Phytomedicine 22 (2015) 621–630

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Supramolecular interaction of 6-shogaol, a therapeutic agent of Zingiber officinale with human serum albumin as elucidated by spectroscopic, calorimetric and molecular docking methods S.R. Feroz a,∗, S.B. Mohamad b,c, G.S. Lee a, S.N.A. Malek a, S. Tayyab a,c,∗∗ a

Biomolecular Research Group, Biochemistry Programme, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia Bioinformatics Programme, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia Centre of Research for Computational Sciences and Informatics for Biology, Bioindustry, Environment, Agriculture and Healthcare (CRYSTAL), Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia b c

a r t i c l e

i n f o

Article history: Received 3 October 2014 Revised 23 March 2015 Accepted 26 March 2015

Keywords: 6-Shogaol Zingiber officinale Human serum albumin Ligand–protein interaction Differential scanning calorimetry Molecular docking

a b s t r a c t Background: 6-Shogaol, one of the main bioactive constituents of Zingiber officinale has been shown to possess various therapeutic properties. Interaction of a therapeutic compound with plasma proteins greatly affects its pharmacokinetic and pharmacodynamic properties. Purpose: The present investigation was undertaken to characterize the interaction between 6-shogaol and the main in vivo transporter, human serum albumin (HSA). Methods: Various binding characteristics of 6-shogaol–HSA interaction were studied using fluorescence spectroscopy. Thermal stability of 6-shogaol–HSA system was determined by circular dichroism (CD) and differential scanning calorimetric (DSC) techniques. Identification of the 6-shogaol binding site on HSA was made by competitive drug displacement and molecular docking experiments. Results: Fluorescence quench titration results revealed the association constant, Ka of 6-shogaol–HSA interaction as 6.29 ± 0.33 × 104 M−1 at 25 ºC. Values of the enthalpy change (−11.76 kJ mol−1 ) and the entropy change (52.52 J mol−1 K−1 ), obtained for the binding reaction suggested involvement of hydrophobic and van der Waals forces along with hydrogen bonds in the complex formation. Higher thermal stability of HSA was noticed in the presence of 6-shogaol, as revealed by DSC and thermal denaturation profiles. Competitive ligand displacement experiments along with molecular docking results suggested the binding preference of 6-shogaol for Sudlow’s site I of HSA. Conclusion: All these results suggest that 6-shogaol binds to Sudlow’s site I of HSA through moderate binding affinity and involves hydrophobic and van der Waals forces along with hydrogen bonds. © 2015 Elsevier GmbH. All rights reserved.

Introduction Zingiber officinale, commonly known as ginger is recognized throughout history for its therapeutic properties (Bode and Zong 2011). Ginger preparations have been found beneficial in alleviating digestive ailments such as dyspepsia, colic, nausea, vomiting, gastritis and diarrhea; in addition to its role in the treatment of colds, arthritis, migraine and hypertension (Bode and Zong 2011; Chrubasik et al. 2005). Due to its prevalence in the diet, investigations about its molecular constituents have led to the discovery of 115 compounds (Bode

∗

Corresponding author. Tel.: +603 7967 5179; fax: +603 7967 4178. Corresponding author. Tel.: +603 7967 7118; fax: +603 7967 4178. E-mail addresses: [email protected] (S.R. Feroz), [email protected] (S. Tayyab). ∗∗

http://dx.doi.org/10.1016/j.phymed.2015.03.016 0944-7113/© 2015 Elsevier GmbH. All rights reserved.

and Zong 2011). Gingerols and their dehydration products, shogaols represent the major class of bioactive compounds found in fresh and dried gingers, respectively; with 6-shogaol (Fig. 1A and B) representing the main component of dried ginger (Bode and Zong 2011; Zick et al. 2008). Several investigations involving animal models have shown anticancer activities exhibited by extracts of ginger and their phenolic compounds against cancers of skin (Katiyar et al. 1996; Park et al. 1998), gastrointestinal tract (Yoshimi et al. 1992), colon (Manju and Nalini 2005) and breast (Nagasawa et al. 2002) in these animals. Recent studies showing significantly higher anticancer potency of shogaols, particularly 6-shogaol, compared to gingerols in human cancer cell lines have led to an increased interest in this family of ginger constituents (Kim et al. 2008; Rhode et al. 2007; Sang et al. 2009). Although researches on the bioactivity of 6-shogaol have been focused mainly on its anticancer potential, a number of reports have also

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Fig. 1. Chemical structure (A) and ball-and-stick model (B) of 6-shogaol.

