A detailed insight into the interaction of memantine with bovine serum albumin: A spectroscopic and computational approach

Journal Pre-proof A detailed insight into the interaction of memantine with bovine serum albumin: A spectroscopic and computational approach

Faisal Ameen, Sharmin Siddiqui, Ishrat Jahan, Shahid M. Nayeem, Sayeed ur Rehman, Mohammad Tabish PII:

S0167-7322(19)36326-3

DOI:

https://doi.org/10.1016/j.molliq.2020.112671

Reference:

MOLLIQ 112671

To appear in:

Journal of Molecular Liquids

Received date:

16 November 2019

Revised date:

1 February 2020

Accepted date:

9 February 2020

Please cite this article as: F. Ameen, S. Siddiqui, I. Jahan, et al., A detailed insight into the interaction of memantine with bovine serum albumin: A spectroscopic and computational approach, Journal of Molecular Liquids(2020), https://doi.org/10.1016/ j.molliq.2020.112671

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© 2020 Published by Elsevier.

Journal Pre-proof

A detailed insight into the interaction of memantine with bovine serum albumin: a spectroscopic and computational approach Faisal Ameena, Sharmin Siddiquia, Ishrat Jahanb, Shahid M. Nayeemb, Sayeed ur Rehmanc, and Mohammad Tabisha* a

Department of Biochemistry, Faculty of Life Sciences, A.M. University, Aligarh, U.P. 202002,

Department of Biochemistry, School of Chemical and Life Sciences, Jamia Hamdard, New

-p

c

Department of Chemistry, Faculty of Science, A.M. University, Aligarh, U.P. 202002, India

ro

b

of

India

*Corresponding author:

Jo

Faculty of Life Sciences

ur

Department of Biochemistry

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Delhi. 110062, India

A.M. University, Aligarh U.P. 202002, India

Email: [email protected]; Tel: +91-9634780818

No. of figures: 11 No. of tables: 3 (+5 Supplementary Tables) Total pages: 41

1

Journal Pre-proof ABSTRACT Memantine is an NMDA receptor antagonist used to treat Alzheimer’s disease. Detailed insight about the interaction between memantine and bovine serum albumin (BSA) was obtained through multi spectroscopic techniques and in silico analysis. UV-visible, steady-state fluorescence spectra, and resonance light scattering (RLS) studies showed the complex formation between memantine and BSA. The binding constant for the BSA-memantine complex was found

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to be in the order of 103 M-1. The local structural changes in BSA induced by memantine were

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detected by synchronous fluorescence spectroscopy (SFS), three-dimensional fluorescence and

-p

circular dichroism. The study of NaCl effect, as well as molecular docking, revealed the

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existence of electrostatic interaction in the complex formation. Binding constant was calculated

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in the presence of metal ions and their different combinations. Site marker fractional occupancy indicated that memantine binds to site IB of BSA. Additionally, the CD-based thermal unfolding

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profile showed that memantine has stabilizing effects on BSA. Molecular dynamics simulations

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with BSA.

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further provided information on the stability and the forces involved in the binding of memantine

Keywords: Memantine; BSA; multi-spectroscopy; CD-based thermal unfolding; MD simulation.

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Journal Pre-proof 1. Introduction Alzheimer’s is a neurodegenerative disorder affecting around 15 million people worldwide and requires prolong medications. Memantine (1-Amino-3, 5-dimethyladamantane) is a cognition enhancer used to treat Alzheimer’s disease. It is an NMDA receptor antagonist (Figure 1a) functions by acting on glutamatergic neurotransmission (rather than working on cholinergic

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receptors). Glutamate binding causes a continuous influx of Ca2+ into the cells resulting in neuronal degradation. Memantine competes with the glutamate and prevents it from binding to

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glutamatergic receptors thus preventing Ca2+ influx into the cells [Danysz and Parsons, 2003.] Among the transport proteins, serum albumin is considered as a model protein for exploring the

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mechanism of protein folding and ligand-binding. The concentration of Human Serum Albumin

lP

(HSA) is 42 mg/ml, and it accounts for approximately 80% osmotic pressure of blood [Akram et al., 2019]. BSA has also been studied (vastly as an alternative to HSA) because of its cheap cost,

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high stability, and high structural homology (76%) with HSA [Majorek et al., 2012; Zhou et al.,

