chlorpromazine: Spectroscopic, calorimetric and computational approaches

Journal Pre-proof In-vitro binding analysis of bovine serum albumin with sulindac/ chlorpromazine: Spectroscopic, calorimetric and computational approaches

Samima Khatun, Riyazuddeen, Faizan Abul Qais PII:

S0167-7322(19)33377-X

DOI:

https://doi.org/10.1016/j.molliq.2019.112124

Reference:

MOLLIQ 112124

To appear in:

Journal of Molecular Liquids

Received date:

15 June 2019

Revised date:

21 September 2019

Accepted date:

11 November 2019

Please cite this article as: S. Khatun, Riyazuddeen and F.A. Qais, In-vitro binding analysis of bovine serum albumin with sulindac/chlorpromazine: Spectroscopic, calorimetric and computational approaches, Journal of Molecular Liquids(2018), https://doi.org/10.1016/ j.molliq.2019.112124

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© 2018 Published by Elsevier.

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In-vitro binding analysis of bovine serum albumin with sulindac/ chlorpromazine:Spectroscopic, calorimetric and computational approaches Samima Khatun1, Riyazuddeen1* and Faizan Abul Qais2 1

Department of Chemistry and 2Department of Agricultural Microbiology, Aligarh Muslim

University, Aligarh, 202002, U.P, India *Corresponding author Email: [email protected]

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Abstract The current study was undertaken to explore the binding of sulindac (SDC), and

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chlorpromazine (CPZ) with the bovine serum albumin (BSA) by fluorescence, isothermal titration calorimetric (ITC), UV-visible, site marker, circular dichroism (CD), and molecular

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docking studies. The fluorescence intensity of native BSA was quenched on increasing

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concentration of SDC/ CPZ in a static manner. The thermodynamic parameters, association constant (Ka), standard enthalpy change (∆Ho), standard entropy change (∆So) and standard

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Gibbs free energy change (∆Go) were obtained from ITC. Site marker experiment highlighted that SDC/ CPZ binds at Sudlow’s site I by using warfarin and ibuprofen probes for site I and

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site II, respectively. The distance between donor, BSA and acceptor, SDC/ CPZ was calculated by applying the FRET theory. Alteration in the secondary structure of BSA by

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SDC/ CPZ was revealed by UV-vis, synchronous and 3D fluorescence studies. The stability of native BSA increases and decreases, respectively in the presence of SDC and CPZ as shown by CD spectroscopy. Molecular docking was performed to further confirm the binding site, amino acid residues and type of interactions involved in the binding process. This study provides an insight into interaction at molecular level between SDC/ CPZ and BSA and will help to understand the thermodynamics and mechanism of drug binding. Keywords: Bovine serum albumin; Sulindac; Chlorpromazine; Spectroscopy; Isothermal titration calorimetry; Computational modeling. 1. Introduction Sulindac (SDC) (Fig. 1a), an indene-type anti-inflammatory agent, is useful in the treatment of acute or chronic inflammatory conditions [1]. SDC has shown an inhibitory effect on tumor growth in gastric, lung, and colorectal cancers in nude mice, with a concomitant decrease in cell growth and an increase in apoptosis [2]. It possesses

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antirheumatic, analgesic and antipyretic properties [3]. SDC is known to prevent relapse and reduce the occurrence of the number and size by as much as 60–70% in patients with familial adenomatous polyposis [4]. It is also of great effective in preventing the formation of intestinal tumors and inherited APC mutation in mouse models [5]. The mechanism of activity of SDC is unknown, but it is thought to act on enzymes COX-1 and COX-2, inhibiting synthesis of activators of inflammatory conditions such as prostaglandins, prostacyclins, and thromboxanes. SDC and its metabolites are potent inhibitors of the uptake and biliary clearance of bile acids in rat and human hepatocytes and also inhibit substrates of rat breast cancer resistance protein, rat and human organic anion-transporting polypeptides,

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and human multidrug resistance-associated protein [1]. SDC can also inhibit metastasis by

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disrupting β-catenin signaling [6].

Chlorpromazine (CPZ) (Fig. 1b) belongs to the family of phenothiazines, which is a

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group of structurally related amphiphilic compounds, recognized for their anti-dopamine activity and widely used as antidepressant tranquilliser in the treatment of psychosis [7].

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Chlorpromazine is a versatile, prototype neuroleptic drug having variety of actions. It acts on the hypothalamus and brainstem reticular formation. It has a remarkable ability to control

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hyperactive and hypomanic states without seriously impairing consciousness. It modifies abnormal behaviours in schizophonic states associated with increase dopaminergic activity in

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the limbic system of the brain [8]. Apart from their activity in neurons, phenothiazines have also been reported to display antiproliferative properties. CPZ was shown to inhibit

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proliferation of leukemia cells in culture without affecting normal lymphocyte viability and it also inhibits proliferation, induces apoptosis in different types of cultured cells including melanoma cells. Thus CPZ potentially used in cancer therapy and terminal cancer symptom alleviation [9]. CPZ stabilizes erythrocyte membranes and inhibits membrane depolarization by the displacement of membrane bound calcium. This property is believed to be associated with the ability of CPZ to inhibit virus-induced membrane fusion [10]. Furthermore, CPZ inhibits calmodulin, which regulates the availability of phosphoinositides via membrane recruitment of potential sequestrators of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) such as myristoylated alanine-rich C kinase substrate (MARCKS). Accordingly, CPZ inhibits clathrin-mediated endocytosis by abrogating the PI(4,5)P2-dependent membrane recruitment of the AP2 adaptor complex. Phenothiazines are also shown to inhibit proliferation and cause apoptosis of tumor cell lines, to abrogate differentiation, and to reverse multidrug resistance. In accord with its effects in vitro, CPZ has been shown to inhibit tumor growth in vivo in a

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number of model systems [11]. CPZ binds to three important blood components: membranes of red blood cells, albumin and lipoproteins [12]. Plasma proteins act as carriers for transportation of drugs and other compounds. Amongst the various plasma proteins, serum albumin is the most abundant protein and it plays a vital role in transportation of many endogenous and exogenous substances such as fatty acids, amino acids, steroids, metals, and drugs at their specific binding sites. Other physiological functions of serum albumin involve its contribution to osmotic blood pressure and pH of the blood, in sequestering oxygen free radicals, and in inactivating various toxic metabolites [13]. The importance of drug-protein binding study lies not only to determine the

