Biophysical and in silico investigations of the molecular association between a potent RNA polymerase inhibitor, thiolutin and human serum albumin

Journal Pre-proof Biophysical and in silico investigations of the molecular association between a potent RNA polymerase inhibitor, thiolutin and human serum albumin

Md. Zahirul Kabir, Zineddine Benbekhti, Nor Farrah Wahidah Ridzwan, Rabiâa Merrouche, Noureddine Bouras, Saharuddin B. Mohamad, Saad Tayyab PII:

S0167-7322(19)36352-4

DOI:

https://doi.org/10.1016/j.molliq.2020.112648

Reference:

MOLLIQ 112648

To appear in:

Journal of Molecular Liquids

Received date:

18 November 2019

Revised date:

25 January 2020

Accepted date:

5 February 2020

Please cite this article as: M.Z. Kabir, Z. Benbekhti, N.F.W. Ridzwan, et al., Biophysical and in silico investigations of the molecular association between a potent RNA polymerase inhibitor, thiolutin and human serum albumin, Journal of Molecular Liquids(2020), https://doi.org/10.1016/j.molliq.2020.112648

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© 2020 Published by Elsevier.

Journal Pre-proof

Biophysical and in silico investigations of the molecular association between a potent RNA polymerase inhibitor, thiolutin and human serum albumin Md. Zahirul Kabira, Zineddine Benbekhtib,c, Nor Farrah Wahidah Ridzwand,

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Rabiâa Merrouchee, Noureddine Bourasb,e, Saharuddin B. Mohamadd,f, Saad

Biomolecular Research Group, Biochemistry Programme, Institute of Biological Sciences,

-p

a

ro

Tayyaba,f*

b

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Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia, Département de Biologie, Faculté des Sciences de la Nature et de la Vie et Sciences de la Terre,

c

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Université de Ghardaïa, BP 455, Ghardaïa 47000, Algeria, Laboratoire Antibiotiques, Antifongiques: Physico-chimie, Synthèse et Activité Biologique,

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Département de Biologie, Faculté des Sciences de la Nature et de la Vie et Sciences de la Terre et de l’Univers, Université de Tlemcen, BP 119, Tlemcen 13000, Algeria, Bioinformatics Programme, Institute of Biological Sciences, Faculty of Science, University of

ur

d

Malaya, Kuala Lumpur, Malaysia,

Laboratoire de Biologie des Systèmes Microbiens (LBSM), Ecole Normale Supérieure de

Jo

e

Kouba, Alger, Algeria, f

Centre of Research for Computational Sciences and Informatics for Biology, Bioindustry,

Environment, Agriculture and Healthcare, University of Malaya, Kuala Lumpur, Malaysia,

*Corresponding author E-mail address: [email protected] (Saad Tayyab).

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Journal Pre-proof Abstract Biomolecular association of thiolutin (TLT), a potent RNA polymerase inhibitor with albumin from human serum (HSA), the main transporter in blood plasma was examined using various biophysical and in silico techniques. An inverse correlation between the Stern-Volmer constant, KSV and temperature predicted TLT-induced quenching as the static quenching, hence suggested

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TLT–HSA complex formation. This was also supported by UV-vis absorption spectral results. A weak binding affinity was registered towards the complex formation, as evident from the binding

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constant, Ka value. Thermodynamic data obtained at different temperatures as well as molecular

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docking analysis revealed participation of hydrophobic and van der Waals forces as well as

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hydrogen bonds in TLT–HSA complexation. Binding of TLT to HSA was found to alter the

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microenvironment around HSA fluorophores (Tyr and Trp), as manifested by three-dimensional fluorescence spectra. Presence of TLT in association with HSA offered protection to the protein

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against temperature-induced destabilization. Competitive site-marker displacement experiments

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using warfarin, phenylbutazone and hemin identified the preferred TLT binding site as Sudlow's site I in HSA, which was also validated by molecular docking analysis. Molecular dynamics

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assessments disclosed that the complex reached equilibrium during simulations, indicating the stability of the TLT–HSA complex.

Keywords: Thiolutin; Human serum albumin; Drug–protein interaction; Fluorescence quenching; Molecular dynamic simulations

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Journal Pre-proof 1. Introduction Thiolutin (TLT) is a naturally existing bicyclic dithiole antibiotic (inset of Fig. 1A), containing a unique pyrrolinonodithiole (4H-[1,2] dithiolo [4,3-b] pyrrol-5-one) skeleton [1]. It is generally known as a potent inhibitor of yeast RNA polymerases [2], as it can completely block both RNA and protein synthesis in yeast whole cells [3]. However, there is no report available in

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the literature on its inhibitory activity against bacterial RNA polymerases [3]. On the other hand, a previous study has shown that TLT inhibits endothelial cell adhesion and suppresses tumor-cell

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induced angiogenesis [4]. This finding might be an indicator for the use of TLT as a promising

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candidate in anti-tumor treatments in future [4].