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2007]. BSA is a globular protein composed of 583 amino acids with 17 disulfide bonds and a

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free cysteine residue that divides BSA into nine loops (L1-L9) [Arumugam et al., 2019]. BSA has three linearly arranged domains, i.e., domain I, II, and III, with each domain having two subdomains, i.e., A and B [Akram et al., 2019]. There are two tryptophan residues in BSA, Trp134, and Trp-212 [Dubeau et al., 2010]. Trp-134 is located in sub-domain IB while Trp-212 is located in sub-domain IIA [Wani et al., 2018]. The structural and functional aspects of serum albumin can be studied in the presence of interacting drugs [Bagoji et al., 2017]. Transportation of drugs to target sites occurs by binding to serum albumin inside the body [Tian et al., 2015a]. Therefore, the absorption, distribution, metabolism, and excretion (ADME) properties of drugs are mainly affected by the extent of binding. Weaker binding of the drug may affect its transport 3

Journal Pre-proof to the target site. Alternatively, stronger binding of drugs to serum albumin may result in a prolonged half-life of the drug within the body. Therefore, optimum binding is crucial for a drug to be effective [Flarakos et al., 2005]. Deficiency in the ADME properties leads to the failure of most of the drugs to clear clinical trials. Therefore, much emphasis is needed for the development of effective in vitro, in vivo, and in silico approaches to fulfil the ADME properties

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of drugs. In this study, we have performed in vitro and in silico experiments to evaluate the binding

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affinity, binding site, and assessment of the type of interactions involved in the formation of the

-p

memantine-BSA complex. The interaction between BSA and memantine was depicted by using

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absorption and fluorescence emission spectroscopy. The consequent perturbations in the local

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structure of BSA in the presence of memantine were also detected by synchronous fluorescence, 3D fluorescence studies, and circular dichroic study. The type of interactions involved and the

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binding site was determined by fluorescence and in silico molecular docking study. The effect of

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metal ions on the binding affinity of BSA was also studied.

2.1 Materials

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2. Materials and methods

Bovine serum albumin (BSA), memantine, ibuprofen, and warfarin were purchased from Sigma Aldrich, Bangalore, India. Bilirubin was purchased from Sisco Research Laboratories (SRL), India. Analytical grade reagents and deionized distilled water were used for performing the experiments. 10 mM sodium phosphate buffer (pH 7.4) was used to perform the experiments. The stock solution of bilirubin was made by dissolving 1.8 mg bilirubin in 1 ml of 10 mM

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Journal Pre-proof NaOH. 10 mM stock solution of memantine was prepared in distilled water and diluted further according to the experimental requirements. 2.2 Methods 2.2.1 UV-Visible spectroscopy The absorption spectra of BSA were recorded with an increasing concentration of memantine

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from 0 µM to 60 µM in 10 mM sodium phosphate buffer pH 7.4. Experiments were performed

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on Shimadzu UV-1800 spectrophotometer using quartz cuvettes of path length 1 cm. Baseline

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correction was done using a 10 mM sodium phosphate buffer.

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2.2.2 Fluorescence studies

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The emission spectra of BSA (5 µM) was recorded in the presence of increasing the

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concentration of memantine (0 µM to 60 µM) on the Shimadzu Spectrofluorometer model RF6000. Excitation was performed at 280 nm, and the emission spectra were recorded between 300-

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450 nm at RT. The slit widths for excitation and emission were set at 5 nm. To further confirm

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the quenching mechanism, types of forces involved in binding and thermodynamic parameters, temperature dependent steady state fluorescence was also performed at 25 , 30

and 37

2.2.3 Synchronous fluorescence The emission spectra of BSA (5 µM) were also recorded in the absence and presence of increasing concentrations of memantine (0-60 µM) in a synchronous mode. BSA was excited at 240 nm, and the emission spectra were recorded from 255-350 nm for tyrosine residue and 300400 nm for tryptophan residue [Siddiqui et al., 2019].

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Journal Pre-proof 2.2.4 Resonance light scattering (RLS) analysis The RLS spectra of BSA (5 µM) were obtained by synchronously scanning the excitation and emission monochromators with increasing the concentration of memantine (0 µM to 10 µM). The spectra were measured and recorded on the Shimadzu spectrofluorometer model RF-6000. BSA was excited at 200 nm, and the difference between emission and excitation wavelength was The excitation and emission slits were set at 5 nm.