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biological effect of a drug but it can also provide necessary information related to the

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therapeutic effect of drugs in pharmacology and pharmacodynamics. The information about the exact location of drug binding site on protein can make us better understand the way of

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distribution of a drug in the body and its use with other drugs and competitive natural catabolites such as bilirubin [14]. Among the family of serum albumins, bovine serum

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albumin (BSA) is a broadly studied model protein not only because of the presence of diverse binding sites and its structural resemblance with human serum albumin but also because of its

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medicinal importance, abundance, low cost, easy availability, remarkable ligand binding properties, and wide acceptance in pharmaceutical industries. BSA, a single chain heart

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shaped globular protein comprises of 583 amino acids and having three domains (I, II and III) which are further sub-divided into A and B [15]. These homologous domains are connected

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by disulfide bonds. Two tryptophan residues namely Trp-134 and Trp-213 located in subdomains IB and IIA, respectively are present in BSA molecule [16] and have intrinsic fluorescence. The two primary binding sites (Sudlow’s site I and site II) are hydrophobic cavities located in subdomains IIA and IIIA, respectively. BSA has 76% structural homology with human serum albumin (HSA) and hence it is being used as a model protein in clinical medicine [17, 18]. The pharmacokinetics parameters of distribution, transportation and excretion of drug depend on the noncovalent binding interactions of drugs with proteins. Exploration of the interaction mechanism between the drug with BSA is of great interest. There are few reports in literature on the binding studies of SDC with HSA by various

chromatographic methods [19, 20]. Zhang et. al. [5] analyzed interaction of SDC with HSA using spectroscopy and molecular modeling techniques. Russeva et. al [3] showed site specific competition study of SDC with site marker for the binding site of HSA. Interaction of BSA with a number of drugs including SDC has been shown by flow injection method and ligand displacement reaction [21, 22]. Effect of urea and guanidine hydrochloride on

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conformation of HSA and BSA and the binding ability of this protein with SDC have been investigated by Równicka-Zubik et. al. which showed that the albumin protein preserved their binding capacity in presence of denaturing agent [1]. But to the best of our knowledge no study is reported in the literature on the detailed and systematic calorimetric, spectroscopic and molecular modeling of SDC with BSA, the model transport protein. The binding studies of CPZ with phospholipid monolayer, lipid bilayers, hemoglobin and human serum albumin by various spectroscopy and equilibrium dialysis method have been reported in the literature [1, 23, 24]. The interaction of CPZ with BSA has been shown by Kitamura et. al [25] through second-derivative spectrophotometric method and by

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Rukhadze et. al [26] by means of active and passive experiments. Silva et al. [12]

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investigated the CPZ interaction with HSA and BSA by fluorescence quenching technique but the thermodynamic analysis, conformational alteration and computational studies of

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BSA-CPZ system have not been investigated so far.

Therefore, it is significant to investigate the complete binding profile of SDC and

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CPZ with BSA to determine the comprehensive energetics and conformational aspects of BSA-SDC and BSA-CPZ systems. In this study, we explored the binding affinity and mode

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of binding between BSA and SDC/ CPZ by UV-Visible and steady state fluorescence method. A comprehensive thermodynamic characterization of BSA-SDC and BSA-CPZ

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complexes has been subsequently obtained by means of isothermal titration calorimetry (ITC). An attempt has also undertaken to unravel the binding site of SDC/ CPZ on BSA by

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site specific displacement studies using standard site probes warfarin and ibuprofen for site I and site II, respectively as well as by molecular docking. Förster’s resonance energy transfer (FRET) theory has been applied to find out the molecular distance (r) between BSA and SDC/ CPZ. The conformational alterations of BSA in presence of SDC/ CPZ have been investigated by synchronous fluorescence, 3D fluorescence and far-UV circular dichroism (CD) techniques. Furthermore, molecular docking was performed to recognize the amino acid residues at binding site BSA for the interaction of SDC and CPZ with BSA. The outcome of this study is expected to provide an insight into the better understanding of binding phenomenon of SDC and CPZ to BSA.

2. Materials and methods 2.1. Materials Bovine serum albumin, sulindac, chlorpromazine, warfarin and ibuprofen were purchased from Sigma Aldrich. All other reagents used were of analytical grade. The 20 mM

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sodium phosphate buffer was prepared by dissolving the disodium hydrogen orthophosphate dihydrate and sodium dihydrogen orthophosphate dihydrate in double distilled water, in 3:1 ratio to maintain pH of the solution at 7.4 [27]. The detailed information of the chemical components used in the present work are given in Table 1. 2.2. Preparation of samples Stock solutions of BSA, SDC, CPZ, warfarin, and ibuprofen were prepared in 20 mM sodium phosphate buffer of pH 7.4. The concentration of BSA was determined 1% spectrophotometrically by using E280nm= 6.6 at 280 nm on Perkin-Elmer (λ-850)

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spectrophotometer [28]. The stock solutions were further diluted with phosphate buffer to

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make working solutions of required concentration. 2.3. Isothermal titration calorimetric (ITC)

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ITC is a well established excellent tool for quickly and directly elucidating the subtle thermodynamic profile of the interaction involved. It is the only technique that allows

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simultaneous determination of all the thermodynamic parameters in a single experiment

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accurately. In order to get information about intermolecular interaction mechanism of SDC and CPZ with BSA, ITC200 microcalorimeter (Micro Cal Inc., Northampton, MA) has been

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used at T = 298.15 K. The sample cell was loaded with BSA whereas the reference cell was filled with 20 mM sodium phosphate buffer solution of pH 7.4. The BSA (30 µM) was titrated with SDC (1 mM) and with CPZ (1 mM) individually by using a rotating stirrer

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syringe keeping stirring speed fixed at 500 rpm. Each experiment consisted of 19 consecutive injections each of which had duration of 4 seconds, and the time interval between the consecutive injections was kept at 120 s [29]. The reference power was set at 0.025 mW. The total heat content, Q of the solution contained in the active cell volume, Vo (determined relative to zero for the non-ligated species) at fractional saturation, Θ is given by [30], Q = n Θ Mt (ΔH) Vo

(1)

where ΔH is the molar enthalpy change of ligand binding, Mt is the total concentration of the protein and n is the number of binding sites in the protein. The heat released/ absorbed from the ith injection, ΔQ(i) for an injection volume, ΔVi is then given by the following equation [31], ΔQ(i) = Q(i) +

dVi

Q(i)+ Q(i−1) [ ] – Q(i-1) V0 2

(2)

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Since BSA has a capacity to offer multiple binding sites to a ligand, we have tried fitting the experimental data to different binding site models using Origin 7.0 software provided with the instrument. The reduced chi-square value and /or least error associated with the binding parameters were used to establish the best fit. Based upon the above fact, single site binding model was adopted to fit the experimental ITC data. In order to correct the data from dilution effects, the control experiments were performed where SDC/ CPZ were titrated into the buffer solution keeping concentration of the drugs same and subtracted from the experimental data.