explored its other pharmacological effects, such as antioxidant and anti-neuroinflammatory activities (Bak et al. 2012; Lantz et al. 2007). The potency of 6-shogaol as an antioxidant and antiinflammatory agent has been attributed to the presence of the α , β -unsaturated ketone moiety in its structure (Dugasani et al. 2010). Human serum albumin (HSA), the major transport protein present in the blood circulation is responsible for shuttling a wide range of compounds, including drugs and metabolites (Peters 1996). It is a single polypeptide chain of 585 amino acid residues, arranged into three domains (I, II and III), which are further compartmentalized into subdomains A and B. The facilitation of ligand transport by HSA depends largely on the presence of two principal binding regions, i.e., Sudlow’s site I and site II, situated within the hydrophobic pockets in subdomains IIA and IIIA of the protein, respectively (Carter and Ho 1994; Sudlow et al. 1975; Sugio et al. 1999). The ability of HSA to significantly interact with molecules of varying structures determines the distribution, delivery, therapeutic efficacy and elimination of a large number of compounds (Olson and Christ 1996). The presence of hydrophobic binding pockets/sites on HSA increases the apparent solubility of lipophilic molecules in the plasma and modulates their delivery to cells (Peters 1996). The effectiveness of drugs/therapeutic compounds as pharmaceutical agents is greatly dependent on their binding to HSA, which governs their stability, delivery to the target site and toxicity during the chemotherapeutic process (Banerjee et al. 2006). Both local concentration of the drug at the target site and duration of the effectual drug contribute to the magnitude of its efficacy in vivo (Lupidi et al. 2010). Furthermore, co-binding of drugs and/or displacement of other ligands as a result of drug–HSA interaction may also lead to alteration

in their respective levels in the circulation (Fasano et al. 2005). In addition, competitive binding of drugs and other ligands also influences their concentrations in the serum and may trigger potential drug-drug interactions (Kragh-Hansen et al. 2002). Therefore, detailed knowledge on the binding of drugs/therapeutic compounds to HSA in terms of their affinities, binding forces and location of binding sites is vital in understanding their pharmacokinetics and pharmacological effects. In the human body, 6-shogaol is extensively metabolized and is detected in the plasma mainly as glucuronide and sulfate conjugates. This biotransformation takes place primarily in the intestinal mucosa and in liver cells (Zick et al. 2008). In addition, several other 6-shogaol metabolites bearing close resemblance to the parent compound have been detected in lower amounts in mice as a result of further metabolism (Chen et al. 2012). Since the binding characteristics of a molecule to HSA are generally governed by its structure, metabolites possessing similar structural features would also show an analogous binding behavior to the original molecule. This is evident from previous investigations, showing comparable HSA binding characteristics of metabolites, including glucuronide conjugates, in terms of binding affinity and location of binding sites, to that of their parent compounds (Bourassa et al. 2011; Khan et al. 2011; Rohacova et al. 2010). Despite extensive documentation of its biological significance, the interaction of 6-shogaol with HSA is yet to be described to the best of our knowledge. In view of the pharmacological importance of this interaction, we present herewith the characteristics of the 6-shogaol– HSA interaction based on spectroscopic, calorimetric and in silico methods.

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Materials and methods Materials Defatted human serum albumin (HSA) (Lot #068K7538V), phenylbutazone (PBZ) (Lot #124K1625) and ketoprofen (KTN) (Lot #BCBG9546V) were obtained from Sigma–Aldrich Co. (St. Louis, MO). All other chemicals used were of analytical grade purity. Extraction and isolation of 6-shogaol 6-Shogaol was isolated in our laboratory from the rhizomes of Zingiber officinale Roscoe, a variety of the common ginger locally known as jahe gajah. The plant name was crosschecked with www.theplantlist.org, accessed on 31 January 2015. The rhizomes were collected from Jogjakarta, Indonesia and were authenticated by Prof. Halijah Ibrahim, a botanist at the Institute of Biological Sciences, Faculty of Science, University of Malaya. A voucher specimen (HI1364) was deposited in the Herbarium of the Institute of Biological Sciences, Faculty of Science, University of Malaya. Rhizomes of the plant were subjected to extraction and fractionation in the same way as described by Malek et al. (2011). Methanol extraction of the freshly grounded sample of the rhizomes (1000 g) was performed and the crude extract was obtained upon solvent evaporation in a rotary evaporator. The crude extract was then successively fractionated with hexane, ethyl acetate and water to yield three different fractions. The ethyl acetate fraction (3.0 g) was then subjected to vacuum liquid chromatography and 11 subfractions were obtained. Using a slightly modified method of Jolad et al. (2005), 6-shogaol (48.0 mg) was isolated from subfraction 5 via semi-preparative high performance liquid chromatography (HPLC) as yellowish oil with light odor of pungency. The identification of 6-shogaol was done by spectrometric and spectroscopic techniques followed by validation of results with those published earlier (Fleming et al. 1999; Jolad et al. 2004, 2005; Kim et al. 2008). HPLC analysis was performed on a Shimadzu liquid chromatography system (Shimadzu Corp., Kyoto, Japan) equipped with LC-10AT VP pump, SCL-10A VP system controller, SPD-M10A VP Photo 4544 Diode Array detector, DGU-12A vacuum degasser and Shimadzu LC Solution software. Chromolith Performance RP-18e columns (Merck KGaA, Darmstadt, Germany) of 100 mm × 4.6 mm and 100 mm × 10 mm dimensions were used for analytical and preparative scale separations, respectively. Analytical procedures The protein stock solution was prepared in 10 mM sodium phosphate buffer, pH 7.4 and its concentration was determined spec% of 5.3 at 280 nm (Wallevik 1973). The trophotometrically using E11 cm stock solutions of 6-shogaol, PBZ and KTN were prepared in ethanol. The working solutions were obtained by diluting the stock solutions to the desired concentration with the above buffer. All absorbance measurements were made on a Shimadzu UV-2450 double beam spectrophotometer (Shimadzu Corp., Kyoto, Japan) using quartz cuvettes of 1 cm path length. The final concentration of ethanol present in the test solutions was less than 1% and had little or no effect on HSA conformation (Taboada et al. 2007).

