Journal of Photochemistry and Photobiology B: Biology 127 (2013) 18–27

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Journal of Photochemistry and Photobiology B: Biology

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Differential binding modes of anti-cancer, anti-HIV drugs belongingto isatin family with a model transport protein: A joint refinementfrom spectroscopic and molecular modeling approaches

1011-1344/$ - see front matter � 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.jphotobiol.2013.06.016

⇑ Corresponding author. Tel.: +91 33 2350 8386; fax: +91 33 2351 9755.E-mail address: nguchhait@yahoo.com (N. Guchhait).

1 These authors contributed equally to this work.2 Present address: Department of Chemistry and Biochemistry, University of

Colorado Boulder, CO 80309, United States.

Debarati Ray 1, Bijan K. Paul 1,2, Nikhil Guchhait ⇑Department of Chemistry, University of Calcutta, 92 A.P.C. Road, Calcutta 700009, India

a r t i c l e i n f o

Article history:Received 13 March 2013Received in revised form 26 June 2013Accepted 28 June 2013Available online 20 July 2013

Keywords:Drug isatin and methyl isatinProtein BSADrug protein interactionFluorescenceCDAutoDock simulation

a b s t r a c t

The present contribution reports a detailed characterization of the binding interaction of two potentialanticancer, anti-HIV drugs isatin (IST) and 1-methylisatin (MI) with model transport protein BovineSerum Albumin (BSA). Thermodynamic parameters e.g., DH, DS and DG for the binding phenomenonhave been evaluated on the basis of van’t Hoff equation to understand the force behind the binding pro-cess. A combined application of steady-state and time-resolved fluorescence spectroscopic techniquessubstantiate the observed drug-induced quenching of intrinsic tryptophanyl fluorescence of the proteinto proceed through a static mechanism. Circular dichroic (CD), synchronous fluorescence and excitation–emission matrix fluorescence spectroscopic techniques have been exploited to delineate the secondaryand tertiary conformational changes in the protein structure induced by the binding of drugs (IST/MI).The probable binding location of the drug molecules within the protein cavity (hydrophobic subdomainIIIA) has been explored from AutoDock-based blind docking simulation. Examination of drug–proteinbinding kinetics using stopped-flow fluorescence technique reveals that the association constants (ka)for IST-BSA and MI-BSA interactions are 1.09 � 10�3 s�1 (±5%) and 1.73 � 10�3 s�1 (±5%), respectively,at the experimental temperature (T) of 298 K. The present study also delves into the effect of drug-bind-ing on the esterase activity of the protein which is found to be reduced in the drug–protein conjugatesystem in comparison with the native protein.

� 2013 Elsevier B.V. All rights reserved.

1. Introduction

Isatin (1H-indole-2,3-dione), belonging to the well-known fam-ily of indole derivatives, was first obtained as a product from chro-mic acid oxidation of indigo dye by Erdmann [1] and Laurent [2] inthe middle of the 19th century. However, a host of sources of isa-tins has been garnered by the nature also as they occur in manyplants like genus Isatis [3], Couroupita guianensis AubI [4] and Cal-anthe discolor LINDL [5]. More interestingly isatin is an endogenousindole which is widely distributed in mammalian brain, peripheraltissues and body fluid and also found as a metabolic derivative ofadrenaline in the human body [6–8]. In recent time, isatins haveformed the nucleus of many-faceted research activities owing tothe multitude of potential applications in clinical and medicinal as-pects. Isatin derivatives opened up a vista of promising prospectsin synthetic organic chemistry owing to their biological and phar-macological properties [3,9]. Isatins and many analogous com-

pounds have formed a prospective avenue of researchsurrounding their anticancer, antioxygenic, anticonvulsant, anti-bacterial properties, and sedative activities [10,11]. In addition,Mannich bases of isatin derivatives have been recognized for theirantirhombotic, antiallergic, muscle relaxing, fibrinolytic activities[10,11]. In fact, some derivatives of isatins (e.g., 5-fluoro-3-substi-tuted-2-oxoindole) are already in use for the treatment of gastroin-testinal stromal tumors [12] and advanced renal cell carcinoma[13], while many other halogenated derivatives are in use for treat-ment of cancer and leukemia [14]. Probably, the most demandingprospect of research surrounding the isatin derivatives has evolvedin the context of their antifungal [15] and antiviral [16,17] activi-ties. Human Immunodeficiency Virus (HIV) is a lentivirus thatcauses Acquired Immunodeficiency Syndrome (AIDS) [18,19], inwhich progressive collapse of the human immune system allowslife-threatening opportunistic infections and cancers to thrive.Increasing infection with HIV has already proven alarming to thehuman society. Moreover, the human race has faced a dire ramifi-cation for the development of drugs with anti-HIV activity whichhas thus been central to medicinal chemistry research. Isatin deriv-atives have claimed their relevance and importance in the field ofanti-HIV activity [20].

D. Ray et al. / Journal of Photochemistry and Photobiology B: Biology 127 (2013) 18–27 19

The present study is based on the interactions of two isatindrugs (viz., isatin (IST) and 1-methylisatin (MI)) with the modeltransport protein bovine serum albumin (BSA). Serum albuminsare abundantly found in blood plasma and belong to one of themost widely studied categories. They function as carrier for numer-ous exogenous and endogenous compounds in the body. The pri-mary structure of BSA is composed of 583 amino acid residuesand the secondary structure is characterized by �66% a-helix[21–25]. BSA contains two tryptophan residues, TRP-134 andTRP-212 of which the former is located in hydrophilic subdomainIB and the latter in hydrophobic subdomain IIA [22].