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Interaction between a drug and the plasma protein can greatly influence the drug’s

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pharmacodynamic and pharmacokinetic properties in terms of distribution, clearance, elimination, efficiency and toxicity throughout the human system [5,6]. The reversible binding of

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a drug to the protein accelerates the effective transport of the drug to its target site through blood

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circulation [7]. Such drug-protein binding studies might be important for therapeutic agent’s

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development [5]. Serum albumin, α-1-acid glycoprotein and lysozyme are commonly used as the model transport proteins in various studies involving ligand binding to the protein [8–10]. Among these proteins, human serum albumin (HSA) is the most abundant extracellular protein in human blood circulation, which functions as the leading transporter for a large number of ligands including drugs. These ligands normally have binding preference to HSA at either of its three well-characterized ligand binding sites, viz., Sudlow’s site I, Sudlow’s site II and site III, which are positioned in subdomains IIA, IIIA, and IB, respectively. Existence of sole tryptophan (Trp214) residue in HSA [11] offers greater advantage in the drug binding studies, using fluorescence spectroscopy. 3

Journal Pre-proof Although several reports describing the antibiotic character as well as inhibition activities of TLT are available in the literature, the interaction of TLT with HSA is still remained to be explored. Therefore, we studied the TLT−HSA interaction, using multi-spectroscopic and computational methods. Various interaction characteristics of TLT binding to HSA such as binding mechanism, binding constant, binding forces involved and location of the drug binding

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site in HSA were attained from this data.

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2. Experimental

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2.1. Reagents

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Albumin from human serum (HSA; lyophilized powder, globulin and essential fatty acid free),

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warfarin (WFN), phenylbutazone (PBZ) and hemin (HMN) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Thiolutin (TLT) was purified according to the procedure described

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2.2. Purification of TLT

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below. Analytical standard samples of all other chemicals were used in this study.

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The bacterium Saccharothrix algeriensis NRRL B-24137 was isolated from a Saharan soil sample collected at the palm grove in Adrar, Algeria [12]. The actinobacterium was maintained on ISP2 (International Streptomyces Project 2) medium composed of malt extract (10 g/l), yeast extract (4 g/l) and glucose (4 g/l). The production of thiolutin (TLT) was conducted in the same medium. The cultures were incubated on a rotary shaker (250 rpm) for 10 days at 30 °C. The extraction of TLT was carried out after centrifugation (5000 g, 20 min) of the culture broth to eliminate biomass. The cell-free supernatant was extracted with the same volume of dichloromethane. The organic layer was dehydrated with Na2SO4 and concentrated to dryness by

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Journal Pre-proof a rotary evaporator under a vacuum at 40 °C. The extract was dissolved in 1 ml of methanol and subjected to TLC (Thin Layer Chromatography) by using solvent system: ethyl acetate-methanol (100-15 v/v). The final purification of TLT was performed by reverse phase HPLC (Agilent 1260) using a C18 column (Uptisphere, UP15WOD, 300 mm × 7.8 mm i.d. Interchim; 5 μm) (Supplementary Fig. 1) and the detection of TLT was carried out by UV at 220 and 390 nm. The purity of TLT (purity ≥ 99%) was established using NMR/mass spectra (Supplementary Figs. 2

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and 3) [13].

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2.3. Protein and drug solutions

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The protein (HSA) crystals were dissolved in 60 mM sodium phosphate buffer, pH 7.4 (PB

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7.4) to prepare HSA stock solution and its concentration was determined spectrophotometrically

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using m of 36 500 M–1 cm–1 at 280 nm [14]. Preparation of TLT, WFN, PBZ and HMN stock solutions involved dissolution of 1.0 mg ml–1 drug crystals in methanol. The PB 7.4 was used to

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dilute these stock solutions for preparing working solutions of the desired concentration. TLT

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and WFN concentrations were determined using m of 11 000 M–1 cm–1 at 388 nm [1] and 13

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600 M–1 cm–1 at 310 nm [15], respectively. 2.4. Absorption spectra

The absorption spectra (230–330 nm) of the protein (10 µM HSA) with increasing TLT concentrations (0–250 µM with 50 µM increments) were recorded spectrophotometrically on a Shimadzu UV-2450 double beam spectrophotometer using a pair of 1 cm path length quartz cuvettes containing the same concentrations of TLT as the reference.

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Journal Pre-proof In a separate experiment, the absorption spectra (295–400 nm) of HSA (3 µM) and its mixture with increasing TLT concentrations (10–80 µM with 10 µM increments) were also acquired. The inner filter effect correction in the fluorescence spectra was made using these spectra. 2.5. Fluorescence spectra A Jasco FP-6500 spectrofluorometer was used to collect the fluorescence spectra of the

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protein and TLT–protein mixtures by placing the sample in a quartz cuvette (path length = 1 cm) and positioning it in the thermostatically-controlled cell holder for 10 min for maintaining the

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sample temperature.

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The protein’s fluorescence spectra (310–400 nm) in the absence and with addition of TLT

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were registered using an excitation wavelength (λex) of 295 nm in the titration experiments.