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2.2.5 Three dimensional fluorescence measurements

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zero, i.e.,

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The three-dimensional fluorescence spectra of BSA (5 µM) and BSA: memantine (1:10) were

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recorded using the Shimadzu Spectrofluorometer (Model RF 6000). The excitation wavelength was fixed from 200 nm to 400 nm, and the emission spectra were recorded from 200 nm to 600

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nm at an interval of 5 nm. The rest of the parameters were fixed as default.

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2.2.6 Circular dichroism (CD) spectroscopy and CD-based thermal unfolding of BSA

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The CD spectra of BSA (5 μM) was recorded using a JASCO CD spectrophotometer. The

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spectral measurements were done from 190 nm to 240 nm. A quartz cuvette of path length 0.2 cm was used. The measurements were made at 1 nm intervals and an average of 2 scans was plotted. The background spectrum of buffer was subtracted from the spectra of BSA and the BSA-memantine complex. The ratio of BSA and memantine were taken as 1:0, 1:1, 1: 3. The CD results were shown in terms of MRE (mean residual ellipticity), and the percentage of α-helical content was further calculated.

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Journal Pre-proof Thermal denaturation of BSA (5 µM) and BSA: memantine (1:1 and 1:3) was performed by scanning the spectra from 20

to 80 . The melting temperatures (Tm) were calculated for BSA

and BSA-memantine complex. 2.2.7 Role of ionic strength The effect of ionic strength on the binding of memantine to BSA was also studied. The

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experiment was performed by titrating 5 µM BSA with increasing concentration of memantine

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(0-60 µM) in the presence of different NaCl concentration (50 µM, 200 µM, 50 mM, and 200

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mM). The excitation wavelength of BSA was fixed at 280 nm, and the emission spectra were

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measured from 300 nm to 450 nm in 10 mM sodium phosphate buffer pH 7.4.

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2.2.8 Effect of metal ions

The effect of metal ion on the BSA-memantine complex was studied by taking a fixed

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concentration of BSA in the presence of metal ions (5 µM each) and increasing the concentration

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of memantine from 0 µM to 60 µM. The excitation wavelength of BSA was fixed at 280 nm, and the emission spectra were measured from 300 nm to 450 nm. The salts of metal ions used in the

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study were CaCl2, MgCl2, ZnCl2, CuCl2, and FeCl3. 2.2.9 Competitive site marker displacement assay and continuous variation analysis (Job’s Plot) Competitive site marker displacement assay was performed by taking a fixed concentration of BSA (5 µM) and memantine (35 µM) followed by addition of increasing concentration of bilirubin, warfarin, and ibuprofen separately (0 µM to 12 µM). The excitation wavelength was

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Journal Pre-proof fixed at 280 nm, and the emission spectra were measured from 300 nm to 450 nm [Afrin et al., 2019]. The continuous variation method or Job’s plot was used for the assessment of binding stoichiometry of the memantine-BSA complex [Job, P., 1928]. The measurements were taken at RT. The molar concentrations of memantine and BSA were varied in a way to achieve a total of

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5 µM. The difference in the fluorescence emission intensity ( F) of BSA with and without

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memantine was plotted as a function of mole fraction of BSA (

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2.2.10 Molecular docking

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Molecular docking was performed using AutoDock 4.0 software. The structure of BSA was downloaded from RCSB (PDB ID: 4F5S), and energy minimization was done using the SWISS

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PDB viewer. A monomeric file was prepared before the energy minimization. The 3D structure

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of memantine was obtained from the PubChem, and the energy was minimized by AVOGADRO. Docking was accomplished using the following steps: (1) BSA structure was

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edited by adding only the polar hydrogen atoms; Kollman charges and AD4 type atoms (2) The

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ligand was edited by detecting and choosing the roots of torsion tree. (3) Grid box was set up consisting of 80 x 80 x 80 points in the x, y, and z directions, and spacing of 0.375 Å was used having a center grid at x = 49.315, y = 31.735 and z = 36.489. (4) A rigid protein was docked with a flexible ligand. (5) 100 runs of docking were accomplished with each run comprising of a population of 150 individuals and 2,500,000 functional evaluations (6) Lamarckian genetic algorithm was applied for docking. (7) Cluster analysis was performed using an RMSD tolerance of 2 Å. The rest of the parameters were fixed with default values. Docked structures were visualized and analyzed using the BIOVIA discovery studio (3.1) program (Dassault Systèmes