The study of binding interactions of isatin drugs with biologicaland biomimicking receptors comprises a germane field of research.However, it is surprising to note that this field of research remainsrather sporadically explored in the literature. Herein, we report aspectral deciphering of the binding interactions of two drugs fromthe isatin family, viz., isatin (IST) and 1-methylisatin (MI) (cf.Scheme 1) with model transport protein bovine serum albumin(BSA). In order to delve into the binding mechanism and natureof the binding forces in the drug–protein interaction process thethermodynamic parameters have been evaluated from van’t Hoffrelationship by applying temperature-dependent fluorescencequenching. An attempt is also undertaken to unravel the effect ofdrug-binding on the protein secondary and tertiary structures soas to rationalize the applicability of the drug molecules as thera-peutic agents. The AutoDock-based ‘blind docking’ strategy hasbeen exploited to delineate the probable binding location of thedrugs within the protein backbone. With a view to the prospectivebiological properties of isatins the characterization of bindinginteraction of IST and MI with a model transport protein appearsto have significance in relation to further development of biomed-icines and the field of safe-engineered drug delivery.

2. Experimental

2.1. Materials

Commercially available isatin and 1-methylisatin (cf. Scheme 1)were obtained from Sigma Chemical Co., USA and used as received.The purity of the compound was established on TLC plate beforeuse. BSA from Sigma Chemical Co., USA was used as received. Trisbuffer was purchased from SRL, India, and 0.01 M Tris–HCl bufferof pH 7.4 was prepared in triply distilled deionized water from aMilli-Q water purification system (Millipore). The solvent appearedvisually transparent and the purity of the solvent was also testedby running the fluorescence spectra in the studied wavelengthrange. Hydrochloric acid (HCl) from E-Merck was used as obtained.

2.2. Instrumentation and methods

2.2.1. Steady state spectral measurementsThe absorption and emission spectra were recorded by Hitachi

UV–Vis U-3501 spectrophotometer and Perkin–Elmer LS55fluorimeter, respectively. All spectra were recorded with appropri-ate background corrections. The concentrations of the protein

N

O

O

H

N

O

O

CH3

Isatin (IST) 1-Methylisatin (MI)

Scheme 1. Schematic molecular structures of the studied drug molecules.

(BSA) and the drugs (IST and MI) used in different experimentshave been specified in the context of the relevant discussions.

Only freshly prepared solutions were used for all spectroscopicmeasurements. The temperature was kept constant at a givenvalue by a recycling water flow accurate upto ±1.0 �C.

2.2.2. Excitation–emission matrix spectraThe excitation–emission matrix spectra (EEMS) or the three-

dimensional fluorescence spectra were recorded by the same Per-kin–Elmer LS55 fluorimeter when the emission wavelength rangewas selected from 200 nm to 500 nm and the initial excitationwavelength was set within 200 nm to 400 nm with 10 nm interval.All other parameters were adjusted to those specified previously[26].

2.2.3. Synchronous fluorescence spectraThe constant wavelength synchronous fluorescence spectra

(CWSFS) presented in this study were monitored on the same Per-kin–Elmer LS55 fluorimeter at a constant protein concentration(10 lM) with varying drugs (IST and MI) composition usingDk = 15 nm and Dk = 60 nm which yield characteristic informationfor the tyrosine (TYR) and tryptophan (TRP) microenviromentwithin the protein [26].

2.2.4. Time-resolved fluorescence decayFluorescence lifetimes were obtained by the method of Time

Correlated Single-Photon counting (TCSPC) on FluoroCube-01-NLspectrometer (Horiba Jobin Yovon) using a light source of nanoLEDat 291 nm and the signals were collected at the magic angle of54.7� to eliminate any considerable contribution from fluorescenceanisotropy decay [26]. The decays were deconvoluted by DAS-6 de-cay analysis software. The acceptability of the fits was judged by v2

criteria and visual inspection of the residuals of the fitted functionto the data. The reported lifetimes data presented in the table arean average of five individual measurements.

2.2.5. Circular dichroismCircular dichroism (CD) spectra were recorded by a JASCO J-815

spectropolarimeter using a cylindrical cuvette of 0.1 cm path-length at 25 �C. The reported CD profiles are an average of four suc-cessive scans obtained at 20 nm/min scan rate with appropriatelycorrected baseline. The concentration of BSA and the drug duringCD measurements are mentioned in the relevant discussion.

2.2.6. Kinetics measurementThe kinetics of associations of the drugs IST and MI with the

protein BSA were monitored by Perkin–Elmer LS55 fluorimeterusing the stopped-flow fluorescence measurement technique. Thedead time of the instrument was found to be 20 ms.

2.2.7. Esterase activity assayThe effect of the drug on the esterase activity of BSA was as-

sayed with the synthetic substrate p-nitrophenyl acetate (PNPA)by following the formation of p-nitrophenol [27] at 37 �C. One unitof esterase activity was defined as the amount of the enzyme (BSA)required to liberate 1.0 lM p-nitrophenol per minute at 37 �C.