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Fluorescence spectra of HSA (3 μM) in the absence and the presence of increasing 6-shogaol concentrations (0–30 μM) were obtained upon excitation at 295 nm to selectively excite the lone Trp residue of HSA and recording the emission spectra in the wavelength range, 310−390 nm. The fluorescence quench titrations were carried out at four different temperatures, i.e., 15, 25, 35 and 45 ºC in the same way as described earlier (Feroz et al. 2012). Three-dimensional (3-D) fluorescence spectra of HSA (3 μM) and 6-shogaol–HSA mixture (4:1 molar ratio) were recorded by monitoring the emission spectra in the wavelength range, 220–500 nm, after exciting the samples from 220 to 350 nm in 5 nm intervals. Fluorescence spectra of appropriate blanks were also recorded in order to subtract the fluorescence contributions of the ligand and the buffer from the fluorescence spectra of the samples. Spectrofluorimetric analysis In order to nullify the inner filter effect, the fluorescence intensity values of the samples were corrected for their absorption at excitation and emission wavelengths using the following relationship (Lakowicz 2006):

Fcor = Fobs 10(Aex +Aem /2)

(1)

where Fcor and Fobs are the corrected and the observed fluorescence intensity values, while Aex and Aem represent changes in the absorbance values due to the addition of ligand at the excitation and emission wavelengths, respectively. The quenching of HSA fluorescence in the presence of increasing 6-shogaol concentrations was analyzed using the Stern–Volmer equation (Lakowicz 2006):

F0 = KSV [Q] + 1 = kq τ0 [Q] + 1 F

(2)

where F0 and F represent the fluorescence intensity values of the protein sample in the absence and the presence of the quencher, respectively, [Q] is the quencher concentration and kq is the bimolecular quenching constant. The value of τ 0 , the fluorophore lifetime of free HSA was taken as 6.38 × 10−9 s (Abou-Zied and Al-Shihi 2008). Values of the association constant, Ka , for the 6-shogaol–HSA system at different temperatures were obtained by treating the titration data according to the following double logarithmic equation (Bi et al. 2004):



log

(F0 − F )





F

= n log Ka − n log

1 ([LT ] − (F0 − F )[PT ]/F0 )



(3)

where n is the Hill coefficient, while [LT ] and [PT ] refer to the total concentration of the ligand and the protein, respectively. The thermodynamic parameters (H, enthalpy change and S, entropy change) for 6-shogaol–HSA interaction were obtained from the van’t Hoff plot using the following equation:

ln Ka = −

H RT

+

S R

(4)

where T is the absolute temperature (273+ ºC) and R is the gas constant (8.3145 J mol−1 K−1 ). The value of the free energy change, G of the binding reaction was subsequently obtained by fitting the H and S values into the following equation:

G = H − T S

(5)

Fluorescence spectroscopy

Differential scanning calorimetry

Fluorescence spectra were recorded on a Jasco FP-6500 spectrofluorometer (Jasco Corp., Tokyo, Japan) equipped with a thermostatically-regulated cell holder using a quartz cuvette of 1 cm path length. The excitation and emission band widths were set at 10 nm each.

Differential scanning calorimetry (DSC) profiles of HSA (15 μM) in the absence and the presence of 6-shogaol (60 μM) were obtained on a Nano DSC microcalorimeter (TA Instruments, New Castle, DE) equipped with 0.3 ml cells. All solutions were degassed under vacuum for 20 min before injecting them into the cells to prevent the

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formation of air bubbles. All experiments were performed at a scan rate of 1 ºC min−1 under 3 atm pressure. The baseline scans were made by filling both sample and reference cells with the same buffer solutions. For sample scans involving 6-shogaol, an equivalent amount of the ligand (6-shogaol) was also included in the reference cell. The excess molar heat capacity, Cp , was determined after baseline correction, using the heat capacity of the protein in its native state as reference. The melting temperature, Tm and the enthalpy of unfolding, H of the protein were estimated using the NanoAnaylze 2.4.1 software. Circular dichroism spectroscopy Circular dichroism (CD) measurements were performed on a Jasco J-815 spectropolarimeter (Jasco Corp., Tokyo, Japan), equipped with a PTC-423S/15 Peltier cell holder for temperature control using a 1 mm path length quartz cuvette under constant nitrogen flow. Ellipticity values at 222 nm of 3 μM HSA in the absence and the presence of 12 μM 6-shogaol were recorded in the temperature range, 25−80 ºC and 25−100 ºC. The solution mixture (4:1) of 6-shogaol and HSA was pre-incubated for 1 h at 25 ºC to achieve equilibrium before CD measurements. For thermal denaturation studies, all samples were kept for 3 min at each temperature before the readings were recorded. For cooling experiments, measurements were made under identical conditions upon reversal of the temperature range (100−25 ºC and 80−25 ºC). Site-specific drug displacement To characterize the binding site of 6-shogaol on HSA, drug displacement experiments were performed using PBZ and KTN as marker ligands for Sudlow’s sites I and II, respectively (Kragh-Hansen et al. 2002). Titration experiments were performed in the absence and the presence of drug/6-shogaol using fluorescence spectroscopy upon excitation at 295 nm. Fluorescence intensity values of HSA (3 μM) as well as 6-shogaol–HSA complex (5:1 molar ratio) were monitored at 338 nm with the addition of increasing drug concentration (PBZ, 0–60 μM; KTN, 0–24 μM). The mixture of 6-shogaol and HSA was incubated for 1 h at 25 ºC prior to the addition of the marker ligands. A further incubation of 1 h at the same temperature was allowed before fluorescence measurements. The displacement experiments were also performed in the reverse order by titrating HSA (3 μM) and drug–HSA complexes (5:1 molar ratio) with increasing concentrations of 6-shogaol (0–30 μM).

