that the drug mainly interacts with protein through hydrophobic interaction that is in agreement with our conclusion of thermodynamic analysis (Table 1). The distance between Trp-214 of HSA and adefovir dipivoxil in minimum energy docking pose, presented by stick model using pymol, rest of the protein is shown by transparent surface (Fig. 10). From the docking calculation, the conformer with minimum binding energy and maximum abundance was 38th run among the 50 runs. From the docking simulation the observed free energy change of binding (ΔG) for the complex HSA+ drug obtained -3.68kcal mol-1. It is lower (as magnitude) than the experimental free energy of binding (-4.3 kcal mol1

) obtained from the fluorescence data. This apparent mismatch in the free energy changes

could be due to the exclusion of the solvent and/or rigidity of some other receptor HSA in the molecular docking studies. Basic formula of binding constant and Gibbs free energy is: ΔG = -RTLnKb

(12)

the binding constant obtained by fluorescence data (1.3 × 103 Lmol-1) matches roughly to the binding constant calculated by docked drug–HSA model (5.1 × 102 Lmol-1). Hence, it can be concluded that drug–HSA docked model is in approximate correlation with our experimental results. 5. Conclusion The interaction of adefovir dipivoxil with HSA was studied using UV–vis, fluorescence and CD spectroscopy and also molecular modeling. The experimental results of fluorescence showed that the quenching of HSA by adefovir dipivoxil is a result of the formation of HSA– 17

drug complex. The thermodynamic parameters (ΔH> 0 and ΔS > 0) hydrophobic forces play main roles in the binding of adefovir dipivoxil to HSA. Synchronous fluorescence spectroscopy indicated that the polarity around the tryptophan residues was increased and the hydrophobicity was decreased, while the microenvironment around the tyrosine residues had no discernable change during the binding process. The absorption spectrum of the HSA shows that the adefovir dipivoxil led to the increase in absorbance of HSA at 280 nm, which indicates formation of a complex between HSA and drug and change in protein conformation. The decrease of α-helical content for HSA and drug indicated that the binding of adefovir dipivoxil to the HSA induced some conformational changes, but the secondary structure of HSA remains predominantly α-helix. Displacement experiments predicted that the binding of adefovir dipivoxil to HSA is located within domain IB: site III. The docking results exhibited that adefovir dipivoxil binds with HSA in site III by hydrophobic forces. Thus results of molecular docking confirm spectroscopic data and provide useful information about drug-HSA interaction.

Acknowledgments Financial support from Razi University Research Center is gratefully acknowledged.

References [1] P. Marcellin, T.T. Chang, S.G. Lim, Adefovirdipivoxil for the treatment of hepatitis B e antigen-positive chronic hepatitis B, N. Engl. J. Med. 348 (2003) 808–16. [2] A.S. Mulato, J.M. Cherrington, Anti-HIV activity of adefovir (PMEA) and PMPA in combination with antiretroviral compounds: in vitro analyses, Antiviral Research 36 (1997) 91-7.

18

[3] R.J. Gilson, K.B. Chopra, A.M. Newell, A placebo-controlled phase I/II study of adefovirdipivoxil in patients with chronic hepatitis B virus infection, J. Viral Hepat, 6 (1999) 387-395. [4] B.P. Kamat, Study of the interaction between fluoroquinolones and bovine serum Albumin, J. Pharm. Biomed. Anal. 39 (2005) 1046–1050. [5] P.N. Naik, S.A. Chimatadar, S.T. Nandibewoor, Study on the interaction between antibacterial drug and bovine serum albumin: A spectroscopic approach, Spectrochim. Acta A 73 (2009) 841–845. [6] P.N. Naik, S.A. Chimatadar, S.T. Nandibewoor, Interaction between a potent corticosteroid drug – Dexamethasone with bovine serum albumin and human serum albumin: A fluorescence quenching and fourier transformation infrared spectroscopy study, J. Photochem. Photobiol. B 100 (2010) 147–159 [7] D.C. Cater, J.X. Ho, Structure and ligand binding properties of human serum albumin, Adv. Protein Chem. 45 (1994) 153–203. [8] C. Wang, Q. Chu, Changyun Chen, Z. Bo, Investigation of the mechanism of binding of thiacloprid to human serum albumin using spectroscopic techniques and molecular modeling methods, Spectroscopy 25 (2011) 113–122. [9] M. Guo, J.W. Zou, P.G. Yi, Z.C. Shang, G.X. Hu, Q.S. Yu, Binding interaction of gatifloxacin with bovine serum albumin, Anal. Sci. 20 (2004) 465–470. [10] S. Sugio, A. Kashima, S. Mochizuki, M. Noda, K. Kobayashi, Crystal structure of human serum albumin at 2.5 °A resolution, Protein Engineering, 12 (1999) 439-446. [11] X. Zhou, Q. Yang, X. Xie, Q. Hu, F. Qi, Z. Ur Rahman, X. Chen, NMR, multispectroscopic and molecular modeling approach to investigate the complexes between C.I. Acid Orange 7 and human serum albumin in vitro, Dyes Pigments 92 (2012) 11001107.