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Different scanning parameters used for fluorescence measurements were kept the same, as described earlier [16].

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The fluorescence spectra of WFN–HSA and WFN–HSA–TLT mixtures were recorded using

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the λex of 335 nm, in the wavelength range of 360–480 nm. The three-dimensional (3-D) fluorescence spectra of 3 µM HSA and 5:1 [TLT]:[HSA]

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mixture were acquired, following the published procedure [16]. 2.6. TLT–HSA binding studies and fluorescence titration data analysis The titration was performed with addition of increasing TLT concentrations (10–80 μM with 10 μM intervals) to the protein (3 μM HSA) taken in different tubes. The solution mixtures (3 ml) were incubated separately at 288 K, 298 K and 308 K for 30 min for equilibrium establishment. The observed fluorescence data were subjected to the inner filter effect correction according to the following relationship [17]: 𝐹𝑐𝑜𝑟 = 𝐹𝑜𝑏𝑠 10(𝐴𝑒𝑥 +𝐴𝑒𝑚)/2

(1) 6

Journal Pre-proof where individual terms have their standard significance [17]. Values of the Stern-Volmer constant (KSV) and the bimolecular quenching rate constant (kq) of TLTHSA system were obtained after analyzing the corrected fluorescence data using the equation (2). 𝐹0 ⁄𝐹 = 1 + 𝐾𝑠𝑣 [𝑄] = 1 + 𝑘𝑞 𝜏0 [𝑄]

(2)

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where F0 and F are the values of the fluorescence intensity of HSA and TLT–HSA mixture,

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respectively; [Q] refers to the quencher (TLT) concentration. The fluorescence lifetime (τ0) value

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of HSA without quencher was taken as 5.6 × 10−9 s [18].

For the determination of the binding constant (Ka), fluorescence data were treated according

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to equation (3).

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log (𝐹0 − 𝐹)⁄𝐹 = 𝑛 log 𝐾𝑎 − 𝑛 log [1 ⁄([𝐿𝑇 ] − (𝐹0 − 𝐹) [𝑃𝑇 ]⁄𝐹0 )]

(3)

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where ?? is the Hill coefficient, while [????] and [????] refer to the total concentration of TLT and

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HSA, respectively [19].

Values of the enthalpy change (ΔH) and the entropy change (ΔS) for TLT–HSA association

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process were retrieved from the van’t Hoff plot (Eq. (4)), using the value of the gas constant (R) as 8.3145 J mol−1 K−1.

ln 𝐾𝑎 = −∆𝐻⁄𝑅𝑇 + ∆𝑆⁄𝑅

(4)

The Gibbs free energy change (∆𝐺) of the binding reaction was calculated by substituting the ∆𝐻 and ∆𝑆 values into the following equation: ∆𝐺 = ∆𝐻 − 𝑇∆𝑆

(5)

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Journal Pre-proof 2.7. Thermal stability Following the procedure reported elsewhere [20], the fluorescence intensity values at 342 nm (FI342 nm) of the protein (3 µM HSA) and 10:1 [TLT]:[HSA] mixture were measured at increasing temperatures (298–353 K with increments of 5 K) to monitor TLT-induced thermal stability of the protein.

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2.8. Site specific marker-induced effects on TLT binding to HSA Experiments involving competitive ligand binding were performed using different site-

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specific markers, viz., WFN and PBZ for site I and HMN for site III [5], following the similar

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procedure as briefed in Section 2.6. The protein (3 μM HSA) solution or site marker–protein

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([WFN] / [PBZ] / [HMN]:[HSA] = 1:1) mixtures were incubated for 30 min at 298 K before

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titration experiments.

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2.9. Molecular docking

The computational docking analyses were made using AutoDock 4.2 and AutoDockTools 4.2

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to predict the binding orientation of TLT on HSA structure. The crystal structure of the protein

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(PDB code: 1BM0; resolution: 2.5 Å) was collected from the Protein Data Bank, whereas Avogadro 1.1.1 software was used to construct the 3-D structure of TLT, followed by energy minimization in MMFF94s force field utilising the steepest descent algorithm [21]. Investigations of binding sites, i.e., I, II and III of HSA was made by conducting independent molecular docking simulations. Analytical tools and other parameters for docking analyses were maintained the same as described in our previously published report [22]. Interactions formed between TLT and HSA binding sites I and III were visualised and captured using UCSF Chimera [23], whereas their 2-D interaction maps were retrieved using LigPlot+ [24].