movement of the ligand. For Sudlow’s site I, the grid boxes were centered at x, y and z coordinates of (35.26, 32.41, 36.46), (5.10, –13.35, 7.44) and (1.33, –10.09, 8.19) for 1BM0, 2BXD and 2BXF, respectively. On the other hand, the grid boxes were centered at (14.42, 23.55, 23.21), (15.23, 4.38, –7.69) and (5.28, 4.64, –10.08) for 1BM0, 2BXD and 2BXF, respectively, for docking at Sudlow’s site II. A total of 100 docking runs were performed for each binding site using Lamarckian genetic algorithm to evaluate the ligand binding energy. In each run, a population of 150 individuals with 27 000 generations and 250 000 energy evaluations were employed, with the operator weights for crossover, mutation, and elitism set to 0.8, 0.02 and 1.0, respectively. A root-mean-square-deviation tolerance of 2.0 A˚ was used for cluster analysis of the docking results. Statistical analysis Experiments were conducted independently at least three times, and all data are presented as the mean ± standard deviation (SD). The statistical differences were analyzed using one way analysis of variance (ANOVA). A value of p value < 0.05 was considered statistically significant. Results and discussion Mechanism of fluorescence quenching Fluorescence spectroscopy has long been utilized in the characterization of ligand–protein interaction due to its high sensitivity toward conformational and microenvironmental changes in a protein. Fig. 2 shows fluorescence spectra of HSA (3 μM) in the wavelength range, 310–390 nm, obtained in the absence and the presence of increasing 6-shogaol concentrations (0–30 μM) at 25 ºC. The emission spectrum with a maximum at 338 nm was originated due to the presence of the solitary Trp residue (Trp-214) of HSA, which was selectively targeted upon excitation at 295 nm. Addition of 6-shogaol to HSA induced a concentration-dependent decrease in its fluorescence intensity, producing 63% quenching at a ligand/protein molar ratio of 10:1 (inset of Fig. 2). While considerable quenching was observed in the presence of 6-shogaol, the emission maximum remained unchanged throughout the titration. Considering Trp as a relatively sensitive fluorophore, it appears that addition of 6-shogaol to HSA had little effect on the polarity of microenvironment in the vicinity of Trp-214 or its

Docking studies ACD/ChemSketch Freeware (Advanced Chemistry Development Inc., Ontario, Canada) was used to draw the structure of 6-shogaol and its geometry was optimized using VegaZZ 2.08 (Pedretti et al. 2002). AutoDock 4.2 (Goodsell et al. 1999) and AutoDockTools 1.5.4 (Sanner 1999) were employed to perform molecular docking, visualization and rendering simulations. In the docking study, non-polar hydrogens of 6-shogaol were merged and its rotatable bonds were defined. Three HSA crystal structures [PDB IDs: 1BM0, 2.5 A˚ resolution (Sugio et al. 1999); 2BXD, 3.05 A˚ and 2BXF, 2.95 A˚ (Ghuman et al. 2005)], were downloaded from the Protein Data Bank (Berman et al. 2000). All water and ligand molecules were removed before the atomic coordinates of the HSA crystal structures were used as input for AutoDockTools. After addition of polar hydrogens to the protein, assignment of Kollman united atom partial charges were made. The HSA structure was fixed at the initial input in a rigid conformation, while the torsional bonds of 6-shogaol were allowed to orientate freely. The grid box in each simulation was defined by a 70 × 70 × 70 grid dimension of 0.375 A˚ grid space and centered so that it enclosed the entire binding site and accommodated the free

Fig. 2. Fluorescence quench titration results of HSA (3 μM) with increasing 6-shogaol concentrations (1–11: 0–30 μM with 3 μM intervals) upon excitation at 295 nm, studied in 10 mM sodium phosphate buffer, pH 7.4 at 25 ºC. Inset shows the decrease in the relative fluorescence intensity of HSA at 338 nm (FI338 nm ) with increasing 6shogaol/HSA molar ratios.

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Table 1 Binding constant and thermodynamic parameters for the interaction of 6-shogaol with HSA, obtained from fluorescence quench titration experiments at different temperatures, pH 7.4. T (ºC)

KSV (M−1 )

Ka (M−1 )

15 25 35 45

(6.47 ± 0.20) × 104 (5.71 ± 0.16) × 104 (5.25 ± 0.23) × 104 (4.47 ± 0.27) × 104

(7.51 ± 0.36) × 104 (6.29 ± 0.33) × 104 (5.56 ± 0.27) × 104 (4.68 ± 0.42) × 104

H (kJ mol−1 )

S (J mol−1 K−1 )