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2.4. UV-visible measurements UV-visible absorption measurements were performed on Perkin-Elmer Lamda-850

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spectrophotometer attached with Peltier temperature programmer-1 (PTP-1) at T = 298.15 K. A fixed concentration of BSA (2 µM) was titrated in a 3 mL cuvette of 1 cm path length with

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varying concentration of SDC and CPZ (0-22 µM each) over the wavelength range of 230-

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330 nm. The buffer solution (pH = 7.4) was used as reference solution for all titrations. The UV-Vis spectra of SDC/ CPZ-buffer solution containing the same concentrations of SDC/

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CPZ as in the sample was taken into consideration to correct background for each titration. 2.5. Fluorescence quenching study

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Fluorescence quenching of native BSA (2 µM) solution in the presence of varying concentration of SDC/ CPZ was measured by monitoring fluorescence spectra in the

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wavelength range of 300 nm to 450 nm after exciting the Trp residue at 295 nm. All fluorescence measurements were recorded on F-2700 fluorescence spectrophotometer (Hitachi) equipped with xenon lamp and 1.0 cm quartz cell. The excitation and emission slit widths both were fixed at 5 nm.

2.6. Synchronous fluorescence Synchronous fluorescence spectra were recorded to observe the molecular microenvironment changes near tryptophan (Trp) and tyrosine (Tyr) residues in BSA in presence of SDC/ CPZ. The constant differences in wavelength, Δλ = 60 nm and 20 nm were maintained between excitation and emission fluorescence monochromators for monitoring

tryptophan and tyrosine microenvironment of BSA, respectively [32]. The concentration of BSA was kept as 2 μM and the concentration of SDC was varied from 0 µM to 10 µM whereas CPZ varied from 0 µM to 30 µM.

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2.7. Three dimensional fluorescence The three dimensional fluorescence spectroscopy has been demonstrated to be a specific method to comprehensively exhibit the fluorescence information of the chromophore and to investigate the characteristic conformational changes of protein [33]. Three dimensional fluorescence spectra of BSA (2 µM) in absence and presence of SDC and CPZ in the molar ratio of 1:0 and 1:5 in each case were acquired by recording the emission spectra in the wavelength range of 220-550 nm with the excitation wavelength in the range of 220350 nm with an increment of 10 nm.

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2.8. Displacement studies To identify the binding site of SDC and CPZ on BSA, site marker competitive

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experiments were performed by using two site probes i.e. warfarin and ibuprofen for site I and site II, respectively [34]. Titration of SDC/ CPZ was carried out by keeping the

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concentration of BSA and the probe in the molar ratio 1:1 (2 μM each) and varying the

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concentration of SDC from 0 μM to 10 μM and CPZ from 0 μM to 20 μM. The fluorescence emission spectra were recorded in a similar way as mentioned in steady state fluorescence

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measurement and the Ksv values of SDC/ CPZ-BSA-probe system were evaluated using Stern-Volmer equation.

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2.9. Fluorescence resonance energy transfer (FRET) Fluorescence resonance energy transfer (FRET) is a non-destructive spectroscopic

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method to monitor the proximity and relative angular orientation of fluorophore. The absorption spectrum of SDC/ CPZ and the emission spectrum of BSA at λex = 295nm (specific for tryptophan excitation) were recorded in the wavelength range of 300 nm to 450 nm to observe whether the donor and acceptor fluorophores are entirely separated or attached to each other.

2.10. CD spectroscopy The far-UV CD spectra of BSA (2 µM) in the absence and presence of SDC/ CPZ in the molar ratio of 1:4 were obtained by using Jasco-815 spectrometer attached with a Peltier temperature

programmer-1

(PTP-1).

The

instrument

was

calibrated

with

d-10-

camphorsulfonic acid. All the CD experiments were run at constant temperature, 298.15 K. The scan speed and response time were as 100 nm·min-1 and 1s, respectively for all measurements. Each spectrum was the average of 2 scans. The spectra were recorded as CD ellipticity in mdeg after baseline subtraction for buffer solution.

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2.11. Molecular docking study The three dimensional structure of BSA (PDB ID: 3V03) was downloaded from the RCSB protein data bank (http://www.rcsb.org/pdb) [35]. The structures of SDC and CPZ were drawn by using Chem Draw 12.0 followed by energy minimization of MM-2 method implemented in Chem. 3D Pro 12.0 and the file was saved in pdb format. Auto Dock vina program was chosen for performing the docking study. To avoid hindrance during docking, all water molecules were removed and polar hydrogen atoms were added. The Lamarckian genetic algorithm (LGA) was applied for minimization using default parameter and grid parameters were set as 96 × 66 × 80 Å points with grid centre x = 8.633, y = 20.892, z =

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101.09 for both BSA-SDC and BSA-CPZ systems to cover all the active site residue.

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Kollman charges were merged to the protein and gasteiger charges were added to the ligand using Auto-Dock Tools (ADT) and then prepared file was saved in PDBQT format [36].

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During the docking, the BSA structure was fixed at the initial input and set to be rigid while all torsional bonds of the SDC/ CPZ were set to be flexible. A total of 10 runs were

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performed and the output files of AutoDock with the lowest binding energy were used for further analysis. The docked pose for the complex was visualized in 2D form by Lig plot

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program [37].