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Journal Pre-proof BIOVIA, Discovery studio modeling environment, San Diego, 2016) as well as the UCSF Chimera molecular visualization software [Pettersen et al., 2004]. 2.2.11 Molecular dynamics simulation GROMACS-5.1.4 packages with the Amber 99 sb - ILDN force field were employed to perform molecular dynamics simulations of the docked structure of the BSA-memantine complex. The

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topology file of memantine was obtained from the ACPYPE interface [W Sousa da Silva and

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Vranken, 2012]. Bound structures were solvated using the TIP3P water model in a cubical box.

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The system was neutralized by adding 16 sodium ions. Similar simulation protocols were used, as reported previously [Kausar and Nayeem, 2018]. 100 ns standard MD was performed, and

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trajectories were stored at every 0.2 ps during the simulation period. The RMSD, total energy,

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and potential energy were calculated with respect to the initial structure using the GROMACS utility. MM-PBSA calculation [Kumari and Kumar, 2014; Nayeem, 2017] was done to calculate

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the binding energy of drug with protein. Hydrogen bonds and hydrophobic interaction between

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BSA and memantine were analysed using LigPlot+ [Wallace et al., 1995]. VMD was used to

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prepare molecular graphics [Humphrey et al., 1996]. 3. Results and discussion 3.1 UV-visible spectroscopy UV-visible absorption spectroscopy has been widely used to analyse the interaction of small molecules with proteins. Conformation changes in proteins induced by small molecules (like drugs) can be easily detected. Figure 1b shows the absorption spectra of BSA in the presence of increasing concentrations of memantine (0 µM to 60 µM). The

*

of aromatic amino acids

account for the absorbance profile of BSA around 280 nm [Gandhi and Roy, 2019]. In the 9

Journal Pre-proof presence of memantine, there was a decrease in the absorbance of BSA with no apparent shift in wavelength, suggesting changes in the structure of BSA. As evident in Figure 1c, the difference in the spectra of the BSA-memantine complex and BSA did not overlap with that of memantine spectra alone. This confirms the formation of a ground-state complex between BSA and

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memantine.

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Figure 1(a) Structure of memantine, (b) UV–visible spectra of BSA (5 µM) in the absence and presence of increasing concentration of memantine (0-60 µM) in 10 mM sodium phosphate buffer pH 7.4, (c) UVvisible absorption spectrum of 5 µM BSA (black), 5 µM memantine (blue), BSA: memantine (1:1) (red) and the difference spectra of (BSA+memantine) – BSA (green).

3.2. Fluorescence studies The intrinsic fluorescence of protein is changed upon interaction with drug molecules. Therefore, the measurement of these changes is employed to unravel the mechanism of drug-protein interactions [Eftink and Ghiron, 1981; Lakowicz, 2013]. Fluorescence quenching is commonly observed upon the interaction of small molecules with proteins providing information regarding 10

Journal Pre-proof the binding mechanism, binding specific-parameters and structural changes in the protein [Pasban Ziyarat et al., 2014]. Thus, fluorescence spectroscopy was performed to study the effect of memantine binding on the fluorescence property of BSA (Figure 2a). A progressive decrease in the fluorescence intensity was observed due to the quenching of BSA fluorescence with increasing concentration of memantine. The observed fluorescence quenching may be because of molecular interactions such as reactions in the excited state, molecular rearrangements, energy

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transfer, or static and dynamic quenching. [Eftink and Ghiron, 1981; Lakowicz, 2013]. This

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linear decrease in the fluorescence intensity was observed with no apparent shift in the emission

-p

wavelength.

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Stern-Volmer quenching constant was calculated using the following equation [Rahman et al.,

and

represents the fluorescence intensities of BSA with and without quencher (i.e.,

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where

(1)

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lP

2018]:

memantine) respectively and

is the Stern–Volmer quenching constant, which represents the

the value of

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quenching efficiency of the drug. The slope obtained from the ( ⁄ ) and [memantine] plot gave which was found to be 2.4*103 M-1, as shown in Figure 2b.