2.3. Molecular modeling: blind docking simulation

The native structure of BSA was taken from the Protein DataBank having PDB ID: 3V03 (DOI: 10.2210/pdb3v03/pdb [28]);Docking studies were performed with AutoDock 4.2 suite of pro-grams which utilizes the Lamarckian Genetic Algorithm (LGA)implemented therein. For docking of IST and MI with BSA, the re-quired files (corresponding to the three-dimensional structure ofthe drugs) for the ligand (IST/MI) were created through the

20 D. Ray et al. / Journal of Photochemistry and Photobiology B: Biology 127 (2013) 18–27

combined use of Gaussian 03W [29] and AutoDock 4.2 [30] soft-ware packages. The geometries of IST and MI were first optimizedat DFT//B3LYP/6-311++G(d,p) level of theory using Gaussian 03Wsuite of programs and the resultant geometries were read in Auto-Dock 4.2 software in compatible file format, from which the re-quired files were generated in AutoDock 4.2. The grid size wasset to 126, 126, and 126 along X-, Y-, and Z-axis with 0.397 Å gridspacing, i.e. in order to recognize the binding sites of IST and MI inBSA blind docking was performed. The AutoDocking parametersused were as follows: GA population size = 150; maximum numberof energy evaluations = 250,000; GA crossover mode = two points.The lowest binding energy conformer was searched out of 10 dif-ferent conformations for each docking simulation and the resultantminimum energy conformation was applied for further analysis.The PyMOL software package was used for visualization of thedocked conformations [31].

3. Results and discussions

3.1. Drug–protein binding interaction: mechanistic assay of theinteraction process

The UV–Vis absorption spectral measurement is a simple butefficient technique to explore structural changes and complex for-mation. The drug (IST)-induced modulation in the absorption spec-tral profiles of BSA is displayed in Fig. 1 (Fig. S1 for MI in theSupporting information). Absorption spectrum of the proteinexhibits a strong band at kabs � 278 nm which arises from threeamino acid residues, viz., TRP, TYR and PHE and a hump at kabs �308 nm is also generated upon increasing the concentration ofboth the drugs, IST and MI. It is to mention in this context thatthe absorption profile of the two drugs (IST and MI) is comprisedof two absorption maxima viz., kabs � 240 nm for IST and kabs �245 nm for MI and another small hump at kabs � 305 nm. The insetof Fig. 1 indicates that the absorption profiles of the protein aloneand 1:1 drug (IST):protein (BSA) complex cannot be directly super-imposed with each other within the experimental error limit. Suchobservation appears to be realizable on the ground of complex for-mation between the drugs (IST and MI) and the protein (BSA) [32–35]. Further confirmation on this issue has been derived from theforthcoming discussions.

Herein, the intrinsic tryptophanyl fluorescence of the proteinBSA is exploited as the actuating tool to follow the drug (IST/MI)–protein (BSA) binding interaction process. Both the drugs arefound to be non-fluorescent in nature whence the drug–proteininteraction is solely examined by monitoring the drug-inducedmodifications in protein fluorescence [21–23,26]. The intrinsic

Fig. 1. Absorption profile of the protein ([BSA] = 10.0 lM) in aqueous buffer(pH = 7.4) in the presence of increasing concentration of the drug IST. Curves(i ? vii) correspond to [IST] = 0, 2.0, 4.0, 6.0, 10.0 and 12.0 lM. Inset showsabsorption spectra for the drugs alone (12.0 lM: –d–), BSA alone (10.0 lM: –N–)and for 1:1 drug:BSA complex (–j–).

fluorescence of BSA mainly arises from the presence of two trypto-phan residues viz., TRP-212 which is buried inside the hydrophobicsubdomain IIA of the protein cavity and TRP-134 in the hydrophilicsubdomain IB which is relatively more exposed to the surroundingenvironment [21–23].

Fig. 2a displays a plot for the variation of the intrinsic fluores-cence of the protein in presence of various concentrations of drugIST (cf. Fig. S2a for MI in Supporting information) at a representa-tive temperature of 298 K. The protein BSA is excited at 295 nmin order to exclusively excite the TRP moiety and the characteristicemission maxima is centered at �346 nm [36]. Upon addition ofthe drugs, IST and MI, the fluorescence intensity of BSA is foundto accompany a regular quenching (cf. Fig. 2a and Fig. S2a). TheTRP residue of the protein is found to be characterized by kmax

em �346 nm (cf. Fig. 2a). This characteristic kmax

em identifies a signaturefor TRP residue embedded in a hydrophobic microenvironment inthe native protein [21–23,26]. The intrinsic fluorescence of serumalbumins is documented to be considerably sensitive to its micro-environments especially around the TRP moieties. Thus the modu-lations of fluorescence properties of the native protein can form aprospective avenue for probing of interaction with a host of exoge-neous ligands and/or drug molecules. In the present study, theinteractions of the drugs (IST and MI) with the transport protein(BSA) are found to be characterized by prominent quenching ofthe intrinsic protein fluorescence with no discernible shift on theemission wavelength.