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2.12. Accessible Surface Area Calculations

Differences between the accessible surface areas (ASAs) of BSA in native and in

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complexed with SDC/ CPZ were calculated using online server from Center for Informational Biology, Ochanomizu University (http://cib.cf.ocha.ac.jp/bitool/ASA)[38]. The structures corresponding to the best docked structure (conformation with minimum energy) were chosen in each case. The change in accessible surface area for residue, i is calculated using the following equation [39],

∆ASAi = ASAiBSA - ASAiBSA−SDC/CPZ

(3)

The amino acids losing >10 Å2 of ASA after interaction with drug are considered to be involved actively in the binding. 3. Results and discussion 3.1. Mode of binding and thermodynamics by ITC ITC is a simple and straightforward method to measure accurate and detailed thermodynamic parameters of molecular interaction. The representative ITC profile for

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titration of SDC/ CPZ with BSA solution at T = 298.15 K and at pH 7.4 is shown in Fig. 2(a) and 2(b). The upper panel represents the raw data for sequential injections of the SDC/ CPZ into the BSA solution and the lower panel shows negative heat deflection in the plot of the amount of heat liberated per injection as a function of the [SDC/CPZ]/ [BSA] molar ratio. The association constant (Ka), standard enthalpy change (ΔH°) and standard entropy change (ΔS°) have been directly obtained after the nonlinear least-squares fitting for the integrated heats by using single site binding model after subtraction of heat of dilution [40]. The experimental data points are best fitted with this model as shown by smooth solid line. Fitting of the data to alternate models yielded poor results suggested that single site binding model is

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best suited for BSA-SDC and BSA-CPZ interaction. The change in standard Gibbs free

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energy (ΔG°) can be calculated using the following equation [41], and the values are summarized in Table 2.

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ΔG° = ΔH°- TΔS°

(4)

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These thermodynamic parameters not only give the energies associated with the reaction, but also confirming binding modes. The negative standard enthalpy change (ΔH°)

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suggests that the binding occurs through exothermic process. According to the thermodynamic model proposed by Ross et al. [42], the various factors like (1) disruption of

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hydrogen-bonded water networks from the binding pocket of BSA and/ or from around SDC/ CPZ, (2) the existence of electrostatic, hydrogen bonding and van der Waals interactions

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among the binding components, lead to the evolvement of heat during the binding process. The ΔH° and ΔS° both values are negative reflects hydrogen bond formation and/ or van der Waals forces in stabilizing the BSA-SDC complex. On the other hand small negative value of ΔH° and positive ΔS° are responsible for electrostatic and hydrophobic interaction for BSACPZ complex formation.

3.2. Effect of SDC/ CPZ on absorption spectra of BSA UV-Vis absorption spectroscopy is a simple and effective method employed to know the complex formation between protein and small molecule like drug. Figs. 3(a) and 3(b) show absorption spectra of BSA in the presence of SDC and CPZ, respectively after subtracting the effect of drug solution. It is evident from the graphs that the peak near 278 nm appearing for BSA is due to the aromatic amino acids (Trp, Tyr, and Phe) [43]. SDC shows small absorption near 278 nm whereas the effect of CPZ near peak maxima is negligible peak of CPZ although a strong peak can be seen at around 250 nm which is the characteristic peak

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of CPZ and do not have any effect on BSA absorption peak maximum as shown in S1 (Supplementary material). On gradual addition of SDC/ CPZ to BSA solution, the intensity of the peak at 278 nm increases indicated that BSA-SDC and BSA-CPZ complexes were formed. The possible quenching mechanism (either dynamic or static) can be distinguished by UV-vis spectroscopy. In dynamic quenching, only the excited state fluorophore is affected by the quencher and so the absorption spectra do not exhibit any change whereas in the case of static quenching, complex formation takes place between ground state fluorophore and quencher [44]. Since the absorption spectrum peak of BSA gradually increases after the addition of SDC as well as CPZ, the quenching mechanism for both the systems are identified

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to be static.

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3.3. Effect of SDC/ CPZ binding on the fluorescence spectra of BSA Fluorescence spectroscopy is a sensitive tool employed to study the interaction of

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small molecules (drug) with biological macromolecules such as protein and also to determine

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the protein conformation and structural changes on drug binding. BSA contains three intrinsic fluorophores such as tryptophan, tyrosine, and phenylalanine. Among them tryptophan

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contributes significantly towards the intrinsic fluorescence quenching [45]. Fluorescence emission spectra of BSA (2 µM) as a result of excitation of Trp fluorophore at 295 nm in the

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presence of SDC/ CPZ (0-12 µM) have been shown in Fig. 4(a) and 4(b), respectively. Appropriate blanks corresponding to the buffer were subtracted to correct the fluorescence

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background. As SDC and CPZ have very low absorbance near excitation and emission wavelength, the inner-filter effect can be considered negligible. It is clearly seen that the fluorescence intensity peak of BSA decreases progressively with increasing concentrations of SDC/ CPZ confirming SDC as well as CPZ quenches the fluorescence of BSA as a result of

complex formation between them. The λmax value of BSA is slight blue shifted with increasing concentration of CPZ which indicated that the microenvironment near Trp fluorophore of BSA became relatively more hydrophobic upon CPZ binding. 3.3.1. Mechanism of quenching Fluorescence quenching may occur due to molecular interactions like ground state complex formation, the excited state interaction, energy transfer, collision [46]. The data obtained from fluorescence measurement of BSA-SDC and BSA-CPZ systems were analyzed by using the Stern-Volmer equation [47],

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= 1 +K sv [Q] = 1+ kq·τo

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(5)

where Fo and F are the steady state fluorescence intensities in the absence and presence of quencher i.e. SDC/ CPZ. Ksv and [Q] are the Stern-Volmer quenching constant and the concentration of quencher, respectively. The kq is the bimolecular quenching rate constant of the quenching reaction and τo is the average lifetime of the fluorophore, Trp (5.7 × 10−9 s) [48]. The data were plotted according to the equation 5 in Fig. 5(a) and 5(b) for BSA-SDC and BSA-CPZ, respectively. It can be seen from Table 3 that the Ksv values were of the order of 105 M-1 indicating strong quenching occurs between BSA and SDC/ CPZ. The kq value

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was found to be in the order of 1013 which is greater than the maximum dynamic quenching

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constant value (2 × 1010 L· mol-1· s−1) [49] revealing that fluorescence quenching of BSA in presence of SDC/ CPZ arises not due to dynamic diffusion but resulted from complex

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formation.