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Journal Pre-proof 2.10. Molecular dynamics The simulation of existing molecular forces within the formed complex structure between TLT and HSA on site I was conducted through molecular dynamics (MD) simulations via GROMACS 2018 [25]. The topology of TLT was obtained through an automated topology builder (ATB) and repository [26]. Preparation of the system includes hydrating the periodic boundary condition with TIP3P water model [27] and adding counter ions to establish a neutral

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system. The system then underwent steepest descent energy minimization using the GROMOS

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54A7 force field [28] with the maximum number of 50,000 steps. The long-range electrostatic

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interactions and the nearest-neighbor search was calculated by particle-mesh Ewald (PME)

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method and Verlet algorithm, respectively [29,30]. The system parameters were maintained at a constant temperature of 300 K by using V-rescale thermostat [31], whereas the pressure was set

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to 1 bar by using Parrinello-Rahman barostat [32]. The system went through equilibration runs

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for 1 ns and followed by the production run with the duration of 50 ns. The trajectory frames

further evaluation.

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generated from the MD simulation were captured at 10 ps intervals, which were later utilized for

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2.11. Statistical analysis

Each experiment was carried out at least three times. The obtained data were transformed into the mean ± standard deviation (SD) according to the mean value of three independent experiments using the following formula: 𝑁

1 𝜎 = √ ∑(𝑥𝑖 − 𝜇)2 , 𝑁 𝑖=1

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Journal Pre-proof where xi is an individual value,  is the mean value and N is the total number of values. Statistical data processing, curve fitting as well as smoothing were made by exploiting the OriginPro 8.5 software (OriginLab Corp., Northampton, USA).

3. Results and discussion

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3.1. Fluorescence titration results and binding constant of TLT–HSA system

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In order to assess the ligand-induced changes in the protein fluorescence, fluorescence spectra of the protein can be monitored in the presence of increasing ligand concentrations. The

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fluorescence spectra of HSA in the absence and with addition of increasing TLT concentrations

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at three different temperatures are depicted in Fig. 1. The protein exhibited an emission (λem)

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peak at 342 nm, which was solely contributed by its lone Trp-214 residue, resided in the pocket of Sudlow's site I (subdomain IIA) of HSA [11]. Addition of increasing TLT concentrations to

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HSA solution quenched the protein’s fluorescence progressively without any change in the λem

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peak (Fig. 1). It is noteworthy that TLT alone did not produce any fluorescence signal in this

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wavelength range (Fig. 1). About 40 % (Fig. 1A), 33 % (Fig. 1B) and 26 % (Fig. 1C) decline in the fluorescence intensity at 342 nm were noticed with the highest TLT concentration at 288 K, 298 K and 308 K, respectively. Reduction in the fluorescence intensity of HSA with addition of TLT clearly suggested the complexation between TLT and HSA, which can be attributed due to the microenvironmental changes around the Trp residue of the protein [33]. Two quenching mechanisms involved in the protein’s fluorescence quenching by its preferred quencher can be characterized either as static quenching and dynamic quenching. These quenching mechanisms can be differentiated based on their temperature dependence [17]. Increase in the temperature weakens the stability of the ligand–protein complex due to the 10

Journal Pre-proof dissociation of noncovalent bonds and hence reduces the KSV in static quenching. On the other hand, higher temperature leads to faster diffusion and thus larger amounts of collisions between the fluorophore and the quencher that increases the KSV in dynamic quenching [17]. Titration experiments were performed at three different temperatures (288 K, 298 K and 308 K) to speculate the mechanism involved in the TLT-induced fluorescence quenching of HSA. The Stern-Volmer plots (Fig. 2A) for TLT–HSA system were obtained after treating the fluorescence

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quenching data according to Eq. (2). The values of KSV, as identified from the slope of these plots

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are listed in Table 1. Decreasing trend in the KSV values (Table 1) with increasing temperature for

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TLT–HSA system clearly characterized TLT-induced quenching of HSA fluorescence as static

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quenching, thus confirming the TLT–HSA complex formation. Furthermore, the calculated kq values (1.50 × 1012 M–1 s–1 at 288 K, 1.14 × 1012 M–1 s–1 at 298 K and 0.86 × 1012 M–1 s–1 at

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308 K) for TLT–HSA system were identified much higher than the value (2 × 1010 M–1 s–1) for

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the maximum dynamic quenching rate constant, reported for diffusion-controlled process [34]. These findings further supported characterization of the static quenching mechanism and thus,

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suggested the TLT–HSA complex formation.

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To determine the binding constant (Ka) for TLT–HSA system, the fluorescence quenching data was treated with Eq. (3), which yielded the double logarithmic plots (Fig. 2B). Values of Ka at three different temperatures were retrieved from these plots and are listed in Table 1. The Ka values were found to fall in the range of 4.32–8.08 × 103 M–1, indicating a weak binding affinity between TLT and HSA. Several earlier reports have shown weak binding affinity for ligand– protein interactions [35–37]. Based on weak binding strength, transport of TLT in the bloodstream and its discharge at the target locus seems to be a feasible process. It is important to note that the drug’s binding affinity for the receptor is significantly higher compared to its 11

Journal Pre-proof affinity for plasma proteins [5]. Additionally, reduction in the Ka value for TLT–HSA system with rising temperature clearly suggested loss of stability of the complex at higher temperature [38,39]. 3.2. Thermodynamics of TLT–HSA system Thermodynamic parameters such as ΔS, ΔH and ΔG for the TLT–HSA binding process were