The phenomenon of fluorescence quenching is conventionallydescribed by the following well-known Stern–Volmer equation[26]:

I0

I¼ 1þ KSV½Q � ¼ 1þ kqs0½Q � ð1Þ

in which I0 is the original fluorescence intensity and I is thequenched intensity, [Q] is the molar concentration of the quencher(here IST and MI), KSV is the Stern–Volmer quenching constant, kq isthe bimolecular quenching rate constant and s0 is the fluorescencelifetime of the fluorophore (here BSA) in the absence of the quench-er. An illustrative example of Stern–Volmer plot for the drug IST in-duced fluorescence quenching of the protein at varioustemperatures is displayed in Fig. 2b (cf. Fig. S2b in the Supportinginformation for MI). The Stern–Volmer plots in both the cases arefound to be linear. Generally a linear Stern–Volmer plot indicatesthe operation of either static or dynamic (collisional) quenching[26,37–39]. Moreover, the Stern–Volmer quenching constant (KSV)values obtained as a function of temperature (vide Table 1) is foundto vary inversely with increasing temperature for IST drug whereasthe same for MI proportionately varies with temperature. Appar-ently these results point toward a static quenching mechanism forthe quenching of tryptophanyl fluorescence of BSA with ISTwhereas for MI it seems to indicate a probable dynamic quenchingmechanism. Thus, in order to delineate the actuating mechanism offluorescence quenching, time-resolved fluorescence spectroscopicresults are meticulously explored. The typical nanosecond-resolvedfluorescence decay profile of the protein (BSA, kex = 291 nm,kmonitored = kmax

em ) with increasing drug concentrations (IST and MI)are displayed in Fig. S3 (Supporting information) and the corre-sponding decay parameters are summarized in Table 2. The biexpo-nential fluorescence decay of the native protein (in the absence ofdrugs) is in well accord with reported literature [26,36].

The multiexponential fluorescence decay (I(t)) is described bythe following expression:

IðtÞ ¼X

i

aisi ð2Þ

in which ai represents the pre-exponential factor (amplitude) corre-sponding to the ith decay time constant, si [26,37–39,40].

Table 2Time-resolved fluorescence decay parameters for intrinsic tryptophanyl fluorescence quenching of the protein (BSA) in the presence of varying concentrations of the drugs (ISTand MI).

[IST] (lM) s1 (ns)a s2 (ns)a a1 ( ± 0.02)a a2 ( ± 0.02)a v2 hsa0ib (ns) hsi0ic (ns)

0 2.32 ± 0.099 6.49 ± 0.012 0.26 0.74 1.06 5.41 6.032 2.31 ± 0.092 6.53 ± 0.011 0.27 0.73 1.05 5.39 6.044 2.23 ± 0.088 6.48 ± 0.011 0.27 0.73 1.14 5.33 6.016 2.18 ± 0.083 6.47 ± 0.012 0.27 0.73 1.06 5.31 6.08 2.13 ± 0.081 6.41 ± 0.011 0.27 0.73 1.03 5.25 5.94

10 2.25 ± 0.074 6.49 ± 0.011 0.28 0.72 1.06 5.30 5.99

[MI] (lM)

0 2.32 ± 0.099 6.49 ± 0.012 0.26 0.74 1.09 5.41 6.032 2.25 ± 0.082 6.40 ± 0.011 0.30 0.70 1.06 5.16 5.864 2.24 ± 0.093 6.40 ± 0.014 0.31 0.69 1.02 5.11 5.846 2.21 ± 0.082 6.37 ± 0.011 0.31 0.69 1.11 5.08 5.818 2.14 ± 0.077 6.36 ± 0.011 0.31 0.69 1.08 5.05 5.81

10 2.21 ± 0.072 6.36 ± 0.011 0.31 0.69 1.10 5.07 5.80

a Error limit in the calculation of time constant and corresponding amplitude.b Amplitude-weighted lifetime.c Intensity-weighted lifetime.

Fig. 2. (a) Spectral profile for quenching of intrinsic tryptophanyl fluorescence of BSA in the presence of varying concentration of the drug IST at a representative temperatureT = 298 K ([BSA] = 10.0 lM, kex = 295 nm, pH = 7.4 in Tirs–HCl buffer). Curves (i ? x) correspond to [IST] = 0, 1.0, 2.0, 3.0, 4.0, 5.0, 7.0, 9.0, 10.0, 11.0 lM. (b) Stern–Volmer plotsfor the quenching of intrinsic tryptophanyl fluorescence of the protein (BSA) in the presence of increasing concentration of the drug IST at varying temperatures (T = 293K: –j–, 298 K: –d–, 313 K: –N–) as specified in the figure legend.

Table 1Summary of binding and thermodynamic parameters for the drug (IST/MI): protein (BSA) interaction process.

Drug T (K) 10�3 KSV ( ± 5%)a (M�1) 10�12 kq ( ± 5%)a (M�1 s�1) 10�3 K ( ± 5%)a (M�1) nb DG (kJ mol�1) DH (kJ mol�1) DS (J mol�1 K�1)

IST 293 22.80 3.78 15.59 0.97 �23.58 �67.85 �151.1298 19.60 3.25 12.79 0.97 �22.82303 19.11 3.17 4.83 0.91 �22.1308 18.56 3.08 3.82 0.88 �21.31313 13.11 2.18 3.10 0.88 �20.56

MI 283 12.19 2.02 14.25 1.07 �22.39 �17.91 15.87288 12.81 2.12 11.12 1.14 �22.47293 13.58 2.25 10.25 0.99 �22.55298 15.28 2.53 9.89 1.05 �22.63308 15.51 2.57 7.24 0.88 �22.79

a Each data is an average of 5 individual measurements.b Binding site according to Eq. (5).