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3.3.2. Binding constant and number of binding sites

For the binding process, the number of binding sites, (n) and binding constant, (Kb)

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have been estimated from the slope and intercept of the following modified Stern-Volmer equation, respectively [50],

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F log[ Fo − 1]= log Kb + n log[Q]

(6)

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The standard free energy change of SDC/ CPZ complexation with BSA can be obtained by using the following equation [51], ΔG° = - RT ln(Kb)

(7)

The plot of log[(Fo/F) - 1] versus log[Q] gives a straight line as shown in Fig. 6(a) and 6(b) for BSA-SDC and BSA-CPZ, respectively. The slope equals to binding stoichiometry which were found to be 1.01 and 0.903 indicates that there was one independent class of binding site on BSA for SDC and CPZ molecules. The intercept on y-axis related to binding constant are listed in Table 3. The magnitude of binding constants are 2.21× 10 5 L·mol-1 and 1.40× 104 L·mol-1, respectively for SDC and CPZ binding with BSA. The slight variation in the magnitude of binding constant values obtained from ITC and fluorescence experiments may be due to the fact that the calorimetric analysis measures a global change in property of the system whereas fluorescence analysis measures local changes around the fluorophore

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(Trp-214) that it may not give information about the protein regions which were far away from tryptophan residues. The negative value of ΔG° for both the cases suggesting that binding processes are spontaneous in nature. 3.4. Conformational investigation by synchronous fluorescence Synchronous fluorescence provides information about microenvironment perturbation around the fluorophores (Tyr and Trp) present in protein upon binding with drug. Any shift in the maximum wavelength describes changes in polarity around the fluorophore [52, 53]. Figs 7 (a) and 7(b) show the synchronous fluorescence spectra of BSA-SDC system when Δλ = 60 nm and Δλ = 20 nm, respectively whereas Fig. 7(c) and 7(d) are for BSA-CPZ system at Δλ =

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60 nm and Δλ = 20 nm, respectively. The fluorescence intensity decreases with increasing

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concentration of SDC as well as with CPZ for both Δλ = 60 nm and Δλ = 20 nm with no

BSA significantly.

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3.5. Three dimensional fluorescence study

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shifting in the wavelength maxima indicating that SDC and CPZ altered the conformation of

The conformational alteration of protein can be investigated by three-dimensional

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fluorescence spectra. Figs 8(a), 8(b) and 8(c) are the 3D graphs with their related contour diagrams for BSA in absence and presence of SDC and CPZ, respectively.

The

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corresponding parameters for both studied systems are listed in Table 4. As it can be seen from Fig. 8 that Peak ‘a’ and peak ‘b’ denote the Raleigh scattering peak (λ ex = λem) and

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second order scattering peak (λem = 2λex), respectively [54]. The very strong peak 1 (λex = 280 nm, λem = 330 nm) mainly reflects the spectral behaviors of tryptophan and tyrosine residues as the maximum emission wavelength and the fluorescence intensity of the residues showed close relation to the polarity of their microenvironment. Apart from peak 1, there is another strong peak, peak 2 (λex= 230.0 nm, λem = 330 nm) that mainly displays the fluorescence spectral behavior of polypeptide backbone structure [55]. It is clear from the fig. 8(d) that the fluorescence intensities of both peak 1 and peak 2 decrease markedly by the addition of SDC as well as CPZ and the reduction is more significant in case of SDC than CPZ indicating the binding of SDC and CPZ to BSA induces conformational changes in protein. 3.6. Characterization of binding site of SDC and CPZ on BSA In order to identify the binding site of SDC and CPZ on BSA, site marker competitive experiments were performed through a basic approach of competition between SDC/ CPZ and site specific marker viz. warfarin and ibuprofen for the binding site of BSA. Site I is a large,

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flexible, multi-chamber cavity within the core of subdomain IIA (residues: 196-383) that comprises all six helices of the sub-domain. Site II in subdomain IIIA (residues: 384-583) composed of six-helices of the corresponding subdomains and exposed to solvent in contrast to site I [56]. Warfarin (3-(a-acetonylbenzyl)-4-hydroxycoumarin), bulky heterocyclic anticoagulant drug and ibuprofen (2-[4-(2-methylpropyl) phenyl] propanoic acid, a nonsteroidal anti-inflammatory agent, have been considered as stereotypical ligands for Sudlow’s sites I and II, respectively [57, 58]. The Stern-Volmer plots for BSA-SDC and BSA-CPZ systems in the absence and presence of site markers are shown in Figs 9(a) and 9(b), respectively and obtained values are listed in Table 5. We observed that Ksv values of

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both systems decreased in presence of warfarin indicating the competition between the SDC/

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CPZ and warfarin for the same binding site i.e. for Site I while the Ksv of SDC/ CPZ on

binding with BSA for site II has not been altered much in presence of ibuprofen. This

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confirmed that SDC/ CPZ binds near site I located in sub-domain IIA of BSA.

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3.7. Energy transfer between BSA and SDC/ CPZ by FRET FRET technique is widely used to determine the proximity of drug and spatial

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distance between a donor and an acceptor during protein-drug interaction. Energy transfer process mainly depends on the magnitude of overlap of emission spectrum of the donor with

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absorption spectrum of acceptor. The efficiency of energy transfer (EFRET) has been calculated by the Förster’s theory [59], F

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EFRET = 1-

Fo

=

R6o

R6o + r6

(8)

Where, Fo and F are the fluorescence intensities of BSA in the absence and presence of SDC/ CPZ, respectively; r is the distance between the donor (BSA) and the acceptor (SDC/ CPZ) molecule and Ro is the critical distance at which the efficiency of energy transfer becomes equals to 50 % and it is determined by the following equation [60],

R6o = 8.79 × 10−25 K 2 n−4 φ J

(9)

Where, K2 and n are the spatial orientation factor of the dipoles and the refractive index of the medium, respectively. The φ represents the fluorescence quantum yield of the BSA in the absence of acceptor and J is the overlap integral between the BSA emission spectrum and the absorption spectrum of SDC/ CPZ. The J value is calculated by the following equation [61]

Journal Pre-proof ∞ F(λ)ε(λ) λ4 dλ J= ∫ 0 F(λ)dλ

14

(10)

Where, F(λ) is the fluorescence intensity of BSA at wavelength λ and ε(λ) is the molar extinction coefficient of SDC/ CPZ at wavelength λ in M-1·cm-1. The emission spectrum of BSA is well overlapped with absorption spectrum of SDC/ CPZ as shown in Fig.10 which indicates that there is a possibility of energy transfer from BSA to SDC/ CPZ. The values of J, EFRET, Ro and r, evaluated according to equations (8-10) using n = 1.336 and φ = 0.118 [62] are listed in Table S1 (Supplementary Table). The values of Ro and r were found to be 2.79

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nm, 3.36 nm and 2.33 nm, 2.77 nm for the BSA-SDC and BSA-CPZ systems, respectively which satisfy the requirement that the average distance between donor and acceptor should be

ro

in the range of 2-7 nm [63]. The reliability of Ro and r values is evident from the fulfilling the

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condition of 0.5 Ro < r < 1.5 Ro [64] indicating that the energy was transferred from BSA to SDC/ CPZ in a non-radiative manner with high probability.