−11.76

52.52

G (kJ mol−1 ) −26.88 −27.41 −27.93 −28.46

Volmer plots (Fig. 3A) which were used to determine the KSV values (Table 1). The inverse correlation between the KSV values and the temperature suggested involvement of static quenching mechanism in the 6-shogaol-induced quenching of HSA fluorescence. In addition, appreciably larger values (0.70–1.01 × 1013 M−1 s−1 ) of the bimolecular quenching constant, kq , calculated for the 6-shogaol–HSA system in comparison to that obtained for a typical diffusion-controlled phenomenon (1010 M−1 s−1 ) further supported the complex formation between 6-shogaol and HSA (Lakowicz 2006). Binding and thermodynamic parameters

Fig. 3. Analysis of the fluorescence quenching data of 6-shogaol–HSA system, obtained at different temperatures. (A) Stern–Volmer plots and (B) logarithmic plots of log (F0 – F)/F against log [1/([LT ] – (F0 – F)[PT ]/F0 )]. Inset of (B) shows the van’t Hoff plot for 6-shogaol–HSA interaction.

accessibility to the solvent (Ladokhin 2000). The quenching of the tryptophanyl fluorescence as shown in Fig. 2 thus, seems to arise due to collisional or static quenching phenomena (Lakowicz 2006). Collisional quenching, a type of dynamic quenching process, results from random encounters between the excited fluorophore and the quencher. On the other hand, static quenching involves the formation of a nonfluorescent ground state complex between the protein and the quencher (Lakowicz 2006). These two mechanisms can be distinguished on the basis of their dependence on temperature, as higher temperatures lead to more frequent collisions (higher extent of collisional quenching) and dissociation of ground state complexes (smaller extent of static quenching) (Lakowicz 2006). In view of this, the fluorescence quench titration experiments were performed at four different temperatures (15, 25, 35 and 45 ºC) to predict the quenching mechanism involved in the 6-shogaol–HSA system. Treatment of the fluorescence data according to Eq. (2) produced Stern–

The fluorescence data were also analyzed according to Eq. (3) to determine the association constant, Ka of the binding reaction. Fig. 3B shows linear double logarithmic plots for 6-shogaol–HSA system, obtained at different temperatures. The values of Ka were calculated from the y-axis intercept of these plots and are listed in Table 1. The Ka value was found to lie between 4.68 × 104 and 7.51 × 104 M−1 within the selected temperature range, which was typical for a moderate affinity binding system (Dufour and Dangles 2005). Such a magnitude of Ka for 6-shogaol–HSA system is helpful as it facilitates both efficient transportation of the ligand in circulation and its release at the target site. Indeed, the binding affinity of 6-shogaol to HSA was found comparable to those (2.7–10.6 × 104 M−1 ) generally observed for many drug–HSA complexes (Kragh-Hansen 1988; Maciazek-Jurczyk et al. 2011; Sueyasu et al. 2000; Sulkowska et al. 2008; Zaton et al. 1988). In view of this, it is reasonable to suggest that once 6-shogaol is in the blood circulation, the ratio of its free/bound concentration, duration of pharmacological efficacy and in vivo half-life would be more or less similar to these drugs. In order to determine thermodynamic parameters associated with the binding reaction, van’t Hoff plot (inset of Fig. 3B) was used to obtain the values of S and H. These values were incorporated into Eq. (5) to calculate G at different temperatures as described in ‘Materials and methods’ section. The values of thermodynamic parameters are listed in Table 1. The positive sign of S (+52.52 J mol−1 K−1 ) and the negative sign of H (−11.76 kJ mol−1 ) showed favorable contribution toward thermodynamic feasibility of the reaction with a G value of −27.41 kJ mol−1 at 25 ºC. The overall 6-shogaol–HSA association can be viewed as proceeding through hydrophobic interactions in between the ligand and the protein (Ross and Subramanian 1981). The partial disordering of the highly organized solvent structures around the ligand and the protein may account for the positive entropic contribution observed in the binding reaction. This is supported by the apolar nature of 6-shogaol due to the presence of a 10-carbon long hydrocarbon chain and benzenoid structure. A variety of short range interactions seem to occur between the closely associated (hydrophobically) ligand and protein molecules. Formation of hydrogen bonds is always accompanied by the liberation of heat, resulting in a negative H value and it is highly likely to occur between 6-shogaol and HSA due to the presence of several polar groups in the ligand. This is supported by our molecular docking results, which predicted hydrogen bonding between the polar moieties of 6-shogaol and the amino acid residues of HSA. van der Waals forces also exhibit similar thermodynamic characteristics as shown by hydrogen bonds (Ross and Subramanian 1981); thus, participation of van der Waals forces

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S.R. Feroz et al. / Phytomedicine 22 (2015) 621–630 Table 2 Three-dimensional fluorescence spectral characteristics of 3 μM HSA and (4:1) 6shogaol−HSA complex at pH 7.4, 25 ºC. System

HSA

6-Shogaol–HSA

Peak ⎧ ⎪ a ⎪ ⎪ ⎨ b ⎪ 1 ⎪ ⎪ ⎩2 ⎧ ⎪ a ⎪ ⎪ ⎨b ⎪1 ⎪ ⎪ ⎩2

Peak position (λex /λem , nm/nm)

