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Colloids and Surfaces B: Biointerfaces 112 (2013) 374–379

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

journa l homepage: www.e lsev ier .com/ locate /co lsur fb

inding study of novel anti-diabetic pyrimidine fused heterocycleso �-lactoglobulin as a carrier protein

ohammad Hossein Mehrabana, Reza Yousefia,∗, Asghar Taheri-Kafranib,arhad Panahic, Ali Khalafi-Nezhadc

Protein Chemistry Laboratory (PCL), Department of Biology, College of Sciences, Shiraz University, Shiraz, IranDepartment of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan 81746-73441, IranDepartment of Chemistry, College of Sciences, Shiraz University, Shiraz, Iran

r t i c l e i n f o

rticle history:eceived 12 June 2013eceived in revised form 2 August 2013ccepted 9 August 2013vailable online 28 August 2013

eywords:-Lactoglobulinyrimidine-fused heterocyclesluorescence intensityircular dichroismhermodynamic parameters

a b s t r a c t

Bovine milk �-lactoglobulin (�-LG) demonstrates significant resistance against both gastric- and simu-lated duodenal digestions. Therefore, it seems a realistic protein candidate for safe delivery and protectionof particularly pH sensitive drugs in stomach. Recently, pyrimidine fused heterocycles (PFHs) revealedinhibitory properties against �-glucosidase (�-Gls) which is an important target enzyme for those drugsplaying significant role in treatment of type-II diabetes and HIV/AIDS infection. The delivery of thesecompounds to small intestine where the enzyme plays its biological function is of great importance.Therefore, in this work the interaction of PFH compounds with �-LG, as a carrier protein has been inves-tigated. Fluorescence, circular dichroism (CD) and UV–vis spectroscopic studies were used to examinethe binding parameters and binding modes of the interaction. Moreover, the effects of PFH complexationon the secondary structures of �-LG were studied. All of these compounds significantly quenched thefluorescence intensity of �-LG due to a ground state complex formation. The binding and thermodynamic

parameters were calculated. While hydrophobic interactions were proved to play significant role in theinteraction of L1, L2 and L3, hydrogen bonding was shown to be important in the complexation of L4. Thesecondary structures of �-LG were preserved upon interaction of these synthetic compounds. Based onthe achieved results, these potentially therapeutic agents can significantly bind to �-LG. Consequently,this protein might be useful for delivery of PFH compounds to small intestine where representing theirpotential ability to inhibit �-Gls and to reduce the postprandial hyperglycemia in diabetic patients.

. Introduction

Bovine whey protein �-lactoglobulin (�-LG) is a small globularrotein (MW = 18 kDa) with known primary, secondary and three-imensional structure but with still unknown biological functions1]. At the physiological conditions, this protein normally exists inimeric form, whereas at acidic pH it dissociates into monomersue to electrostatic repulsion between the subunits [2]. �-LG is aell-known member of the hydrophobic transporter superfamily,

ermed lipocalin. According to the crystal structure of this trans-orter, its three dimensional structure consists of eight anti-parallel-sheets, forming a �-barrel calyx which is responsible for the

ajor binding properties of this protein [3,4]. �-LG demonstrates

ignificant affinity towards specific binding to a wide variety of

∗ Corresponding author. Tel.: +98 7116137617/7116137665;ax: +98 7112280916.

E-mail address: ryousefi@shirazu.ac.ir (R. Yousefi).

927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.colsurfb.2013.08.013

© 2013 Elsevier B.V. All rights reserved.

hydrophobic ligands such as retinoids, steroids, fatty acids, vita-mins and cholesterol [5–9].

This protein is an exceptional transporter due to its stability inacidic milieu of the stomach. At pH 2.0, it reversibly dissociatesto monomers, while its native structure remains intact [10]. Fur-thermore, the native structure of �-LG is considerably resistant topepsin degradation at low pH of stomach [11,12]. Because of theseexceptional properties, it is possible to detect intact �-LG in smallintestine, where most of its digestion occurs [13]. Therefore thisprotein may serve as a carrier for safe delivery of especially non-soluble ligands which their target molecules are existed in the smallintestine.