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3.8. Effect of SDC/ CPZ on CD spectra of BSA

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CD spectroscopy is a sensitive technique to gain an insight into the conformational alteration in protein as a result of drug binding. The CD spectra of BSA in the absence and presence of SDC/ CPZ exhibit two negative bands centered at 208 nm (π-π* transition) and

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222 nm (n-π* transition) which are the characteristics of alpha helix rich protein, BSA [65]. Upon binding of ligand, intermolecular forces responsible for sustaining the secondary and

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tertiary structure may get reorganized resulting in conformational alteration in the protein. Fig. 11 shows the far-UV CD spectra of BSA in absence and presence of SDC and CPZ, respectively. The observed ellipticity is converted to mean residue ellipticity (MRE) by using

the following equation [66],

MRE =

Θobs (mdeg)

(11)

10×n×C×l

Where, Θobs is observed ellipticity (mdeg) and n is the total number of amino acid residues in BSA (583). C is the molar concentration of BSA and l is the path length (in cm). The αhelical content of BSA-SDC and BSA-CPZ systems were calculated from the following expression [67], % α⎼helix = (

MRE222nm −2340 30300

) × 100

(12)

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15

The % α-helix of native BSA is 67.22 %. The value increases to 74.62 % in the presence of SDC (4 µM) and decreases to 63.80 % in presence of CPZ (4 µM), illustrating that the secondary structure of native BSA get stabilized as a result of SDC binding and CPZ unfolds the native conformation of BSA. However, the shape of peaks and the position of peak maximum do not alter and it suggested that BSA retains its identity (i.e. remains predominantly α-helix in nature) even after binding with SDC/ CPZ molecule [68]. 3.9. Molecular docking studies of BSA-SDC and BSA-CPZ interaction The experimental observations were further validated by molecular docking study to

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substantiate the preferred binding site on the protein. The heart shaped BSA has three domains as domain I ( 1–195), domain II (196–383) and domain III (384–583) with further

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subdivisions into A and B. The subdomain IIA (Sudlow’s site I) and IIIA (Sudlow’s site II) are the principal regions of drug binding with BSA [56]. In the present study, the different

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possible conformations of SDC and CPZ that binds with BSA have been determined using

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Auto dock Vina program. It resulted in nine conformations with varying Gibbs free energy values and we selected the best fit docked conformation with lowest binding energy. As we

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can observe from Fig. 12 that SDC binds with the sub-domain IIA of BSA which is also confirmed by site marker experiment. Inside the interaction cavity, two hydrogen bonds were

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formed between SDC and Ser191, Ala290 amino acid residues of BSA which has been substantiated by ITC results. It forms hydrophobic interaction with Tyr149, Glu152, Arg194,

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Arg198, Trp213 Arg217, Leu237, His241, Arg256, Leu259, Ile263, Ser286, and Ile289 as given in Table 6. On the other hand, it is revealed from Fig. 13 that BSA-CPZ complex is stabilized at the site I of BSA by hydrophobic interaction with Tyr149, Glu152, Ser191, Arg198, Trp213 Arg217, Lys221, Arg256, Leu259, Ile289 and Ala290 residues is concurrent with those obtained from fluorescence and ITC techniques.

3.10. Change in the SASA of BSA after complexed with SDC/ CPZ The accessible surface areas of the residues were calculated for BSA and BSA-SDC/ CPZ complexes. Comparisons of the ASA changes caused by binding provide insights of the goodness of packing of amino acid residues in a protein structure and their importance with respect to drug binding. The native BSA had a total ASA of 27,716.956 Å2, which was reduced to 27,435.411 Å2 and 27,433.191 Å2 after binding with SDC and CPZ, respectively. This large reduction in accessible surface area provides significance about the effectiveness of SDC/ CPZ binding to BSA. Residues that lose more than 10 Å2 of accessible surface area

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16

upon switching from the uncomplexed to complexed state are considered to be actively involved in the interaction [69]. Table 7 shows amino acid residues which lost ΔASA values more than 10 Å2 after complex formation with SDC and CPZ. For example, Ala290 in BSASDC complex showed an ASA reduction from 59.333 to 1.021 Å2 due to hydrogen bond formation, and among other interacting residues, Arg residues both in BSA-SDC and BSACPZ complexes also showed large reductions in ASA. It is seen from Table 7 that most of residues whose ASA change more than 10 Å2 are belong to subdomain IIA of BSA and few are from subdomain IB. This finding confirmed that SDC and CPZ bind effectively and

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tightly to site IIA of BSA. 4. Conclusion

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We have examined in-vitro interaction of BSA with SDC and CPZ at physiological condition (pH=7.4) using spectroscopic, calorimetric and computational methods.

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Fluorescence results illustrated that SDC/ CPZ quenches the fluorescence intensity of BSA in

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a static manner which is also substantiated by UV-visible findings. Thermodynamic parameters (ΔH° and ΔS°) obtained from ITC measurements suggested that SDC binding

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occurs mainly through hydrogen bonding and hydrophobic interactions whereas electrostatic interactions and hydrophobic interactions play significant role in stabilizing BSA-CPZ

na

complex. The binding processes are exothermic in nature as ΔH° values are negative in both cases. Competitive binding study of BSA in presence of SDC/ CPZ by using warfarin and

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ibuprofen as site probes revealed that both SDC as well as CPZ bind at Sudlow’s site I (in sub-domain IIA) of BSA. The synchronous and 3D fluorescence spectroscopy shows microenvironmental changes near Trp and Tyr residues. The molecular distance, r calculated from FRET study indicates that donor (BSA) and acceptor (SDC/ CPZ) are in close proximity to each other. Analysis of secondary structure by far-UV CD technique clearly indicated that SDC has stabilized the secondary structure of BSA whereas unfolding occurs in presence of CPZ. In addition, the molecular docking results showed that SDC forms two hydrogen bonds one with each, Ser191 and Ala290 and hydrophobic interaction occur with other amino acids whereas CPZ molecule enters the hydrophobic cleft of subdomain IIA (Sudlow’s site I) near Trp213 which substantiated our ITC and site marker experiment results. This study not only provides important insights into the binding of SDC and CPZ with BSA but also it will provide a useful direction in the pharmacology and clinical medicine fields. Acknowledgement