19

[12] S. Naveenraj, S. Anandan, Binding of serum albumins with bioactive substances– Nanoparticles to drugs, J. Photochem. Photobiol.C 14 (2013) 53– 71. [13] 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. [14] P.N. Naik, S.A. Chimatadar, S.T. Nandibewoor, Pharmacokinetic study on the mechanism of interaction of sulfacetamide sodium with bovine serum albumin: a spectroscopic method, Biopharma. Drug Dispos. 31 (2010) 120-128. [15] S. Tabassum, W. M. Al-Asbahy, M. Afzal, F. Arjmand, R. H. Khan,Interaction and photo-induced cleavage studies of a copper basedchemotherapeutic drug with human serum albumin:spectroscopic and molecular docking study, Mol. BioSyst. 8(2012) 2424−2433. [16] F. Arjmand, P. Tewatia, M. Aziz,R.H. Khan, Interaction studies of a novel Co(II)-based potential chemotherapeutic agent with human serum albumin (HSA) employing biophysical techniques, Med. Chem. Res. 19 (2010) 794-807. [17] W. Yu , L. Shi, G. Hui, F. Cui, Synthesis of biological active thiosemicarbazone and characterization of the interaction with human serum albumin, J. Luminescence, 134 (2013) 491-497. [18] M. Li, E.M. Auley, Y. Zhang, L.L. Kong, F. Yang, Z.P. Zhou, X.Y. Wu, H. Liang, Comparison of binding characterization of two antiviral drugs to human serum albumin, Chem. Bio.Drug Design, 83 (2014) 576-582. [19] N. Shahabadi, S. Hadidi, Study on the interaction of antiviral drug ‘Tenofovir’ with human serum albumin by spectral and molecular modeling methods, Spectrochim. Acta A 138 (2015) 169-175.

20

[20] N. Shahabadi, M. Falsafi, Experimental and molecular docking studies on DNA binding interaction of adefovirdipivoxil: Advances toward treatment of hepatitis B virus infections, Spectrochim.ActaA 125(2014) 154–159. [21] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Springer Science+ Business Media, New York, 3rd edn, 2006. [22] F.L. Cui, Y.H. Yan, Q.Z. Zhang, G.R. Qu,J. Du, X.J. Yao, A study on the interaction between 5-Methyluridine and human serum albumin using fluorescence quenching method and molecular modeling, J. Mol. Mod. 16 (2010) 255-262. [23] M. Weert, L. Stella, Fluorescence quenching and ligand binding: A critical discussion of a popular methodology, J. Mol. Struct. 998 (2011) 144–150. [24] T. Chatterjee, A. Pal, S. Dey, B.K. Chatterjee, P. Chakrabarti, Interaction of Virstatin with Human Serum Albumin: Spectroscopic Analysis and Molecular Modeling, PLoS One, 7 (2012) 37468. [25] Y.J. Hu, Y. Liu, R.M. Zhao, Spectroscopic studies on the interaction between methylene blue and bovine serum albumin, J. Photochem. Photobiol. A 179 (2006) 324–329. [26] Y. Shi, H. Liu, M. Xu, Z. Li, G. Xie, L. Huang, Z. Zeng, Spectroscopic studies on the interaction between an anticancer drug ampelopsin and bovine serum albumin, Spectrochim. ActaA 87 (2012) 251– 257. [27] Y.J. Hu, Y. Liu, L.X. Zhang, R.M. Zhao, S.S. Qu, Studies of interaction betweencolchicine and bovine serum albumin by fluorescence quenching method, J. Mol. Struct. 750 (2005) 174–178. [28] J. Liu, J. Tian, X. Tian, Z. Hu, X. Chen, Interaction of isofraxidin with human serum albumin, Bioorg. Med. Chem. 12 (2004) 469–474.