Fluorescence intensity of a fluorophore may be quenched as a result of dynamic or static quenching [Siddiqui et al., 2019]. Dynamic quenching occurs because of molecular diffusion in solution, while the formation of a complex in the ground state is a result of static quenching. The mechanism of quenching was confirmed by calculating the value of biomolecular quenching constant from the following equation [Siddiqui et al., 2019]: (2) 11

Journal Pre-proof where

is the average lifetime of a fluorophore without quencher is typically near 10-8 s was found to be 2.4*1011 M-1 s-1 at RT. The value of

[Rahman et al., 2018]. The value of

was higher than the value of the maximum scatter collision quenching constant, i.e., 2*1010 M-1 s1

[Rehman et al., 2015]. This indicated that the probable quenching mechanism of BSA with

memantine may be initiated by the complex formation in the ground state and not because of dynamic collision. and the number of binding sites ‘n’ were also determined using the

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The binding constant

(3)

where

and

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following equations [Afrin et al., 2019; Essemine et al., 2011]:

(4)

are the fluorescence intensities in the absence and presence of quencher; ΔF is

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the change in fluorescence intensities before and after the addition of quencher and

and the number of binding sites ‘n’ was calculated

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binding constant. The binding constant

is the

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from the intercept and slope respectively of the plot of log (ΔF/F) versus log [memantine] and were found to be 3.85*103 M-1 and 0.88 respectively (Figure 2c) at RT.

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Figure 2(a) Emission spectra of BSA (5µM) in the absence and presence of increasing concentrations of memantine (0-60 µM) in 10 mM sodium phosphate buffer pH 7.4, (b) Stern-Volmer plot for F0/F vs [memantine], (c) modified Stern-Volmer plot for log (F0-F/F) vs log [memantine] (d) synchronous fluorescence spectra of BSA (5 µM) with increasing concentration of memantine (0-60 µM). The difference between excitation and emission wavelength is △λ= 60 nm, and (e) synchronous fluorescence spectra of BSA (5 µM) with increasing concentration of memantine (0-60 µM).The difference between excitation and emission wavelength is △λ= 15 nm.

Interaction of memantine with BSA is associated with changes in the microenvironment of BSA can be studied by monitoring the shift in the emission maxima. The shift is based on the changes in the polarity around the micro-environment of the chromophore [Makarska-Bialokoz and Lipke, 2019]. Therefore, changes are determined by scanning the excitation and emission monochromators in synchronous mode while maintaining a constant wavelength interval (△λ) i.e., for tyrosine (△λ=15 nm) and for tryptophan (△λ=60 nm) residue [Tian et al., 2015b]. On subsequent addition of memantine, quenching in the emission spectra with no apparent shift in 13

Journal Pre-proof the wavelength of maximum emission of tryptophan was observed, as shown in Figure 2d. While, the emission spectra of tyrosine residue was not significantly quenched without any shift in the emission maxima, as shown in Figure 2e. From the results, it can be inferred that memantine binds to BSA near tryptophan residues.

3.3 Determination of quenching mechanism and thermodynamic parameters

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To further confirm the quenching mechanism, the Stern-Volmer constant and binding constant at

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different temperatures were calculated. From figure 3a, it can be observed that the value of , 30

, and 37

-p

was found to be 2.59*103 M-1, 1.81*103 M-1, and 1.15*103 M-1 at 25

value with increase in temperature is because increase in

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respectively. The decrease in the

temperature disfavour the interaction between BSA and memantine, suggesting static type of

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quenching [Liu et al., 2018] which in turn decreases the binding constant with increase in

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temperature, as shown in figure 3b. The thermodynamic parameters were calculated from the Vant Hoff’s plot as shown in table 1. The values of enthalpy change and entropy change were

and

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values of

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found to be -26.1 kcalmol-1 and -70.59 calK-1mol-1, respectively, as shown in figure 3c. When

interaction. If values of interaction. If the value of

then the predominant force of stabilization is electrostatic and and

then the stabilizing force is hydrophobic then the stabilizing forces are hydrogen bonding

and van der Waals interaction [Kumar and Asuncion, 1993]. The negative values of enthalpy and entropy suggests that the BSA-memantine complex is stabilized by hydrogen bonding and van der Waal’s interaction. The large negative value of enthalpy suggests that the interaction is enthalpically driven and entropy is unfavourable for this interaction [Ross and Subramaniam,

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Journal Pre-proof 1981]. Free energy of the interaction was also calculated from the value of binding constant using the following equation [Siddiqui et al., 2019]: 0

=

(5)

Table 1 Various parameters for the BSA- memantine complex formation.