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The authors are thankful to Chairman, Department of Chemistry, A.M.U, Aligarh for providing the necessary facilities to carry out this research work. The PURSE and FIST grants from DST and SAP (DRS-II) from UGC are acknowledged. One of the authors (S.K.) gratefully acknowledges to UGC, New Delhi for awarding Maulana Azad National Fellowship (MANF). References

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Chemicals

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Table 1 Compounds used in this study with their sources and purity. Provenance

Mass fraction puritya

Bovine serum albumin

Sigma-Aldrich

0.98

Sulindac

Sigma-Aldrich

0.98

Chlorpromazine hydrochloride

Sigma-Aldrich

0.99

Warfarin

Sigma-Aldrich

0.98

Ibuprofen

Sigma-Aldrich

0.98

Di-sodium hydrogen orthophosphate

Merck

0.98

Merck

0.98

dehydrate (Na2HPO4·2H2O) Sodium dihydrogen orthophosphate dehydrate (NaH2PO4·2H2O) a

Purity as stated by the supplier.

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Table 2 Thermodynamic parameters and association constants for the binding of BSA with SDC and CPZ analyzed from ITC at pH= 7.4 (20 mM). System

Ka/ (L· mol-1)

ΔH°/ (kJ· mol−1)

TΔS°/ (kJ· mol−1)

ΔG°/ (kJ· mol−1)

BSA-SDC

1.25 × 105

-139.8

-110.7

-29.05

BSA-CPZ

1.40 × 104

-3.15

20.92

-24.06

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Table 3 Binding parameters of BSA-SDC and BSA-CPZ systems obtained from fluorescence experiment at T = 298.15 K, P = 101.13 kPa and pH= 7.4 (20 mM). BSA-SDC system

BSA-CPZ system

Stern-Volmer constant (Ksv)/ (L·mol-1)

1.92 × 105

1.576 × 105

3.37 × 1013

1.92 × 1013

re

Quenching constant (kq)/ (L·mol-1·s-1)

5.102 × 104

1.01

0.903

-30.63

-26.97

na

Gibbs free energy change (ΔGo)/ (kJ·mol-1)

2.21 × 105

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Binding constant (Kb)/ (L·mol-1) Stoichiometry (n)

-p

Parameter

Peak No.

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Table 4 Three-dimensional fluorescence spectral characteristics of BSA, BSA-SDC and BSA-CPZ systems. Peak position λex/λem (nm/nm)

Stokes Shift ∆λ (nm)

BSA (1:0)

BSA-SDC (1:5)

BSA-CPZ (1:5)

Fo

F

F/ Fo

F

F/ Fo

Peak 1

280/330

50

640.5

120.5

0.19

411.6

0.64

Peak 2

230/330

100

284.0

52.0

0.18

147.7

0.52

Table 5 Effect of site probes (warfarin and ibuprofen) on the interaction of SDC/ CPZ with BSA at T = 298.15 K, P = 101.13 kPa and pH= 7.4 (20 mM). System

Ksv w/o site

Ksv with

Ksv with

probes

warfarin

ibuprofen

R2

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BSA-SDC

1.922 × 105

8.55 × 104

1.737 × 105

0.99

BSA-CPZ

1.096 × 105

3.883 × 104

9.839 × 104

0.99

Table 6 Molecular docking results of BSA-SDC and BSA-CPZ systems using Autodock Vina software. Systems

Amino acid residues

Force involved

Ser191 , Ala290

Hydrogen bonding

Tyr149, Glu152, Arg194, Arg198,

Hydrophobic interaction

Trp213 Arg217, Leu237, His241,

ro

Arg256, Leu259, Ile263, Ser286,

Trp213 Arg217, Lys221, Arg256,

Hydrophobic interaction

-31.08

lP

Leu259, Ile289, Ala290.

re

BSA-CPZ

-p

Ile289. Tyr149, Glu152, Ser191, Arg198,

-36.12

of

BSA-SDC

Binding energy kJ·mol-1

System

BSA-SDC

Jo ur

na

Table 7 Changes in the ASA (Å2) values of the interacting residues of BSA before and after binding with SDC and CPZ. ASA (Å2) in complex 0.567

ΔASA (Å2)

Domain

Tyr149

ASA (Å2) in BSA 16.802

16.235

IB

Ser191

15.324

3.607

11.717

IB

Arg194

82.83

35.193

47.637

IB

Arg198

28.202

8.627

19.575

IIA

Trp213

40.404

29.723

10.681

IIA

Arg217

68.368

37.834

30.534

IIA

Arg256

14.707

2.497

12.21

IIA

Leu259

13.505

2.156

11.349

IIA

Ile289

15.362

1.97

13.392

IIA

Ala290

59.333

1.021

58.312

IIA

Tyr149

16.802

0.567

16.235

IB

Residues

Journal Pre-proof Glu152

12.502

0.308

12.194

IB

Ser191

15.324

3.859

11.465

IB

Arg198

28.202

8.213

19.989

IIA

Trp213

40.404

28.893

11.511

IIA

Arg217

68.368

41.509

26.859

IIA

Lys221

23.28

14.882

8.398

IIA

Arg256

14.707

2.497

12.21

IIA

Leu259

13.505

2.156

11.349

IIA

Ile289

15.362

0.00

15.362

IIA

of

BSA-CPZ

26

Jo ur

na

lP

re

-p

ro

(a)

Fig. 1. Chemical structure of (a) Sulindac, (b) Chlorpromazine.

(b)

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27

(b) Time/ (min)

Time (min) 0

10

20

0

30

0.00

10

20

30

0.00

µWatts

µWatts

-2.00 -4.00

-0.80

-6.00 -8.00

-1.20

0.0 -8.4 -16.7 -25.1 -33.5 -41.9 -50.2 -58.6 -67.0 -75.3 -83.7 -92.1 -100.5 -108.8

kJ mol of injectant

-3.00

-5.00

ro

-1

of

-4.00

-6.00

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0

0

1

2

3

4

5

6

Molar Ratio

lP

re

Molar Ratio

-p

-1

kJ mol of injectant

-0.40

na

Fig. 2. ITC isotherm for the interaction of BSA with (a) SDC and (b) CPZ. The upper panel shows calorimetric response as successive injections of SDC/ CPZ into the sample cell containing BSA. The bottom panel represents integrated heats of interaction as a function of [SDC/ CPZ] ⁄ [BSA] molar ratio.