Various biological activities such as anticancer, anti-inflammatory, antioxidant, antiviral and antimicrobial activitieshave been ascribed to pyrimidine fused heterocycles (PFHs)[14–16]. These compounds consist of a pyrimidine ring which in

turn is a crucial chemical moiety, as it is existed in a wide numberof alkaloids, drugs, antibiotics and antimicrobial agents [17]. Also,the simple bioactive PFHs such as purine and pteridine can befound in the structure of natural compounds [18,19]. Furthermore,

M.H. Mehraban et al. / Colloids and Surfaces

sdRd�riMeiTta�t

ptaiaa

2

2

cTpaogCac

2

aHific

Fig. 1. The molecular structure of L1 , L2 , L3 and L4 .

ome PFH derivatives have been used as potassium conservingiuretics [20], anti-leukemic [21] and anti-allergic drugs [17].ecently, it has been proved for the first time that some PFHerivatives demonstrate significant inhibition against mammalian-glucosidase (�-Gls) enzyme. Inhibition of this crucial enzyme

esults in the reduction of postprandial hyperglycemia whichs the most popular strategy for treatment of diabetic patients.

oreover, �-Gls inhibition interferes with synthesis of viralnvelope which in turn reduces the viral infectivity, and thereforet is a useful strategy in treatment of HIV/AIDS infection [22,23].he mammalian �-Gls is located in the brush border membrane ofhe small intestine [23]. Regarding to the needs for secure deliverynd protection against acidic milieu of stomach, it is essential for-Gls inhibitors to be delivered safely to the small intestine where

hey exert their pharmaceutical activities.In the present study the interaction of four PFHs as novel and

otentially anti-diabetic compounds (Fig. 1) with �-LG were inves-igated, using circular dichroism (CD), ultraviolet–visible (UV–vis)nd fluorescence spectroscopy. The aim of this study was tonvestigate the protein’s ability for binding to these potentiallynti-diabetic molecules. The obtained results may have importantpplications in drug delivery and drug design procedures.

. Materials and methods

.1. Materials

�-LG variant A was isolated from the milk of a homozygousow according to method of Mailliart and Ribadeau Dumas [24].he homogeneity of the protein preparation was assessed by higherformance gel permeation chromatography and SDS-poly acrylmide gel electrophoresis. The preparations of �-LG obtained werever 98% pure. All other materials and reagents were of analyticalrade, and the solutions were made using double-distilled water.hemicals for the synthesis of ligands were purchased from Flukand Aldrich chemical companies and used without further purifi-ation.

.2. General method for the synthesis of PFH ligands

In order to synthesis the PFH ligands, a mixture of barbituriccid (0.26 g, 2 mmol), aldehyde (1 mmol), amine (1 mmol), and

3PW12O40 (0.04 g, 2 mol%) in ethanol (5 mL) at 80 ◦C was stirred

nto a canonical flask, for 12 h. As completion of the reaction con-rmed by TLC (eluent EtOAc/MeOH), the reaction mixture wasooled to room temperature. Then, the precipitated product was

B: Biointerfaces 112 (2013) 374–379 375

filtered and washed with water (2 × 10 mL) and EtOH (2 × 5 mL) toafford the pure product. The synthesized compounds L1–L4 werealso characterized, using 1H NMR, 13C NMR, infrared (IR), andmass spectroscopy. The data clearly confirmed that these moleculeswere created successfully. As the purity of synthetic compoundsexamined using elemental analysis, they were highly pure (>97%).The detailed description of the synthetic procedure and inhibitionagainst �-Gls enzyme are under publication somewhere else.