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analysed to characterize the intermolecular forces associated with the stabilization of the complex. Therefore, dependence of the binding constant (Ka) on temperature was studied using

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the van't Hoff equation (Eq. (4)). Values of ΔS and ΔH, as obtained from the van't Hoff plot

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(inset of Fig. 2B) and ΔG, using Eq. (5) are summarized in Table 1. Four common noncovalent

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forces, involved in ligand-protein complex formation include electrostatic interactions,

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hydrophobic interactions, van der Waals forces and hydrogen bonds. Different sign and magnitude of the values of ΔS and ΔH can predict the nature of the intermolecular forces,

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involved in the ligand–protein association [40]. Negative sign of ΔG values (Table 1) manifested

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that the binding process was feasible and spontaneous at all temperatures. A positive ΔS value (+5.35 J mol–1 K–1) for the TLT–HSA system projected contribution of both hydrophobic and

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electrostatic interactions. However, a larger magnitude of ΔH value (– 23.08 kJ mol–1) ruled out the participation of electrostatic interactions, as such interactions are usually accompanied by a ΔH value close to zero [40]. On the other hand, the negative ΔH value obtained for TLT–HSA system may predict the involvement of van der Waals interactions and hydrogen bonds [40]. Therefore, hydrophobic and van der Waals interactions as well as hydrogen bonds can be assumed as the leading intermolecular forces in stabilizing the TLT–HSA complex. 3.3. Absorption spectral analysis of TLT–HSA system UV–vis absorption spectral analysis was made to validate the TLT–HSA complex formation 12

Journal Pre-proof and support our intrinsic fluorescence results, described in Section 3.1. Ligand-induced alterations in the absorption spectrum of the protein can be seized as an indicator for the static quenching mechanism and ligand-protein complexation [17]. Changes in the absorption spectrum of the protein (HSA) with added TLT are illustrated in Fig. 3. Appearance of an absorption maximum at 278 nm in the protein’s absorption spectrum was due to the presence of Trp and Tyr residues in HSA [41]. Increase in the protein’s absorption peak upon TLT addition

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clearly indicated TLT-induced microenvironmental changes around the protein’s chromophores,

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which suggested complexation between TLT and HSA and supported characterisation of TLT-

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induced protein quenching as static quenching.

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3.4. Three-dimensional fluorescence spectral analysis of TLT–HSA system

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The 3-D fluorescence spectra of the protein with added TLT were assessed to check the probable influence of TLT on the microenvironment around protein fluorophores (Trp and Tyr).

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These spectra (free HSA and TLT–HSA mixture) along with their contour maps (top) are

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depicted in Fig. 4, while the related spectral properties, i. e., peak position and fluorescence intensity values are summarized in Table 2. Among the four peaks (Fig. 4A), two Rayleigh

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scattering peaks, i. e., the first-order scattering peak, ‘a’ (λex = λem) and the second-order scattering peak, ‘b’ (2λex = λem) are frequently observed in the protein’s 3-D fluorescence spectra [17,42]. The two other main peaks, peak ‘1’ (λex = 280 nm) and peak ‘2’ (λex = 230 nm) characterized the fluorescence spectral behaviour of the protein’s Trp and Tyr residues. Noticeable quenching in the fluorescence intensity values of peak ‘1’ (~21 %) and peak ‘2’ (~20 %) were detected with TLT addition to the protein solutions (Fig. 4B, Table 2). These results clearly indicated detectable changes in the microenvironment around Trp and Tyr residues of HSA due to the complexation between TLT and HSA. 13

Journal Pre-proof 3.5. Protein’s thermal stability analysis upon TLT binding The protein’s thermal stability was studied by comparing the temperature-induced differences in the fluorescence signal at 342 nm (FI342 nm) of HSA and TLT–HSA mixture. The variations in the FI342

nm

of the protein as well as TLT–protein mixture with respect to

temperature is illustrated in Fig. 5. Presence of TLT in the reaction mixture produced lesser decline in the fluorescence intensity values at higher temperatures in comparison with those

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noticed with HSA alone (Fig. 5). Quantitatively, the fluorescence intensity of HSA and TLT–

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HSA mixture exhibited ~ 70 % and ~ 58 % quenching in the FI342

nm,

respectively, at the

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maximum temperature (353 K). The observed smaller loss in the intensity value of TLT–HSA

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mixture clearly indicated enhanced thermal stability of the protein upon TLT binding [43].

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Higher temperature seems to be needed to break the intermolecular forces, existing in the TLT– HSA complex [44]. These results were in line with several earlier reports that suggested

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improvement in the protein's thermal stability upon ligand binding [45–47].