21

[29] X. Wang, X. Xie, C. Ren, Y. Yang, X. Xu, X. Chen, Application of molecular modeling and spectroscopic approaches for investigating binding of vanillin to human serum albumin, Food Chem. 127 (2011) 705–710. [30] X.Z. Feng, R.X. Jin, Y. Qu, X.W. He, Studies on the ion effect on the binding interaction between HP and BSA, Chem. J. Chin. Univ. 17 (1996) 866–869. [31] M. Guo, J.W. Zou, P.G. Yi, Z.C. Shang, G.X. Hu, Q.S. Yu, Binding interaction of gatifloxacin with bovine serum albumin, Anal. Sci. 20 (2004) 465-470. [32] J. Zhao, X. Jiang, X. Liu, F. Ren, Investigation of the effects of temperature and ions on the interaction between ECG and BSA by the fluorescence quenching method, Arch. Biol. Sci. 63 (2011) 325-331. [33] J. Toneatto, G.A. Argüello, New advances in the study on the interaction of [Cr(phen)2(dppz)]3+ complex with biological models; association to transporting proteins, J. Inorg. Biochem. 105 (2011) 645–651. [34] J.L. Yuan, Z. Lv, Z.G. Liu, Z. Hu, G.L. Zou, Study on interaction between apigenin and human serum albumin by spectroscopy and molecular modeling, J. Photochem. Photobiol. A 191 (2007) 104–113. [35] Z. Cheng, R. Liu, X. Jiang, Spectroscopic studies on the interaction between tetrandrine and two serum albumins by chemometrics methods, Spectrochim. Acta A 115 (2013) 92–105. [36] Q.L. Zhang, Y.N. Ni, S. Kokot, Molecular spectroscopic studies on the interaction between Ractopamine and bovine serum albumin, J. Pharma. Biomed.Anal. 52 (2010) 280-288. [37] F. Ge, L. Jiang, D. Liu, C. Chen, Interaction between Alizarin and Human Serum Albumin by Fluorescence Spectroscopy, Anal. Sci. 27 (2011) 79-84.

22

[38] Y. Yuea, J. Liu, J. Fan, X. Yao, Binding studies of phloridzin with human serum albumin and its effect on the conformation of protein, J. Pharma. Biomed.Anal.56 (2011) 336–342. [39] F. JanatiFard , Z. MashhadiKhoshkhoo , H. Mirtabatabaei , M.R. HousainDokht , R. Jalal, H. EshtiaghHosseini, M.R. Bozorgmehr, A.A. Esmaeili, M. JavanKhoshkholgh, Synthesis, characterization and interaction of N,N0-dipyridoxyl (1,4-butanediamine) Co(III) salen complex with DNA and HSA, Spectrochim.Acta A 97 (2012) 74–82. [40] T. Foِrster, O. Sinanoglu, Modern Quantum Chemistry, vol. 93, Academic Press, New York, 1996. [41] N. Shahabadi, A. Khorshidi, N. HossinpourMoghadam, Study on the interaction of the epilepsy drug, zonisamide with human serum albumin (HSA) by spectroscopic and molecular docking techniques, Spectrochim. Acta A 114 (2013) 627–632. [42] Y. Li, X. Yao, J. Jin, X. Chen, Z. Hu, Interaction of rhein with human serum albumin investigation by optical spectroscopic technique and modeling studies, Biochim. Biophys.Acta, 1774 (2007) 51–58. [43] P. Sevilla, J.M. Rivas, F. García-Blanco, J.V. García-Ramos, S. Sánchez-Cortés, Identification of the antitumoral drug emodin binding sites in bovine serum albumin by spectroscopic methods, Biochim. Biophys.Acta, 1774 (2007) 1359–1369. [44] S. Jana, S. Dalapati, S. Ghosh, N. Guchhait, Binding interaction between plasma protein bovine serum albumin and flexible charge transfer fluorophore: A spectroscopic study in combination with molecular docking and molecular dynamics simulation, J. Photochem. Photobio.A 231 (2012) 19– 27.