2.3. Fluorescence measurements

Fluorimetric studies were carried out on a Cary-Eclipse spec-trofluorimeter (Carry-100 Varian, Sydney, Australia). A �-LGsolution of 0.1 mg/mL was prepared in 50 mM sodium phosphate(NaPi) buffer, pH 7.0. The ligands stock solutions were preparedby dissolving them in 0.1 M NaOH, at a final concentration of5 mM, and then diluted in the above mentioned buffer to obtainthe desired concentrations. The intrinsic fluorescence experimentswere performed at constant �-LG concentration, titrating witheach ligand in the range of 0–50 �M. The emission spectra wererecorded between 300 and 500 nm, with an excitation wavelengthof 290 nm. The maximum fluorescence intensity (about 330 nm)was used to calculate the binding constant parameters, accordingto the previous publication [5]. To analyze the interaction of �-LGand synthetic compounds, quenching constant Ksv was determined,using Stern–Volmer equation [25]:

F0

F= 1 + Ksv[L] (1)

where F0 and F are relative fluorescence of �-LG in the absenceand presence of the ligands, respectively, [L] is the concentrationof the quencher, and Ksv is the quenching constant. The bindingconstant and the number of binding sites on �-LG can be calculatedaccording to the following equation [26]:

LogF0 − F

F= Log KA + n Log[L] (2)

where F0 and F are the fluorescence emission intensities of proteinwithout and with ligand, respectively, [L] is ligand concentration,KA is the binding constant, and n is the apparent molar ratio of[L]/[�-LG] complexes.

Thermodynamic parameters are the main clues to confirm thebinding force of the interaction namely hydrogen bonds, van derWaals force, hydrophobic bonds, electrostatic interactions, etc.When the enthalpy change (�H�) is not varied significantly overthe temperature range studied, its value and that of entropy (�S�)can be determined by van’t Hoff equation:

In KA = −�H�

RT+ �S�

R(3)

The free energy change (�G�) can be calculated according to thefollowing equation:

�G� = −RT ln KA (4)

where R is the gas constant and T is the temperature (K).

2.4. Circular dichroism measurements

The CD spectra of �-LG and ligands were recorded on anAviv dichrograph model 215 (Proterion Corp., USA). Far-UV region(195–260 nm) was selected to investigate the �-LG secondarystructure changes using 1 mm path cuvettes. The concentration

of the protein was constant (0.35 mg/mL), while the ligand con-centrations varied between 20 and 80 �M. Each spectrum wasthe accumulation of five successive measurements and baselinewas corrected by subtracting buffer spectrum. The results were

3 rfaces B: Biointerfaces 112 (2013) 374–379

ed

[

w(low

2

t1wcctalaab[

woape

K

2

r2

3

3

tiiottiorsis3t2

dt

Fig. 2. The fluorescence emission spectra of �-LG in the presence of various con-centrations of L1–L4 (A–D) in 50 mM NaPi buffer, pH 7.0 at 298 K. A constantconcentration of �-LG (0.1 mg/mL) and different concentrations of each ligand wereused. Inset: log [(F0 − F)/F] vs. log[Ligands] as per Eq. (2) at different temperatures.

76 M.H. Mehraban et al. / Colloids and Su

xpressed as molar residue ellipticity [�] (deg cm2/dmol) which isefined as the following equation [27]:

�] = MRW × �obs

10 × d × C(5)

here MRW is the mean amino acid residue weight of the �-LG1 1 4), �obs is the observed ellipticity in degrees at a given wave-ength, d is the length of the cuvette in cm and C is the concentrationf the protein in mg/mL. The secondary structure alteration of �-LGas predicted using CDNN software.