Intensity

230/230 → 350/350 250/500 280/336 235/331

19.18 → 79.06 125.96 560.17 308.49

230/230 → 350/350 250/500 280/333 235/325

13.82 → 56.21 93.40 348.47 184.74

produced significant reduction in the fluorescence intensity, as 38% and 40% quenching was observed for peaks 1 and 2, respectively. This was accompanied by a blue shift (3 and 6 nm for peaks 1 and 2, respectively) in the emission maximum. These changes in the fluorescence characteristics (fluorescence quenching and blue shift in the emission maxima) observed in the presence of 6-shogaol were suggestive of tertiary structural alteration in the protein induced by 6-shogaol binding. Similar changes in the protein fluorescence characteristics upon ligand binding have been reported earlier (Abdollahpour et al. 2011; Feroz et al. 2012, 2013; Freitas et al. 2012). It is interesting to note that such blue shift in the emission maximum was not observed upon ligand binding when the protein was excited at 295 nm (Fig. 2). Hence, it seems plausible that tertiary structural alterations in HSA upon 6-shogaol binding did not involve structural features around Trp-214 to a significant extent. Thermal stability

Fig. 4. Three-dimensional spectral projections and corresponding contour maps of (A) 3 μM HSA and (B) 6-shogaol–HSA (4:1) complex, recorded in 10 mM sodium phosphate buffer, pH 7.4 at 25 ºC. (For interpretation of the references to the colour map in this figure, the reader is referred to the web version of this article.)

in the binding process cannot be excluded. While positive S value of the reaction points toward the involvement of electrostatic forces, this seems rather improbable in practice. Analysis of 6-shogaol structure using ALOGPS 2.1 software (Tetko et al. 2005) predicted a pKa value of 9.8. Hence, at the experimental pH of 7.4, its only ionizable group (–OH) remained in protonated form (neutral compound) which eliminates the role of electrostatic interactions in the 6-shogaol–HSA system. Conformational alterations In order to obtain information on the 6-shogaol-induced structural changes in HSA, 3-D fluorescence spectroscopy was employed to analyze protein samples in the absence and the presence of 6-shogaol. The 3-D fluorescence spectra and corresponding contour maps of free HSA and 4:1 6-shogaol-bound HSA are shown in Fig. 4A and B, respectively. The characteristics of these spectra are given in Table 2. Peaks labelled as ‘a’ and ‘b’ refer to the Rayleigh and the second-order scattering peaks, respectively (Lakowicz 2006). The peaks designated as 1 and 2 represent protein fluorescence peaks due to the presence of Trp and Tyr residues and provide information about the tertiary structure of the protein. Addition of 6-shogaol to the protein in a 4:1 molar ratio

Many proteins have shown changes in their thermal unfolding behavior in the presence of their ligands due to complex formation (Gonzalez et al. 1999; Layton and Hellinga 2010). Thus, ligand–protein interaction can also be studied by measuring thermal stability of a protein in the absence and the presence of its ligand. DSC results. DSC is a well suited technique to examine conformational transitions in proteins in response to change in temperature. Proteins in their ligand-bound states have been found more stable, characterized by a higher Tm value compared to their native forms (Anraku et al. 2004; Lupidi et al. 2010; Celej et al. 2006). This increase in the stability of a protein in the ligand-bound form can be attributed to the coupling of the binding and unfolding equilibria (Shrake and Ross 1990). Fig. 5 shows variation in the excess molar heat capacity, Cp , of HSA (15 μM) with temperature, observed in the absence and the presence of 6-shogaol (60 μM). While both endotherms exhibited a single transition peak, the Tm value of the protein was shifted from 63.6 to 68.1 ºC in the presence of 6-shogaol. Furthermore, the unfolding enthalpy was found to increase from 743.8 to 850.4 kJ mol–1 . Such differences in these parameters clearly indicated that binding of 6-shogaol to native HSA afforded a higher thermal stability to the protein. CD spectral results. The protein stability against heat denaturation in the absence and the presence of 6-shogaol was also examined using CD spectroscopy by recording the ellipticity value at 222 nm (CD222 nm ), a characteristic signal of the α -helical structure in proteins (Kelly et al. 2005). Since HSA has a high (67%) α -helical content (Peters 1996), any decrease in CD222 nm value upon heat treatment can be used to probe the thermal stability of the protein. Fig. 6A shows variation in the CD222 nm value of HSA (3 μM) upon heating and cooling in the temperature range, 25–100 ºC, as obtained in the absence and the presence of 12 μM 6-shogaol (same ligand/protein molar ratio as used in DSC experiments). Thermal denaturation profiles of HSA as well as its complex with 6-shogaol displayed cooperative transitions up to 94 ºC. However, the denaturation curve of 6-shogaol–HSA

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Fig. 5. DSC scans of thermal denaturation of 15 μM HSA in the absence and the presence of 6-shogaol at 4:1 ligand/protein molar ratio, obtained in 10 mM sodium phosphate buffer, pH 7.4.

Fig. 7. Site-specific marker ligand displacement. (A) and (B) depict the decrease in the relative fluorescence intensity at 338 nm (FI338 nm ) of HSA (3 μM) and 6-shogaol–HSA (5:1) complex with increasing concentrations of PBZ (0–60 μM) and KTN (0–22.5 μM), respectively. Effect of increasing concentrations (0–30 μM) of 6-shogaol on the relative FI338 nm of HSA (3 μM) and its complexes (5:1) with PBZ and KTN are shown in (A ) and (B ), respectively.