D. Ray et al. / Journal of Photochemistry and Photobiology B: Biology 127 (2013) 18–27 21

This implies an intensity-weighted average lifetime hsi0i to bedefined as [26,37–39,40]:

hsi0i ¼P

iais2iP

iaisið3Þ

and an amplitude-weighted average lifetime hsa0i as [26,37–39,40]:

hsa0i ¼P

iaisiPiai

ð4Þ

The operation of dynamic quenching mechanism should havebeen reflected through the lowering of the individual decay timeconstants with increasing quencher concentration, and hence theintensity-weighted average lifetime (hsi0i) [26,37–39,40]. In thepresent study, the individual decay time components of the proteinare found to be only nominally influenced in the presence of thedrugs (IST and MI) (cf. Table 2). The data compiled in Table 2 alsoreflect the constancy of the intensity-weighted average life time(hsi0i) of BSA with increasing concentration of both the drugs

22 D. Ray et al. / Journal of Photochemistry and Photobiology B: Biology 127 (2013) 18–27

(IST and MI) which in turn unequivocally establish the inoperative-ness of dynamic quenching mechanism in the presently studiedcases.

Further, the nature of quenching mechanism (static or dynamic)can be established from the analysis of the magnitude of the bimo-lecular quenching constant (kq), (ca. Eq. (1)). The calculated kq val-ues (cf. Table 1) are found to be remarkably higher (two orders ofmagnitude higher) than the maximum threshold for diffusion-con-trolled process (2 � 1010 M�1 s�1) [26,40]. This result, coupled withthe aforementioned discussion on the results of time-resolvedfluorescence decay experiments, leads to negate the possibility ofdynamic quenching and attests the operation of static mechanismfor the observed quenching with both the drugs IST and MI [26,32–35,36,39,40].

Further, it is interesting to note in the present context that thedeconvoluted time-resolved fluorescence decay parameters of theprotein as a function of the concentration of the drugs (IST andMI) as comprised in Table 2 reflect the invariance of the pre-expo-nential factors (amplitude) corresponding to the individual life-times. This seemingly simple kind of observation dictates theinoperativeness of Förster’s resonance energy transfer (FRET) fromthe TRP residue of the protein to the drugs as an actuating mecha-nism resulting in fluorescence quenching of the protein [26,40,41].The resulting constancy in the amplitude-weighted average life-time (hsa0i) of BSA in the presence of the drugs (cf. Table 2) mani-fests a consequence of nonoccurrence of FRET [26,40].

3.2. Binding parameters

Quantitative evaluation of the binding efficiency of drugs toprotein is important as it characterizes an important pharmacoki-netic parameter. The therapeutic efficiency of drugs is affected bytheir degree of binding to the proteins present in blood plasma[42]. Therefore, it is imperative to evaluate the binding constant(K) and the free energy change (DG) for the drug–protein (BSA)interaction process. The following equation has been employedfor the determination of the drug–protein binding constant in thepresent case [43]:

logI0 � I

I

� �¼ log K þ n log½Q � ð5Þ

in which K is the binding constant and n is the number of bindingsites, I0 and I represent the fluorescence intensity of BSA, respec-tively, in the absence and presence of the drugs. The double loga-rithmic plots of log½I0�I

I �vs: log½Drug� at various temperatures arepresented in Fig. 3 and the corresponding results are collected inTable 1. Generally static quenching as induced by ground state com-plex formation is characterized by an inverse relationship of K withtemperature [26,39]. Here also the binding constant (K) for the drug

Fig. 3. Double logarithmic plot of log[(I0 – I)/I] vs. log[Drug] for elucidation of the bindin(T = 283 K: –j–, 293 K: –N–, 308 K: –d–) interaction at different temperatures. Here I0 dthe drugs (IST/MI) and the I terms denote the same in the presence of varying concentr

(IST and MI)–protein(BSA) binding interaction decreases withincreasing temperature which again corroborates to the oper-ation of static quenching mechanism as discussed above (videSection 3.1).

3.3. Thermodynamic parameters: elucidating the nature of the bindingforces

In a broad sense, the underlying forces governing the drug–pro-tein binding interaction process can be manifold, such as ionic/hydrophobic interaction, hydrogen bond or van der Waal’s forcesand so forth. The thermodynamic parameters e.g., enthalpy change(DH), entropy change (DS) associated with the drug–protein inter-action process can be employed as some potential tools for deci-phering the actuating binding force(s) [39,44]. For this purposethe temperature dependence of binding constant has been investi-gated and the relevant data have been summarized in Table 1. Inorder to identify the nature of interactions between the studieddrugs with the protein the thermodynamic parameters (e.g., DH,DS and DG) have been evaluated from the following van’t Hoffequation (assuming no significant variation of enthalpy withinthe range of temperature studied) [32–35,39,44,45]:

ln K ¼ �DHRTþ DS

Rð6Þ

Here R is the universal gas constant, T is the Kelvin temperature.The free energy change (DG) of the process is then estimated

from the following relationship:

DG ¼ DH � TDS ð7Þ

The calculated thermodynamic parameters are collected inTable 1 and the representative van’t Hoff plot (ln K vs. 1/T) is dis-played in Fig. 4. Ross and Subramanian [44] have previously dem-onstrated the identification of various interaction forces from theanalysis of thermodynamic parameters in the binding interactionprocess. Also an excellent review on the topic has recently been re-ported by Homans [45]. Briefly, the model of interaction can besummarized as follows on the basis of the thermodynamics data:

(i) DH � 0, DS > 0 correspond to hydrophobic forces.(ii) DH < 0, DS < 0 correspond to van der Waals interaction,

hydrogen bond formation.(iii) DH < 0, DS > 0 correspond to electrostatic/ionic interactions.