.5. UV–vis absorption measurements

The UV–vis spectra were recorded on a T90+ UV–vis spectropho-ometer instrument (PG instrument, UK). A quartz cuvette withcm path length was used and the absorption spectra of �-LGere recorded in the absence and in the presence of various con-

entrations of each ligand (5–50 �M). The binding constants werealculated based on the previous publication [28]. It is assumedhat the interaction between the ligand (L) and �-LG (B) can form

single complex BL (1:1). It was also assumed that the Beer’saw is followed. A wavelength is selected at which the molarbsorptivities, εB (molar absorptivity of the protein) and εBL (molarbsorptivity of the complex) are different. As Bourassa reported, theinding constant can be calculated by using the following equation28]:

b

�A= 1

PtKb�εBL[L]+ 1

Pt�εBL(6)

here b is the light path length (1 cm), Pt is the total concentrationf the protein; Kb is the binding constant and �εBL is the molarbsorptivity of the ligand/protein complex. The double reciprocallot of 1/�A versus 1/[L] is linear and the binding constant can bestimated from the following equation:

b = interceptslope

(7)

.6. Protein assay

The protein concentration was determined spectrophotomet-ically, using a molar excitation coefficient of 17,600 M−1 cm−1 at78 nm [29].

. Results and discussion

.1. Fluorescence quenching results

Fluorescence quenching refers to any process that may decreasehe emission intensity of a fluorophore due to a variety of molecularnteractions. Based on the dependency on temperature and viscos-ty, fluorescence quenching mechanisms can be either dynamicsr statics. As the temperature rises, the diffusion of quencher tohe fluorophore increases and the collisional encounter betweenhem is more probable; accordingly the quenching constants willncrease. However, in static quenching, the quencher and flu-rophore can form a ground state complex; subsequently theise of temperature inversely correlates to the quenching con-tant [25]. In order to determine the quenching mechanism of thenteraction between �-LG and the PFH ligands, the fluorescencetudies were carried out at various temperatures of 298, 304 and10 K. Fig. 2 shows the fluorescence emission spectra of �-LG inhe absence and presence of various concentrations of L1–L4 at

98 K.

As shown in this figure, the fluorescence intensity of �-LGecreased with increasing the ligand concentration. Fig. 3 showshe plots of fluorescence intensity and also F0/F versus ligand

concentration, at different temperatures of 298, 304 and 310 K.To achieve the Stern–Volmer constants (KSV), these curves wereobtained according to the Eq. (1).

The Stern–Volmer constant values of the interaction between �-LG and PFH ligands are summarized in Table 1. It is obvious that theKsv decreases with increasing temperature for all ligands, provingthe involvement of static quenching in the interaction of �-LG with

these synthetic compounds.

M.H. Mehraban et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 374–379 377

–D) w

3

itnS[utownel

dil

TB

Fig. 3. The Stern–Volmer plots for the binding of L1–L4 (A

.2. Calculation of binding parameters

Based on the assumption that small molecules can interactndependently with the macromolecule, there will be n substan-ive binding sites on the protein. The number of binding sites

and the binding constant KA were calculated based on thecatchard method, using Eq. (2). The inset of Fig. 2 demonstrates log(F0 − F)/F] vs. log[Ligand], utilizing Eq. (2). Table 1 shows the val-es of Ka and n which obtained respectively from the intercept andhe slope of this linear plot (inset of Fig. 2). The mean values of n asbtained from the linear plots at temperature range of 298–310 Kere 1.14, 1.23, 1.32 and 1.23 for L1, L2, L3 and L4, respectively. Thevalues indicated that �-LG interacts with the PFH ligands, formingquimolar complexes. The order of binding affinity of the studiedigands due to their interaction with �-LG was L4 > L1 > L3 > L2.

The ligand L4 with a bi-pyrimidine heterocycle structureemonstrates the highest affinity towards �-LG which may clar-

fy the important role of PFH moiety in the complexation of theseigands. The fluorescence intensity of �-LG is mainly because of

able 1inding parameters for the interactions of �-LG with four PFHs ligands at various temper

Entry T Ksv (L mol−1) KA (L mol−1) n

L1 298 0.069 × 106 22.38 × 103 1.29304 0.037 × 106 25.70 × 1O3 1.08310 0.034 × 106 26.30 × 103 1.06

L2 298 0.243 × 106 11.30 × 103 1.9304 0.077 × 106 14.l2 × 103 1.38310 0.062 × l06 22.38 × 103 1.25

L3 298 0.065 × 106 11.99 × l03 1.73304 0.037 × l06 13.49 × 103 1.21310 0.030 × 106 26.91 × 103 1.02

L4 298 0.4 × 106 300 × 103 0.5304 0.09 × 106 20.41 × 103 1.36310 0.07 × 106 2.23 × 103 1.89

ith �-LG at various temperatures of 298, 304 and 310 K.