Jo ur

(a)

Absorbance

0.8

(b) (b) 1.6

1.2

Absorbance

1.2

0.8

0.4 0.4

0.0

0.0 240

260

280

Wavelength/ nm

300

320

240

260

280

300

320

Wavelength/ nm

Fig. 3. UV-visible absorbance spectra of BSA in the absence and presence of (a) SDC and (b) CPZ at T = 298.15 K and pH 7.4.

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(b)

(a)

200

160 Fluorescence Intensity

Fluorescence Intensity

200

120

100

80

40

0

0 350

400

300

320

340

of

300

450

360

380

400

420

Wavelength/ nm

ro

Wavelength/ nm

re

-p

Fig. 4. Fluorescence quenching spectra of BSA in the presence of (a) SDC (0-12 µM) and (b) CPZ (0-12 µM) at T = 298.15 K and pH 7.4. (b)

3.0

Fo/F

na

2.4

0.0 0

2

Jo ur

1.2

4

6

[SDC]/ (mM)

8

Fo/F

lP

(a)

1.5

0.0 10

0

2

4

6

8

10

12

[CPZ]/ mM

Fig. 5. The Stern-Volmer plots for the quenching of tryptophan of BSA by (a) SDC (b) CPZ at T= 298.15 K and pH = 7.4.

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(b)

(a)

29

0.4

log[Fo/F-1]

log[Fo/F-1]

0.4

0.0

0.0

-0.4

-0.4 -0.8 -6.0

-5.6

-5.2

-4.8

-5.7

log[SDC]

-5.4

-5.1

of

log[CPZ]

ro

Fig. 6. The modified Stern-Volmer plots of (b) BSA-SDC and (d) BSA-CPZ systems for the determination of n and Kb. (b)

-p

(a) (a)

(a)

(a)

)

lP

600

na

400

200

0 280

300

Jo ur

Fluorescence Intensity

re

800

)

320

340

Wavelength/ (nm)

360

380

200 Fluorescence Intensity

(a)

100

0 320

340

360

Wavelength/ (nm)

380

Journal Pre-proof (c)

30

(d)

(a)

(a)

)

) 200

Fluorescence Intensity

400

100

200

0

0 280

300

320

340

360

380

of

Fluorescence Intensity

600

320

Wavelength/ (nm)

340

360

380

-p

ro

Wavelength/ (nm)

re

Fig. 7. Synchronous fluorescence spectra of BSA-SDC system at (a) ∆λ = 60 nm (b) ∆λ = 20 nm and for BSA-CPZ system at (c) ∆λ = 60 nm (d) ∆λ = 20 nm.

Jo ur

na

lP

(a)

1

(a)

)

(a)

a (a)

)

) 2 (a)

)

350 300 EX(nm) 250

b (a)

)

300 400 500 EM(nm)

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(b) (a)

) 100 350 300 EX(nm) 250

0 300 400 500

ro

(c)

of

EM(nm)

(a)

-p

)

400 300

lP

re

350 300 EX(nm) 250

Fluorescence Intensity

0 EM(nm)

(d) (a)

Peak 1 Peak 2

) 400

200

0 BSA

100 300 400 500

na Jo ur 600

200

BSA-SDC

BSA-CPZ

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Fig. 8. Three-dimensional fluorescence spectra of (a) BSA (b) BSA-SDC complex (c) BSACPZ complex along with their contour diagrams and (d) Bar diagram representing 3D fluorescence intensity of peak 1 and peak 2 of the systems. (a)

(b)

(a)

(a)

)

)

3

3

2

Fo/F

Fo/F

2

1

1

of

w/o probes warfarin ibuprofen

w/o probes warfarin ibuprofen

0

0 0

2

4

6

8

0

10

5

10

15

20

[CPZ]/ (mM)

ro

[SDC]/ (mM)

Jo ur

na

lP

re

-p

Fig. 9. Stern-Volmer plot for the fluorescence of (a) BSA-SDC and (b) BSA-CPZ system in the absence and presence of site probes (warfarin and ibuprofen).

Fig. 10. Overlapping of fluorescence spectrum of donor (a) BSA with absorption spectrum of acceptor, (b) SDC and (c) CPZ.

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0

CD/ mdeg

-10

-20

BSA BSA-SDC BSA-CPZ

-30

200

210

220

230

240

ro

of

Wavelength/ nm

-p

Fig. 11. CD spectra of BSA, BSA-SDC and BSA-CPZ complexes monitored by far-UV CD at T = 298.15 K and pH= 7.4.

(b)

re

(a) (a)

)

Jo ur

na

lP

)

(a)

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34

(c) (a)

-p

ro

of

)

)

na

(a)

Jo ur

(a)

lP

re

Fig. 12. (a) Molecular docked model of SDC binding with BSA with lowest energy conformation. (b) Molecular surface representation of SDC binding in the hydrophobic pocket of sub-domain IIA of BSA, (c) A 2D representation of BSA-SDC complex by Ligplot program. (b) (a)

)

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(c) (a)

-p

ro

of

)

Jo ur

na

lP

re

Fig. 13. (a) Molecular docked model of CPZ binding with BSA with lowest energy conformation, (b) Detailed view of binding of CPZ in the hydrophobic pocket of sub-domain IIA of BSA, (c) A 2D representation of BSA-CPZ complex by Ligplot program.

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36

Declaration of interests

The authors declare that they have no known competing financial interests or personal

Jo ur

na

lP

re

-p

ro

of

relationships that could have appeared to influence the work reported in this paper.

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37

`Highlights:

of ro -p re



lP



na



SDC and CPZ quench the fluorescence spectra of BSA in a static manner. SDC and CPZ bind to BSA through hydrogen bonding and hydrophobic interaction respectively. Binding site of SDC and CPZ on BSA is site I proved by site marker and docking methods. SDC stabilized the native structure of BSA whereas CPZ unfolds it revealed by CD spectroscopy. The secondary structure of BSA is altered by SDC and CPZ.

Jo ur

 