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3.6. Identification of TLT binding locus on HSA

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The molecular docking cluster analysis of TLT–HSA interaction initially indicated that TLT preferred to bind to the binding sites I and III of HSA compared to site II (Section 3.7, Fig. 7). Therefore, we targeted sites I and III of HSA for site-specific marker displacement experiments to further support the docking cluster analysis results. Various site-specific markers, viz., WFN and PBZ for site I and HMN for site III were used to perform competitive displacement investigations for exploring the possible binding locus of TLT in the protein. Fig. 6A shows the fluorescence spectra of 1:1 [WFN]:[HSA] mixture acquired in the absence and with addition of increasing TLT concentrations. It is worth mentioning that except free WFN, TLT and HSA alone or TLT–HSA (1:1) mixture were devoid of any significant fluorescence 14

Journal Pre-proof signal in the selected wavelength range. The fluorescence intensity value of the WFN–HSA complex progressively quenched with rising TLT concentrations, showing ~ 35 % reduction in the FI386 nm at the highest TLT concentration (inset of Fig. 7A) along with blue shift (4 nm) in the λem. These results clearly suggested the competition between TLT and WFN for the same ligand binding site, i.e., Sudlow's site I of the protein. Titration experiments were also performed using PBZ and HMN to corroborate the

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experimental results, obtained with WFN–HSA mixture (Fig. 6A). Titration results of HSA and

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1:1 [PBZ]/[HMN]:[HSA] mixtures with added TLT concentrations are displayed in Fig. 6B. A

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gradual loss in the intensity value at FI342 nm of HSA and [PBZ]/[HMN]:[HSA] mixtures were

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seen with continuous increments of TLT concentrations. However, [PBZ]:[HSA] mixture

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produced lesser decrease in the FI342 nm compared to that noticed with [HMN]:[HSA] mixture (Fig. 6B). These findings clearly revealed improved defence against TLT-induced reduction in

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the fluorescence signal in the presence of PBZ compared to HMN. Hence, competitive displacement results were inclined more towards preference of TLT binding at Sudlow’s site I of

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HSA. In view of it, the preferred locus of TLT binding in the protein appears to be Sudlow's site

Section 3.7.

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I, which was further supported by the molecular docking analysis, as described below in the

3.7. Molecular docking analysis of TLT–HSA system In order to identify and visualise the formed interactions between TLT and the primary binding sites of HSA at the atomic level, molecular docking analysis was performed. Based on the docking cluster analysis, the most populated cluster consisted of 51, 27, and 48 members for sites I, II and III, respectively (Fig. 7). The mean binding energy for these clusters was – 25.48 kJ mol–1 for site I, – 19.41 kJ mol–1 for site II and – 25.06 kJ mol–1 for site III. These initial 15

Journal Pre-proof analyses indicated that TLT preferred to bind to site I and site III of HSA instead of site II. The lowest binding energy recorded were – 25.52 kJ mol–1, – 21.59 kJ mol–1 and –25.271 kJ mol–1 for sites I, II and III, respectively. A low binding energy corresponds to the stability of TLT at the respective sites, which is dictated by the formed interactions. Based on the conformation generated through the lowest binding energy conformation, the interactions of TLT and HSA on sites I and III were evaluated (Fig. 8A). TLT formed two H-bonds after binding to site I (Fig. 8B)

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and only one H-bond at site III (Fig. 8C). The details of the interacting atoms together with the

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H-bond distances are shown in Table 3. The contributions of hydrophobic and polar interactions

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towards binding energy at sites I and III were evaluated through LigPlot+ and are shown in Figs.

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8D and E, respectively. As evident from the figures, orientation of TLT at site I allowed relatively higher hydrophobic and polar interactions compared to those observed at site III.

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Therefore, these results indicated that TLT had relatively higher preference to bind to site I than

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site III of HSA, as the formed complex at site I would be relatively more stable.

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3.8. Molecular Dynamics (MD) simulations analysis of TLT–HSA system MD simulations offer comprehensive insights on the molecular behaviour and interactions at

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the atomic level in its natural environment [48]. Therefore, MD simulations were made to study the structural behaviour and flexibility of HSA upon the binding of TLT. Since the simulation frames were captured at every 10 ps, a total of 5,000 trajectory frames were generated within the 50 ns production run and were used for analysis. Throughout the 50 ns run, the system temperature and pressure averaged at 300.001 K and 1.061 bar, respectively. Furthermore, the total energy within the 50 ns simulation averaged at  1.552106 kJ mol–1 (Fig. 9A), which indicated that the system was stable within the set range. In general, TLT remained bound to site I of HSA throughout the 50 ns MD simulation (Fig. 9A). 16

Journal Pre-proof This signified the stability of the formed complex. The overall stability of TLT on site I of HSA was assessed based on the root-mean-square deviation (RMSD). The RMSD at time t2 with respect to a given reference structure at time t1 was calculated as [49]: 𝑁

1 RMSD (𝑡1 , 𝑡2 ) = [ ∑ ||𝑥𝑖 (𝑡2 ) − 𝑥𝑖 (𝑡1 )||2 ] 𝑁

1 2

𝑖=1

where xi (t) is the position of atom i at time t, and N is the total number of atoms in the molecule.