Trp19 which is located inside of hydrophobic calyx of the protein[30]. The fluorescence intensity decreases with increasing ligandconcentration to the �-LG solution. Consequently, it can be pro-posed that the protein hydrophobic pocket (calyx) probably is themain binding site in the complexation of these PFH ligands with�-LG.

3.3. Determination of thermodynamic parameters

The driving forces of the interaction between small moleculesand macromolecules are hydrogen bonding, electrostatic inter-actions, hydrophobic and van der Waals forces. The majorcontributing forces in the interaction can be obtained by determin-ing the thermodynamic parameters. Accordingly, these parameterswere determined using Eqs. (3) and (4). According to the rules

summarized by Ross and Subramanian, when �H and �S havepositive values, the hydrophobic interactions are the main actingforces in the complexation, while negative values of �H and �Sarise from the van der Waals force and hydrogen bonding [31]. The

atures of 298,304 and 310 K.

�H (kJ mol−1) �G (kJ mol−1) �S (J mol−1 K−1)

10.37 −24.81 0.12−25.66−26.23

43.63 −23.12 0.22−24.15−25.81

51.47 −23.26 0.25−24.03−26.29

−463.8 −36.95 −1.43−25.08−19.83

378 M.H. Mehraban et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 374–379

Table 2Calculated binding parameters of PFHs/�-LG complexes using UV/vis spectroscopy.

Entry Kb (M)−1 �ε (M cm)−1

L1 7.9 × 103 4.6 × l03

L2 2.5 × 103 5.9 × l03

L 3.2 × 103 8.7 × 103

vsa�iTswe

3

Uoaprotmdio

3

ittaL2fse�stetti

�t

Fig. 4. The UV–vis spectra of �-LG (8 �M) with increasing concentrations of L1–L4

TC

3

L4 9.5 × 103 4.4 × l03

alues of �G, �H and �S which obtained from eqs.3 and 4 are pre-ented in Table 1. Considering these values, van der Waals forcend hydrogen bonding were observed for the interaction of L4 with-LG. However, the hydrophobic interactions were the major act-

ng forces during physical binding of L1, L2 and L3 to this protein.he negative values of �G proved that all of the interactions werepontaneous. On the contrary to ligand L4, ligands L1, L2 and L3ere interacted more spontaneously as a function of temperature

levation.

.4. UV spectra and binding parameters

The PFH/�-LG binding constants were also calculated using theV–vis spectroscopic method. As described previously, using a plotf 1/(A − Ao) versus 1/[L] according to Eq. (6), where A0 is the initialbsorbance of free �-LG and A is the absorbance of PFH/�-LG com-lexes, the binding constant can be obtained using Eq. (7) by theatio of the intercept to the slope (Fig. 4). One binding site for allf the PFHs on the protein was conceived. The order of binding ofhe PFHs is consistent with the obtained results from fluorescence

easurements. Also, L4 with a bi-pyrimidine fused heterocycleemonstrates the highest affinity for binding to �-LG and the bind-

ng affinity is according to the following order: L4 > L1 > L3 > L2. Thebtained results are presented in Table 2.