*103 M-1

kcalmol-1

calK-1mol-1

kcalmol-1

298.15K

2.59

2.59

4.67

-26.1

-70.59

-5.05

303.15K

1.81

1.81

3.04

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*1011 s-1

of

*103 M-1

-70.59

-4.7

310.15K

1.15

1.15

0.872

-26.1

-70.59

-4.2

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lP

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-p

-26.1

Figure 3(a) Stern-Volmer plot at temperatures 298.15 K (25

(37

(b) modified Stern-Volmer plot at temperatures 25 , 30

, 303.15 K (30 and 37

and 310.15 K

and (c) Vant Hoff’s plot.

3.4 Resonance light scattering (RLS) analysis To explore the knowledge regarding the aggregation of bio-macromolecules after its interaction with drug, RLS can be used. This technique is highly susceptible to the weak binding forces such as electrostatic, hydrophobic and hydrogen bond [Xiao et al., 2007]. The RLS spectrum of the 15

Journal Pre-proof BSA-memantine complex is shown in Figure 4. It can be seen that the free molecules, i.e., BSA (5 µM) and memantine (10 µM), display weak RLS signals. When increasing concentrations of memantine (0-10 µM) were added to a fixed amount of BSA (5 µM), the intensity of RLS was continuously increased [Xiao et al., 2007]. According to the RLS theory, the RLS intensity is related to the dimensions of the particle and is directly proportional to the square of molecular volume. Larger, the molecular volume stronger is the light scattering signals [Xiao et al., 2007].

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Increase in the RLS intensity may be either because of the conformational changes in BSA

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exposing the hydrophobic residues to form aggregate with drug through hydrophobic interaction;

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or BSA-memantine complex developing larger sizes compared to BSA alone; or due to

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precipitation of memantine on BSA at a certain concentration, which in turn caused an increase in RLS intensity [Amani et al., 2011]. This increase in the RLS intensity suggests an increase in

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ground state [Siddiqui et al., 2019].

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the molecular volume of BSA due to complex formation between memantine and BSA at the

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Journal Pre-proof Figure 4 Rayleigh light scattering (RLS) spectra of BSA (5 µM) with increasing concentrations of memantine (0-10 µM) and memantine alone (10 µM) in 10 mM sodium phosphate buffer pH 7.4.

3.5 Three dimensional fluorescence measurements Changes in the protein structure and conformation on interaction with drugs can also be studied by 3D fluorescence spectra [Manea et al., 2019]. The 3D spectral profile of BSA was explored in the absence and presence of memantine. The spectral profile of BSA exhibits three different

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peaks (A, B and C). Peak A, peak B, and peak C attribute to the Rayleigh scattering peak, tryptophan, and tyrosine residues [Bolattin et al., 2016] and polypeptide backbone structure

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arising due to π π* transition of electrons, [Makarska-Bialokoz and Lipke, 2019] respectively.

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As seen in Figure 5a and 5b, on the addition of memantine, the fluorescence intensity of peak A,

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peak B, and peak C was decreased. The decrease in the intensity of peak A may be due to a

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decrease in the diameter of protein, which exhibits a decreased Rayleigh scattering effect, thus confirming the complex formation between BSA and memantine. Also, the decrease in Peak B

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and Peak C indicates changes in the microenvironment around tryptophan and tyrosine residues

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of BSA and conformation of the polypeptide backbone, respectively [Makarska-Bialokoz and Lipke, 2019]. The observations suggest that the binding of memantine induces conformational

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changes in BSA. The contour plot of BSA alone (5 µM) and BSA: memantine (1:10) molar ratio is shown in Figures 5c and 5d, respectively. Changes in peak A and peak B intensities in the presence of memantine can better be understood from the 5 μM BSA intensity bar graph with and without 50 μM memantine as shown in Figure 5e.

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Figure 5(a) 3D plot of BSA alone, (b) 3D spectra of BSA: memantine (1:10), (c) contour plot of BSA (5 µM), (d) BSA: memantine (1:10), and (e) bar graph for fluorescence intensity of BSA: memantine molar ratios (1:0 and 1:10).