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The RMSD for the TLTHSA complex, carbon-alpha (C-alpha) of HSA, and TLT were

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computed against the initial input structure and were plotted using Xmgrace, as shown in Fig. 9B.

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The overall RMSD pattern showed the stable TLTHSA complex, HSA, and TLT within the 50

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ns period. There were only minute fluctuations with a small deviation (< 2 Å) on HSA seen in

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the ~ 2 ns period of the simulation. Meanwhile, the RMSD of TLT revealed that the position and

0.337 Å.

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conformation of TLT at site I of HSA was stable throughout the simulation bearing an average of

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Inspection of HSA structure profile was made by evaluating the root-mean-square fluctuation (RMSF) and radius of gyration (Rg). RMSF provides a measure of residue displacement in the

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protein averaged over the simulation period [50]. The computed RMSF of HSA was generated using Xmgrace and is shown in Fig. 9C. The high fluctuation seen between residue 53–55 was expected since the residues reside next to a loop structure, which is known for their high degree of flexibility [51]. To ensure the structure of HSA did not disintegrate during the simulation, the structure compactness was evaluated through Rg, shown in Fig 9D. The Rg was calculated as [52]: 1

2 2 ∑𝑁 𝑖=1 𝑚𝑖 𝑟𝑖 𝑅𝑔 = [ 𝑁 ] ∑𝑖=1 𝑚𝑖

17

Journal Pre-proof where mi denotes the mass of atom i, ri denotes the position vector of atom i from the centre of mass, and N is the total number of atoms in the molecule. The values of Rg, obtained for HSA implied that the structure remained consistently compact with an average of 2.621 nm over the period of 50 ns. This indicated that the conformation of HSA neither expanded nor collapsed upon binding with TLT. The formation of hydrogen bonds (H-bonds) between HSA and TLT was also evaluated (Fig.

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9E). Since H-bonds are a non-covalent interaction, it is expected to see their formation and

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deformation over the period of simulation. The maximum number of H-bonds that can be formed

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between HSA and TLT was 4. Although the majority number of the formed H-bonds was 1, it

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can be said that the formation of H-bonds between TLT and HSA was frequent over the simulation period. Two H-bonds formation can be seen at the initial instances, as obtained from

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the molecular docking analysis and became more prevalent after ~ 43 ns of the simulation.

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Evidently, the formation of H-bonds would stabilise the complex, hence allowing and ensuring

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4. Conclusions

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that TLT remained within site I of HSA.

The results acquired from this study confirmed the TLT–HSA complex formation with weak binding affinity, and the complex was stabilized by hydrophobic and van der Waals forces along with hydrogen bonds. Whereas perturbations in the microenvironment around HSA fluorophores were manifested upon association with TLT, thermal denaturation of HSA was significantly defended. The TLT binding locus was identified in the proximity of Sudlow's site I (subdomain IIA) of HSA.

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Acknowledgements This work was financially supported by the University of Malaya Frontier Research Grant (FRG) 2017 (FG025-17AFR). Md. Zahirul Kabir gratefully acknowledges the financial assistance from the University of Malaya in the form of post-doctoral research fellowship. The authors thank the Dean, Faculty of Science and the Head, Institute of Biological Sciences, University of Malaya

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for providing necessary facilities.

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Disclosure statement

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No potential conflict of interest was reported by the authors.

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Journal Pre-proof Legends to Figures Fig. 1. The fluorescence quenching titration results of HSA (top spectrum) with increasing concentrations (spectra ‘2–9’) of TLT, as obtained at (A) 288 K, (B) 298 K and (C) 308 K. Different experimental conditions were: [HSA] = 3 M; [TLT] = 1080 M with 10 M intervals; λex = 295 nm; buffer = PB 7.4. The dotted line depicts the fluorescence spectrum of

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80 M TLT. Inset in (A) shows the 2-D structure of TLT.

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Fig. 2. (A) Stern-Volmer plots and (B) Double logarithmic plots for TLT−HSA system, as

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obtained from the fluorescence quenching titration results shown in Fig. 1, at three different

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temperatures. The van’t Hoff plot for the TLTHSA system is shown as the inset in (B).

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Fig. 3. UV-vis absorption spectra of free HSA (spectrum ‘1’) and with increasing TLT concentrations (spectra 2–6). The experimental conditions were: [HSA] = 10 M; [TLT] =

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50250 M with 50 M intervals; buffer = PB 7.4; T = 298 K.