.5. CD spectroscopy analysis

Further experiments were carried out with circular dichro-sm to investigate the secondary structure changes of �-LG dueo its interaction with PFH ligands. Circular dichroism is a sensi-ive and accurate method for exploring protein structural changesnd interaction with small molecules. Far-UV CD spectra of �-G (195–260 nm) were obtained in the absence and presence of0, 40 and 80 �M of L1–L4. The CD spectra prove the structuraleatures which can be attributed to the �-helical and �-sheettructural elements (data not shown). Moreover, the negativellipticity at 217 nm of these spectra proves the high portion of-sheet in �-LG structure. The results of deconvolution of CDpectra were used to give the most close assessment on the con-ent of �-LG secondary structures [32]. The secondary structurelements of �-LG in the absence and presence of different concen-rations of L1, L2, L3 and L4 were evaluated by deconvolution ofheir corresponding CD spectra. The obtained results are presented

n Table 3.

As shown in this table, native �-LG contains 17% �-helix, 42.96%-sheet and 40.14% random coils which are in good agreement with

he previous literatures [33–35]. The interaction of �-LG with PFH

able 3ontent of secondary structure of �-LG upon interaction with PFH ligands at pH 7.0 in 50

Concentration (�M) L1 L2

� helix � sheet Random coil � helix � sheet Rand

0 17 42.96 40.14 17 42.96 40.1420 17.3 42.9 39.8 17.2 42.8 4040 17 42.8 40.2 17.35 42.75 39.960 17.2 42.8 40 16.7 43.1 40.2

(A–D) in 50 mM NaPi buffer pH 7.0. Inset: a plot of 1/A − A0 vs. 1/ligands concentra-tions for (A) L1/�-LG, (B) L2/�-LG, (C) L3/�-LG and (D) L4/�-LG. The binding constantKb is the ratio of the intercept and the slope.

ligands does not significantly affect folding state of the protein;furthermore upon complexation of PFHs with �-LG, no importantalteration in the secondary structures was observed. Therefore, it

is suggested that �-LG conformation almost remained intact andno perturbation in the protein structure was established due to theinteraction with PFH ligands.

mM phosphate buffer.

L3 L4

om coil � helix � sheet Random coil � helix � sheet Random coil

17 42.96 40.14 17 42.96 40.1417.7 42.4 30.9 16.6 43 40.417.7 42.4 39.9 17 42.9 40.117.8 42.5 39.7 16.8 42.9 40.3

rfaces

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M.H. Mehraban et al. / Colloids and Su

. Conclusion

In this study, four PFH derivatives with already provednhibitory properties against �-Gls were used to study their inter-ction with bovine �-LG. These PFH derivatives are potentiallymportant to control postprandial hyperglycemia (PPHG) in type-IIiabetes and for treatment of HIV/AIDS infection. �-LG speculateso be a reasonable protein carrier of these inhibitor moleculesor delivering them to the small intestine where their targetnzyme (�-Gls) is existed. Different spectroscopic methods (fluo-escence, CD and UV–vis spectroscopy) were applied to investigatenteraction between the PFH derivatives and �-LG. The resultsemonstrated that all of these ligands can interact with �-LGhrough static quenching. According to the thermodynamic datahich obtained from the interaction of these ligands with �-LG,ydrophobic interactions proved to play a major role in binding of1, L2 and L3, while the hydrogen bonding and van der Waals forcesere the acting force in the complexation of L4. While L4 revealed

he strongest affinity towards �-LG, L2 displayed the weakest bind-ng affinity to this carrier protein. The n values indicate that �-LGnteracts with PFH ligands, forming equimolar complexes. Further-

ore, the structure of �-LG remained intact and no secondarytructure alteration was observed in this protein upon interactionith the PFH derivatives. It is worth to mention that L4 with theighest binding constant for �-LG also demonstrated the strongest

nhibitory activity against mammalian �-Gls. Based on the achievedesults, these compounds show the desirable features of functionalharmacological agents, as they have the potentiality to be deliv-red to their target enzyme (�-Gls) by the aid of �-LG.

cknowledgments

We would like to thank financial support of Iran National Scienceoundation (INSF)-Grant no. 88001894. Also the financial supportsf research council of Shiraz University are gratefully acknowl-dged.

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.013.08.013.

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