Scrutiny of the results presented in Table 1 thus reflects the pre-dominance of van der Waal’s interaction/ hydrogen bonding inter-action as the responsible factor for IST–BSA binding. This inferenceis well supported from blind docking simulation result whichshows a hydrogen bonding interaction between the oxygen atom

g constant of the (a) IST–BSA (T = 293 K: –d–, 298 K: –j–, 313 K: –N–), (b) MI–BSAesignates the intrinsic tryptophanyl fluorescence intensity of BSA in the absence ofations of the respective drugs.

Fig. 4. van’t Hoff plot of lnK vs. 1/T for elucidation of the thermodynamicparameters for interaction of the drugs IST (–j–) and MI (–N–; inset) with theprotein (BSA). K (in M�1) denotes the binding constant at a given temperature (T inKelvin).

D. Ray et al. / Journal of Photochemistry and Photobiology B: Biology 127 (2013) 18–27 23

of IST drug with the hydrogen atom of SER-428 residue of protein(H bond distance 2.1 Å as found from Autodock-based blind dock-ing simulation, elaborately discussed in upcoming Section 3.6). Onthe other hand, the as-obtained low negative DH and positive DSvalues for MI–BSA interaction suggest the combined effect ofhydrophobic association and electrostatic attraction. This result isfound to be in reasonable harmony with the binding force of MIwith other biological receptor [46].

3.4. Conformation investigation

3.4.1. Circular dichroism (CD) spectroscopyThe far-UV CD spectrum of native BSA is characterized by two

minima at �208 nm and �222 nm, corresponding to a a-helix richsecondary structure of the native protein (BSA) (vide Fig. S5 in theSupporting information). The change of protein secondary struc-ture following interaction with the drugs has been studied fromthe modulations in the far-UV CD spectra of native BSA in the pres-ence of increasing concentration of the drugs (IST and MI). The far-UV CD spectral profile of BSA (cf. Fig. S5) reflects a decrease of CDsignal in response to increasing drug concentration without signif-icant shifting of the peak positions. These results primarily indicatesome sort of conformational change in BSA secondary structure asa result of interaction with the studied drug molecules. The lower-ing in the negative ellipticity points towards a decrease in thea-helical content which dictates unfolding of the peptide strand[25,36–39].

The modification in the BSA secondary structure in the presenceof the drugs has been quantified from the observed CD results at298 K. The details of the quantitative analysis [38,47,48] of theCD results are elaborated in the Supporting information (Sec-tion S5). The estimated a-helicity content of native BSA in Tris-HCl buffer (pH = 7.40 and at T = 298 K) comes out to be �66(± 2)%, which is in reasonable agreement with literature reports[21–23,26,36,38,46–48]. A loss in the a-helicity content (from�66 (± 2)% in the free protein to �63.8 (± 2)% in the presence of6.0 lM IST and to �64.4 (± 2)% in the presence of 6.0 lM MI) ofthe protein upon binding to the drugs is also evident from thequantitative analysis of the far-UV CD spectral data (cf. Table S1in the Supporting information). A close perusal of the quantitativeanalysis of the CD spectral results also delineate that the perturb-ing effect of drug binding on the native protein conformation (asestimated on the basis of loss of a-helicity) is relatively greaterwith IST as compared with MI. Drawing on this, a consistent reflec-tion of the effect is also seen in the context of drug binding effect

on the functionality (in terms of esterase activity) of the proteinas discussed in forthcoming Section 3.5.

3.4.2. Constant wavelength synchronous fluorescence spectroscopy(CWSFS)

The synchronous fluorescence spectra of BSA in the presence ofvarying drug (IST) concentration are shown in Fig. 5 (Fig. S4 of sup-porting information displays the effects for MI) [49]. The constantwavelength SFS technique involves simultaneous scanning of exci-tation and emission monochromators while maintaining a con-stant wavelength interval between them. According to the theoryput forward by Miller [50], when the wavelength interval (Dk) isfixed at Dk = 60 nm and Dk = 15 nm, the SFS yields characteristicinformation of TRP and TYR residue of the protein, respectively[39,47–50]. Fig. 5 shows representative synchronous fluorescencespectral profiles of the protein with increasing drug (IST) concen-tration for Dk = 15 nm and Dk = 60 nm. As seen in the figure,increasing IST concentration accompanies a little blue-shift of bothtyrosine (from kem � 292 nm in free BSA (i.e., absence of drug) tokem � 290 nm in the presence of the drug, cf. Fig. 5a) and trypto-phan (from kem � 285 nm in free BSA to kem � 283 nm in the pres-ence of the drug, cf. Fig. 5b) emission maxima (Fig. S4 of Supportinginformation displays the effects for MI). This blue-shift signifiesthat the fluorescent aromatic residues of the protein (TYR andTRP) are moved from a polar hydrophilic environment within thefree protein cavity to a more hydrophobic environment after inter-action with the drug molecule. This observation corroborates toour previous proposition (vide Section 3.4.1) that upon interactionwith the drug the secondary structure of the native protein is par-tially modified in such a manner the aromatic TRP and TYR resi-dues become buried as compared with the native proteinconformation [39,47–50].