bic patches (Chi et al. 2003). This was supported by the results of the cooling experiment where the CD222 nm value of HSA remained unchanged upon cooling to 25 ºC. In fact, protein aggregates were visible in the cuvette. In contrast, the 6-shogaol–HSA complex showed no aggregation beyond 94 ºC and significant recovery (58%) in the CD222 nm signal was observed upon renaturation at 25 ºC. These observations clearly suggested a higher thermal stability of HSA conferred by 6shogaol binding. In view of the aggregation phenomenon observed at higher temperatures, the experiments were also performed in the temperature range, 25–80 ºC (Fig. 6B). As evident from the figure, HSA showed partial refolding in the cooling experiment, characterized by 50% regain in the CD222 nm signal. On the other hand, 71% reversal in the CD222 nm value of HSA was achieved in the presence of 6-shogaol at 25 ºC. Both DSC and CD results thus, conclusively demonstrated the stabilizing effect of 6-shogaol on the thermal stability of HSA. Fig. 6. Thermal stability profiles of HSA (3 μM) and 6-shogaol–HSA complex (4:1) monitored using CD values at 222 nm (CD222 nm ) in 10 mM sodium phosphate buffer, pH 7.4 in the temperature range, 25–100 ºC (A) and 25–80 ºC (B). The spectral signals obtained in the cooling experiments are shown by the smaller symbols.

complex showed a relatively lesser loss in the CD222 nm value compared to HSA, demonstrating relatively higher resistance against thermal denaturation. Thermal denaturation curves yielded the value of Tm as 64 and 67 ºC for HSA in its free and 6-shogaol-bound forms, respectively, which were akin to those obtained from the DSC experiments. Increase in Tm value of HSA observed in the presence of 6-shogaol was also suggestive of higher protein stability. At temperatures above 94 ºC, a drastic decrease in the CD222 nm value of HSA was observed which can be ascribed to the formation of protein aggregates due to interactions among the unfolded hydropho-

Location of binding site Many physiological ligands as well as a large number of drugs have been shown to bind to HSA through one of the two high affinity binding sites, namely, Sudlow’s site I (located in subdomain IIA) and site II (placed in subdomain IIIA) (Carter and Ho 1994; Kragh-Hansen et al. 2002; Sudlow et al. 1975). Based on their affinities, these drugs are often used as marker ligands for these sites. In view of this, drug displacement experiments were performed using PBZ and KTN as probes for sites I and II respectively, in order to identify the probable binding site of 6-shogaol on HSA. Fig. 7A shows the effect of increasing PBZ concentrations (0– 60 μM) on the fluorescence intensities of HSA (3 μM) and 6-shogaol– HSA (5:1) complex. As evident from the figure, addition of PBZ to HSA

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led to a continuous decrease in the fluorescence intensity at 338 nm, reaching to 73% quenching at 60 μM PBZ. On the other hand, presence of 6-shogaol in the mixture interrupted the quenching phenomena, thus led to only 43% quenching at 60 μM PBZ. These results indicated interference in PBZ–HSA interaction at site I by 6-shogaol, as both ligands seem to compete for the same binding site on the protein. To validate these observations, displacement experiments were also performed with HSA and PBZ–HSA mixture using 6-shogaol as the titrant (Fig. 7A ). Whereas a 63% reduction in the fluorescence intensity of HSA was observed at a 6-shogaol/protein molar ratio of 10:1, PBZ–HSA (5:1) system showed only 45% quenching. These displacement results suggested location of 6-shogaol binding site on HSA to be either site I or in its vicinity. In order to assess the involvement of site II in the interaction between 6-shogaol and HSA, competitive displacement experiments were also repeated using KTN as the marker ligand. As revealed by the displacement results presented in Fig. 7B and B , less pronounced differences in the fluorescence quenching were observed upon replacing PBZ with KTN. Fig. 7B shows 14% difference in the KTN-induced quenching of HSA, observed in the absence and the presence of 6shogaol at 22.5 μM KTN concentration, while an even smaller difference (9%) was noticed upon titrating HSA and the KTN–HSA complex with 6-shogaol at 30 μM concentration (Fig. 7B ). Although these results also indicated the involvement of site II in 6-shogaol–HSA interaction, there was noticeably less interference by KTN to 6-shogaol– HSA binding and vice versa. The drug displacement results hence, pointed toward site I as the preferred binding locus of 6-shogaol on HSA, with site II having a lower affinity. Molecular docking Docking studies of 6-shogaol–HSA interaction were performed to predict the binding of 6-shogaol to the two main ligand binding sites of HSA, and to validate the drug displacement results. Multiple crystal structures of HSA (1BM0, 2BXD and 2BXF) were analyzed to validate the docking workflow. The HSA crystal structure 1BM0 represented the protein with empty sites, while 2BXD and 2BXF are reported as complexes with warfarin and diazepam, respectively (Ghuman et al. 2005). Since HSA is known to bind to warfarin at site I and diazepam at site II, docking analysis using these structures could reveal the binding preference of 6-shogaol to these sites on HSA. The docking analysis of the 6-shogaol–1BM0 complex (Fig. 8) revealed a total of 33 conformation clusters for site I, with the majority of the clusters being single- or double-membered. The highest populated cluster contained 20 out of 100 conformations and was also the most energetically favorable cluster, possessing a mean binding energy of −23.27 kJ mol−1 . A similar clustering pattern dominated by single-membered clusters was also observed for site II, where a total of 49 conformation clusters were populated. The two highest populated clusters had the same number of conformations (11 conformations each) and comparable mean binding energies of −17.04 kJ mol−1 and −16.66 kJ mol−1 . The fewer conformational clusters and lower binding energy associated with docking at site I thus, strongly suggested the preference of 6-shogaol to site I and supported the experimental findings. Clustering analysis of the 6-shogaol–2BXD and 6-shogaol–2BXF complexes (Fig. 8) also showed similar results in terms of the binding energy at site I, strengthening our conclusion about site I of HSA as the primary binding site of 6-shogaol. However, comparison of the clustering results for site II revealed that the mean binding energy was significantly lower and the number of conformations in the highest populated cluster for site II was also larger than its counterpart for site I, when 6-shogaol was docked to 2BXF. In view of these observations, it appears that binding of diazepam to HSA might have induced a change in the geometry of site II, which allowed 6-shogaol to interact more favorably to HSA at this binding site.