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Fig. 4. Three-dimensional fluorescence spectra along with contour maps of (A) free HSA and (B)

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5:1 [TLT]:[HSA] mixture. Various experimental conditions were: [HSA] = 3 M; [TLT] = 15 M; buffer = PB 7.4; T = 298 K. Fig. 5. Bar diagram showing the influence of temperature on the FI342 nm value of free HSA and 10:1 [TLT]:[HSA] mixture. Various experimental conditions were: [HSA] = 3 M; [TLT] = 30 M; buffer = PB 7.4; T = 298–353 K with 5 K intervals. Fig. 6. (A) Diagram showing quenching effect of increasing TLT concentrations (spectra 2–9) on the fluorescence spectrum of 1:1 [WFN]:[HSA] mixture (spectrum 1). The spectra of 3 μM WFN

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Journal Pre-proof (spectrum ‘a’), 3 μM HSA (spectrum ‘b’), [TLT]:[HSA] (26:1) mixture (spectrum ‘c’) and 80 μM TLT (spectrum ‘d’) are also included. Insets shows quenching in the FI386 nm value of WFN– HSA complex with increasing TLT concentrations. (B) Quenching in the FI342 nm value of the protein and 1:1 [PBZ / HMN]: [protein] mixtures with increasing TLT concentrations. Various experimental conditions were: [HSA] = [WFN / PBZ / HMN] = 3 M; [TLT] = 080 M with 10 M intervals; λex = 335 nm (for WFN–HSA mixture) and λex = 295 nm (for PBZ / HMN–

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HSA mixtures); buffer = PB 7.4; T = 298 K.

Fig. 7. Cluster analysis of the AutoDock docking runs (a total of 100) of TLT to the ligand

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binding sites, viz., I, II and III of HSA (1BM0).

Fig. 8. (A) Binding orientation of TLT (rendered in sticks) in HSA binding sites I and III based

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on the lowest binding energy. Different colours depict domain I (orange), domain II (sky blue),

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domain III (green) of the protein. Binding sites were enlarged to display the formed hydrogen bonds (green lines) between the amino acid residues of HSA (rendered in yellow stick) and TLT

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at sites I (B) and III (C) of HSA. LigPlot+ diagrams manifesting the formed hydrophobic and

and III (E).

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polar interactions between TLT atoms and the amino acid residues of the protein at sites I (D)

Fig. 9. The XMgrace generated graphs for MD simulation analysis, as obtained throughout the 50 ns MD simulations. (A) total energy of the system, (B) RMSD of the complex in black, Calpha of HSA in red and TLT in green, (C) RMSF for HSA, (D) computed radius of gyration (Rg) of HSA and (E) hydrogen bonds formation between TLT and HSA.

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Table 1. Binding constants and thermodynamic parameters for TLT–HSA interaction, as obtained

(K)

(M–1)

(M–1)

288

8.41 ± 0.06

8.08 ± 0.14

298

6.40 ± 0.09

5.87 ± 0.11

308

4.82 ± 0.07

4.32 ± 0.08

ΔS

ΔH

ΔG

(J mol–1 K–1)

(kJ mol–1)

(kJ mol–1)

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Ka × 103

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Ksv × 103

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+ 5.35 ± 0.13

– 24.62 ± 0.12

– 23.08 ± 0.10

– 24.67 ± 0.14

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– 24.72 ± 0.11

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in PB 7.4 at three different temperatures.

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Table 2. Three-dimensional fluorescence spectral characteristics of 3 µM HSA and [TLT]:[HSA] mixture, as obtained in PB 7.4 at 298 K.

Peak

Peak position

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System

Intensity

230/230→350/350

22.1→68.8

b

250/500

66.6

280/338

277.9

230/335

71.7

a

230/230→350/350

25.1→290.4

b

250/500

230.6

1

280/337

220.6

2

230/334

57.0

1

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[TLT]:[HSA] = 5:1

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HSA

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λex / λem (nm/nm)

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Table 3. Predicted hydrogen bonds formed between atoms of amino acid residues of HSA (1BM0) and

HSA residue : atom

Distance (Å)

Arg-222 : HH11

O

2.06

Ile-290 : O

H

2.11

Tyr-138 : OH

H

2.04

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Site I

Site III

TLT atom

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HSA binding sites

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TLT at binding sites, i. e., I and III, as obtained from the molecular docking analysis.

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Fig. 1.

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Fig. 2.

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Fig. 3.

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Fig. 4.

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Fig. 5.

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Fig. 6.

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Fig. 7.

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Fig. 8.

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Fig. 9.

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Author Statement Md. Zahirul Kabir: Methodology, Binding data curation, Analysis and Writing original draft Zineddine Benbekhti: Binding data curation

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Nor Farrah Wahidah Ridzwan: Computational data curation and Writing

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Rabiâa Merrouche: Purification of TLT

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Noureddine Bouras: Supervision of TLT purification and Reviewing

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Saharuddin B. Mohamad: Supervision, Computational data analysis

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Editing

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Saad Tayyab: Conceptualization, Supervision, Data analysis, Reviewing and

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Journal Pre-proof Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Graphical Abstract

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Journal Pre-proof Highlights 

Quenching of the protein fluorescence by TLT is characterized as static quenching.



Hydrophobic and van der Waals forces along with hydrogen bonds stabilize the TLT−HSA complex. TLT binding produces alteration in the microenvironment around protein’s fluorophores.



Thermal stability of HSA is increased upon TLT binding.



Site I is predicted as the preferred TLT binding site in HSA.

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

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