The argument on conformational modulation of the native pro-tein upon interaction with the drugs is further evidenced from thestudy of excitation–emission matrix fluorescence spectroscopy(EEMS) which suggests the occurrence of specific interaction be-tween BSA and the drugs resulting in some micro-environmentaland conformational changes in BSA [39,51,52]. The relevant discus-sion is elaborated in the Supporting information (vide Section S6).

Thus the present score of observations corroborate to previousfindings in respect of conformational change of the protein uponinteraction with the drugs.

3.5. Esterase activity

So far we have discussed the binding parameters and variousthermodynamic parameters related to the drug–protein bindinginteraction, most probable binding mechanism and consequentlythe influence of drug binding on the conformation of the nativeprotein. Therefore, it is pertinent at this stage to explore the mod-ification of biological activity of BSA following interaction with thestudied drug molecules (IST and MI). Any disruption in the nativeprotein conformation will influence its functionality also. The mod-ification in the activity of BSA in the presence of the drugs can bemonitored by the well established esterase activity of the protein[27]. In this measurement the absorbance of p-nitrophenol(kabs = 400 nm) as generated from p-nitrophenyl acetate upontreatment of BSA is monitored. The effect of the drugs (IST andMI) on the esterase activity of BSA is depicted in Fig. 6. It is seenfrom the figure that upon binding with the drugs the esteraseactivity of BSA decreases, but the lowering of activity is not muchsignificant. Therefore, the above results are consistent with the re-sults of the CD experiment that the native protein structure is notseverely perturbed in the presence of the drugs IST and MI [27,48].However, it is intriguing to note that the relative lowering of theesterase activity of the protein (for a given amount of the protein,

Fig. 5. Constant wavelength synchronous fluorescence spectral (CWSFS) profiles of the protein ([BSA] = 10.0 lM) in the presence of varying concentrations of the drug IST at(a) Dk = 15 nm (–s–) and (b) Dk = 60 nm (–h–) as specified in the figure legends. Curves (i ? ix) correspond to [IST] = 0, 1.0, 2.0, 3.0, 4.0, 5.0, 7.0, 9.0, 10.0 lM. T = 298 K, andpH = 7.40 in Tris-HCl buffer.

Fig. 6. Relative variation in esterase activity of BSA in the presence of the drugs (ISTand MI; [Drug] = 10 � 10�5 M). The represented profile shows the effect of additionof the drugs on the release of p-nitrophenol in the reaction between BSA andp-nitrophenyl acetate (PNPA) as monitored by following the absorbance of liberatedp-nitrophenol at kabs = 400 nm and using e = 17, 700 M�1 cm�1. [BSA] = 25.0 lM,[PNPA] = 50.0 lM, T = 37 �C, pH = 7.4 in Tris-HCl buffer.

24 D. Ray et al. / Journal of Photochemistry and Photobiology B: Biology 127 (2013) 18–27

towards a given amount of the substrate with all experimentalconditions and instrumental settings being conserved) is greaterfor IST in comparison to MI. This is, however, understandable inconnection with the effect of drug binding on the extent of pertur-bation of the native protein conformation which is relatively great-er for IST compared with MI (as revealed from CD spectral results,cf. Sections 3.4.1 and S5 in the Supporting information).

3.6. Modeling of drug binding site in protein: blind docking simulation

In order for understanding the efficacy of a biologically active drugmolecule to function as a therapeutic agent, knowledge of its bindinglocation in the model transport protein environment is very crucialand important. Herein, the drug (IST and MI) binding sites in BSA havebeen explored on the basis of docking simulation performed accord-ing to the protocol described in Section 2.3. With an eye to evaluate anunbiased result in this respect, the AutoDock-based blind dockingsimulation has been employed. The strategy of AutoDock-based blinddocking includes a search over the entire surface of the protein forbinding sites (and simultaneously optimizes the conformations ofthe peptides [30,38,39,48,53,54] whereby indulging in an unbiasedresult, and hence has rightfully been described as ‘‘very encouraging’’in a recent review [54]. The docked poses displayed in Fig. 7 reveal thehydrophobic subdomain IIIA of the protein to be the favorable bindingsite for both the drugs IST and MI, though the drug molecules are notfound to be deeply embedded into the binding pocket [38]. Thisobservation is also in consensus with the fact that the principal hydro-phobic binding sites in BSA are located in domains II and III, while do-main I, characterized by a net negative charge, can serve as anappropriate binding site for cationic drugs [38]. As is usual in a blind

docking simulation protocol we obtained number of binding sitesand the corresponding free energies from which the resultantminimum energy conformation was used for further analysis[30,38,39,48,53,54]. Compelling evidence for probable bindinglocation of the drugs (IST and MI) in subdomain IIIA of BSA wasderived from the observation that for binding of IST and MI in thedomains I and II of the protein the binding energy and inhibitionconstant values were quite high in comparison to that for bindingin the hydrophobic binding site subdomain IIIA. The binding of drugsIST and MI in subdomain IIIA is found to be characterized by favorablebinding energy of –2.69 kcal/mol and –2.78 kcal/mol, respectively.