23

Table.1. The binding constants, number of binding sites (n), thermodynamic parameters and Stern– Volmer dynamic quenching constants of HSA-drug system at different temperatures with λex = 280 nm.

T(K)

KSv×10-3

283

4.6

298

310

Kq×10-12

Kb×10-3

∆G0(kJmol-1)

4.6

0.35

-13.7

4.0

4.0

1.3

-18.0

2.9

2.9

4.2

-21.4

24

∆H0(kJmol-1)

66.62

∆S0(Jmol-1K-1)

283.92

n

R2

1.02

0.99

1.12

0.99

1.22

0.99

Table.2. Docking Summary of HSA with adefovir dipivoxil by the AutoDock Program Generating Different Ligand Conformers Using a Lamarkian GA rank

run

Binding energy

a

Ki

(kcalM-1)

Ligand

Cluster

Reference

efficiency

rmsd

rmsd

1

38

-3.68

2.0mM

-0.11

0.00

38.41

2

43

-3.52

2.63 mM

-0.1

0.00

51.4

3

40

-2.85

8.21mM

-0.08

0.00

42.78

4

35

-2.57

13.08mM

-0.08

0.00

40.68

5

32

-2.54

13.81mM

-0.07

0.00

42.57

6

45

-2.28

21.31mM

-0.07

0.00

40.57

7

30

-2.27

21.64mM

-0.07

0.00

40.02

8

39

-2.19

24.68mM

-0.06

0.00

41.36

9

27

-2.15

26.75mM

-0.06

0.00

41.95

10

23

-1.95

37.29mM

-0.06

0.00

59.45

a

Ki is the inhibition constant.

25

Fig. 1. Chemical structure of adefovir dipivoxil.

160 140

Intensity

120 100 80 60 40 20 0 285

335

385

435

485

Wavelength (nm)

Fig. 2. The influence of adefovir dipivoxil concentration on the fluorescence intensity of HSA at λex = 280 nm. Conditions: C [HSA] = 10−5 mol L−1; C [drug] = 0-9.5×10−5 mol L−1.

26

Fig. 3.SternVolmer plots for the fluorescence quenching of HSA by adefovir dipivoxil at different temperatures at λex = 280 nm.

27

Fig .4. Synchronous fluorescence measurements for HSA in the presence of adefovir dipivoxil with Δλ = 15 nm (a) and Δλ = 60 nm (b), respectively.

28

Fig. 5. UV absorption spectra of HSA in the presence of different concentrations of adefovir dipivoxil at pH 7.4 and room temperature. C[HSA] = 3× 10-5molL-1, and C[drug] = 0.0 6.0×10-5 molL-1 (ri = [drug] / [HSA] = 0.0-2.0).

Fig. 6. Spectral overlap of adefovir dipivoxil absorption (a) with HSA fluorescence (b); C[HSA] =10-5 mol L-1 , C[drug] = 10-5 mol L-1 (T = 298 K).

29

0 200

210

220

230

240

250

-1

-2

-3

CD(mdeg)

HAS(α%=54.32) ri=0.25(α%=54.63

-4

ri=0.50(α%=53.95) ri=0.75(α%=52.53)

-5

ri=1.0(α%=51.56) ri=1.5(α%=51.15)

-6

-7

-8

-9

Wavelength(nm)

Fig. 7. CD spectra of HSA in the absence and presence of adefovir dipivoxil (ri = [drug] / [HSA]=0-1.5). C[HSA]= 2.2× 10-5molL-1 , and C[drug] = 0.0-3.2 ×10-5molL-1.

30

Fig. 8. Modeling of structure of HSA and adefovir dipivoxil. The subdomains was displayed.

Fig. 9. The hydrophobic and hydrophilic amino acid residues surrounding the adefovir dipivoxil

31

Fig. 10. The distance between Trp-214 of adefovir dipivoxil in minimum energy docking pose presented by stick model using pymol; B.

32

Highlights -The interaction of adefovir dipivoxil, drug for the treatment of HIV and HBV with human serum albumin (HSA) is investigated. - The drug bound to HSA by hydrophobic force and induced some conformational changes in HSA. - The study of molecular docking showed that adefovir dipivoxil could bind to the site III of HSA mainly

33