Fig. 8. Cluster analysis of the docking of 6-shogaol to three different crystal structures of HSA (1BM0, 2BXD and 2BXF) at binding sites I and II. A total of 100 docking runs were performed for each site.

Fig. 9. Binding orientations of the lowest docking energy conformations of 6-shogaol at sites I and II of HSA (1BM0). Domains I, II and III of the protein are represented in red, blue and green, respectively. The zoomed-in view of the binding sites show the hydrogen bonds (turquoise lines) formed between 6-shogaol and amino acid side chains of HSA (yellow). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

The predicted binding models showing minimum docking energy for both sites I and II, were then used to analyze the binding orientation (Fig. 9). The binding site was defined as the collection of amino acid residues present within 5 A˚ radius from the ligand. 6-shogaol binding site at site I was largely comprised of a hydrophobic cleft, walled by the amino acids residues: Tyr-150, Glu-153, Phe-157, Ala191, Ser-192, Ser-193, Lys-195, Gln-196, Lys-199, Leu-219, Arg-222, Phe-223, Leu-234, Leu-238, His-242, Arg-257, Leu-260, Ile-264, Ser287, His-288, Ile-290, Ala-291 and Glu-292. Although hydrophobic

S.R. Feroz et al. / Phytomedicine 22 (2015) 621–630 Table 3 Distance of predicted hydrogen bonds between amino acid residues of HSA and 6-shogoal. HSA

6-Shogaol

Tyr-150: HH Gln-196: HE21 Arg-257: HE

O (carbonyl) O (hydroxyl) O (carbonyl)

˚ Distance (A)

Site I 1.88 2.20 1.86

Site II Lys-414: HZ2

O (hydroxyl)

1.94

interactions between nonpolar amino acid residues and 6-shogaol contributed significantly towards the docking stability in the simulation, hydrogen bonding also had a major influence in stabilizing the complex. Three hydrogen bonds were predicted between 6-shogaol and HSA at site I, involving Tyr-150, Gln-196 and Arg-257 (details are listed in Table 3). On the other hand at site II, the ligand was orientated outward (toward the surface), with its binding pocket bordered by the following residues: Leu-398, Lys-402, Asn-405, Ala-406, Lys409, Arg-410, Tyr-411, Lys-413, Lys-414, Leu-491, Glu- 492, Val-493, Asp-494, Thr-540, Lys-541 and Lys-545. Furthermore, only a single hydrogen bond was predicted, linking Lys-414 and the hydroxyl oxygen of 6-shogaol (Fig. 9 and Table 3). In summary, the present study provides a detailed description of the interaction of a bioactive ginger constituent, 6-shogaol with HSA in terms of binding mode, thermodynamic characteristics, effect on protein conformation and thermal stability, as well as binding location. Studies on the binding of such therapeutic compounds to HSA are of great importance in understanding chemico-biological interactions for clinical research and drug design. These results can be helpful in providing insights into the pharmaceutics of 6-shogaol and other structurally similar compounds. Conflict of interest The authors declare that they have no conflicts of interest to disclose. Acknowledgments This work was financially supported by the High Impact Research MoE Grant UM.C/625/1/HIR/MoE/SC/02 approved by the Ministry of Education, Government of Malaysia and the University of Malaya. The financial assistance from the University of Malaya to S.R.F. in the form of University of Malaya Postgraduate Research Fund (PG073/2013B) is highly appreciated. We thank the Dean, Faculty of Science and the Head, Institute of Biological Sciences, University of Malaya for providing the necessary facilities. References Abdollahpour, N., Asoodeh, A., Saberi, M.R., Chamani, J., 2011. Separate and simultaneous binding effects of aspirin and amlodipine to human serum albumin based on fluorescence spectroscopic and molecular modeling characterizations: a mechanistic insight for determining usage drugs doses. J. Lumin. 131, 1885– 1899. Abou-Zied, O.K., Al-Shihi, O.I., 2008. Characterization of subdomain IIA binding site of human serum albumin in its native, unfolded, and refolded states using small molecular probes. J. Am. Chem. Soc. 130, 10793–10801. Anraku, M., Tsurusaki, Y., Watanabe, H., Maruyama, T., Kragh-Hansen, U., Otagiri, M., 2004. Stabilizing mechanisms in commercial albumin preparations: octanoate and N-acetyl-l-tryptophanate protect human serum albumin against heat and oxidative stress. Biochim. Biophys. Acta 1702, 9–17. Bak, M.J., Ok, S., Jun, M., Jeong, W.S., 2012. 6-Shogaol-rich extract from ginger upregulates the antioxidant defense systems in cells and mice. Molecules 17, 8037– 8055.

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