Also the minimum energy docked conformation has been fur-ther exploited to locate the protein residues in near vicinity ofthe drug binding sites. As seen from Fig. 7 the protein residues inimmediate vicinity (within 5.0 Å) at the drug binding locus areSER-428, TYR-451, THR-190, LEU-454, LEU-189, ALA-193, ARG-458, and ILE-455 for IST and MI (cf. Fig. 7). Therefore, the above dis-cussion (scrutinizing the protein residues around the bound drugs)points toward the fact that both the drugs reside on subdomain IIIAof BSA while the drug IST is also involved in hydrogen bondinginteraction with SER-428 (hydrogen bonding distance 2.1 Å, thedotted line in Fig. 7a). This result along with the experimentalobservation (vide Section 3.3) strongly supports the role of hydro-gen bonding interaction as a key binding force for IST–BSA interac-tion. However, given the same binding location for both the drugs(IST and MI) within the protein the absence of similar intermolec-ular hydrogen bonding interaction in the MI–BSA system may beargued to be due to the methyl crowding around the nitrogen atomof MI which hinders the carbonyl oxygen atom to form hydrogenbonding with SER-428 residue of BSA.

3.7. Drug (IST/MI)–protein (BSA) binding kinetics

In the study of interaction of small drug molecules with rele-vant biological and biomimicking targets the kinetics of interactionis considered to have vital diagnostic importance [48]. The kineticsof the drug (IST/MI)–protein (BSA) association reaction have beenstudied by monitoring the quenching of protein fluorescence atkem = 346 nm following interaction with the drugs IST and MI.Fig. 8 displays the representative fluorescence traces for the timecourses describing the processes of drug–protein interaction. Thefluorescence trace has been computer fitted by a nonlinear regres-sion to the following equation [48]:

IðtÞ ¼ A expð�katÞ þ C ð8Þ

in which I(t) is the fluorescence intensity at time t, A is amplitudecorresponding to the apparent association rate constant ka and Cis the fluorescence intensity at equilibrium.

Fig. 7. Stereo view of the docked conformation of the drugs (a) IST and (b) MI with the protein BSA. The drugs (IST/MI) are shown in Corey–Pauling–Koltun (CPK) model. Thelower panels in each case display the protein residues in near vicinity (within 5.0 Å) of the drugs over a molecular surface representation of the protein. The dotted linedenotes hydrogen bond. Color scheme: white for hydrogen atom, red for oxygen atom, blue for nitrogen atom and carbon atoms are green for drug molecules and yellow forprotein residues. The pictures have been prepared by PyMOL software package [31]. (For interpretation of the references to colour in this figure legend, the reader is referredto the web version of this article.)

Fig. 8. Fluorescence kinetic profiles for the interaction of the drugs (a) IST and (b) MI with the protein (BSA). The represented profile describes the time courses offluorescence quenching of BSA upon interaction with the drugs. The gray profile designates the raw data and the solid red line is the fitted line. kex = 295 nm,kmonitored = kem = 346 nm, T = 298 K, pH = 7.4 (Tirs–HCl buffer), [Drug] = 14 � 10�5 M, [BSA] = 2.0 lM.

D. Ray et al. / Journal of Photochemistry and Photobiology B: Biology 127 (2013) 18–27 25

The drug (IST/MI)–protein (BSA) association kinetics are foundto be characterized by a rate constant of ka (±5%) = 1.09 �10�3 s�1 for IST-BSA interaction and ka (±5%) = 1.73 � 10�3 s�1

for MI-BSA interaction at the experimental temperature ofT = 298 K. Here the observed fluorescence kinetic traces are fittedto a monoexponential decay function according to the pseudo-firstorder reaction kinetics model within the presently employedexperimental window in which the drug concentration is orderof magnitude higher than the protein concentration [48].

4. Summary and conclusions

The present study describes a spectral deciphering of the bind-ing interaction of two potential anti-HIV drugs belonging to theisatin family, viz., IST and MI with a model transport protein BSA.The evaluation of the thermodynamic parameters points out thedifferential modes of interaction of the two drugs. A careful inves-tigation from time-resolved fluorescence decay experiment dem-

onstrates that the quenching process is governed by staticquenching mechanism. The large magnitude of the quenching rateconstant (typically two orders of magnitude higher) in comparisonto the maximum threshold of diffusion-limited process also aids infavor of a static mechanism. Further, the results of our series ofexperiments on conformation investigation of the protein follow-ing drug binding lead to conclude conformational changes of thenative protein. This juxtaposes well with the observation of lower-ing of functionality of the protein upon drug binding. The Auto-Dock-based blind docking simulation study recognizes thehydrophobic subdomain IIIA of the protein as to be the probablebinding site for both the drugs (IST and MI) with commendableconformity to the experimental findings.

Overall, with a view to the wide range of prospective biologicalapplications of the drugs IST and MI, the characterization of thestrength, binding site and thermodynamics and kinetics parame-ters of the binding interaction of the drugs with a model transportprotein BSA, is of potential. We are optimistic that extension of thework to relevant biological and biomimicking targets should be

26 D. Ray et al. / Journal of Photochemistry and Photobiology B: Biology 127 (2013) 18–27

important from the standpoint of understanding the biological andmedicinal applications of these drugs.

Acknowledgments

DR acknowledges the University Grant Commission, Govt. of In-dia for research fellowship. NG acknowledges the Department ofScience and Technology, Govt. of India (Project No. SR/S1/PC/26/2008) and UPE Laser group for financial assistance. The authorsalso acknowledge the instrumental facility of Indian Associationfor the Cultivation of Science, Calcutta, India for providing accessto CD measurements.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.jphotobiol.2013.06.016.

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