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3.6 Circular dichroism (CD) spectroscopy and CD-based thermal unfolding of BSA

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Upon interaction with small molecules like drugs, the secondary structure of proteins is altered. Circular dichroic spectroscopy is a sensitive method to assess these structural alterations. The CD spectral profile of BSA exhibit two bands at 208 nm and 222 nm with negative intensities. The band at 208 nm arises due to the transition of electrons from π the transition of electrons from n

* and at 222 nm due to

* in the far UV region (190-260 mm). Both bands are the

characteristics of the α-helical structure of the protein [Arumugam et al., 2019]. In order to consider changes in the secondary structure of BSA after memantine interaction, the CD spectra of BSA is reported in varying BSA: memantine molar ratio (1:0, 1:1 and 1:3). Upon the addition

18

Journal Pre-proof of memantine, an increase in the intensity of both the bands was seen, thereby suggesting the conformational changes in BSA (Figure 6a). The CD results were expressed as molar residual ellipticity (MRE) by converting ellipticity into MRE by using the following equation [Bolattin et al., 2016]: (6) where Cp is the molar concentration of BSA, n represents the number of amino acid residues in

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BSA (i.e., 583). Pathlength is denoted as ‘l,’ which is equal to 0.2 cm.

-p

)

(7)

re

%α helix = (

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The % alpha helicity was also calculated using the following equation [Afrin et al., 2019]:

The α-helical content for 1:0, 1:1, and 1:3 molar ratio of BSA: memantine was found to be 54.29

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ur

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BSA from the native conformation.

lP

%, 55.4 %, and 55 %, respectively. Therefore, indicating variations in the secondary structure of

Figure 6(a) Circular dichroic spectra of BSA (5 µM) with increasing concentration of memantine (0-15 µM) in 10 mM sodium phosphate buffer pH 7.4, and (b) thermal stabilization of BSA on memantine binding of molar ratios 1:0, 1:1 and 1:3.

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Journal Pre-proof The stability of the tertiary structure of proteins depends on specific conditions of pH and temperature. Proteins are wholly or partially denatured because of changes in any of these conditions. It has been observed that the melting temperature (Tm) of proteins often increases in the presence of drugs. This increase in the melting temperature is because of the disturbance in the tertiary structure of a protein to attain equilibrium between the native and denatured states during thermal unfolding, which is coupled to the ligand-binding equilibrium [Shrake and Ross,

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1990]. Due to complex formation, the thermal unfolding profile of most of the proteins in the

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presence of drugs is changed [Precupas et al., 2019]. The stability of BSA was determined by

-p

estimating the melting temperature in the absence and presence of memantine. A plot of

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ellipticity at 222 nm and temperature with the molar ratio of BSA and memantine 1:0 and 1:1 and 1: 3 is shown in Figure 6b. The ellipticity loss at 222 nm was monitored to measure the

lP

thermal unfolding of BSA. Thermal denaturation curves yielded the value of Tm for the BSA-

62.43

and 61.06

,

na

memantine complexes. The Tm for molar ratios 1:0, 1:1, and 1:3 were calculated as 59.58

, respectively. From Figure 6b, it can be seen that the presence of

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memantine expanded the transition, causing a noticeable increase in Tm value as compared to

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the native BSA. At higher temperatures, the denaturation of BSA is caused by the devitalizing of hydrophobic and polar interactions [Rabbani et al., 2014]. Hence, memantine binding to BSA resulted in the increased thermal stability of BSA.

3.7 Role of ionic strength The interaction of memantine with BSA may originate from either electrostatic or hydrophobic interaction. To establish the binding between memantine and BSA was electrostatic or not, BSA fluorescence was measured in the presence of increasing concentrations of memantine at 20

Journal Pre-proof different concentrations of NaCl (0, 0.05, 0.2, 50, and 200 mM at pH 7.4). The binding mode was distinguished by increasing ionic strength. The presence of a higher concentration of salt does not alter the hydrophobic interaction but strongly affects the electrostatic interaction. If only hydrophobic interaction existed, the binding constant in the absence and presence of NaCl would not change. But if both types of interaction are present, the binding constant will change in the absence and presence of NaCl [Bolel et al., 2012].

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The magnitude of the binding constant was calculated in the absence and presence of salt

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using equation 4. At lower NaCl concentration, i.e., at 0.05 and 0.2 mM NaCl, the binding

-p

constant in the presence of NaCl did not change significantly, indicating the presence of

re

hydrophobic interaction between memantine and BSA. But as the concentration of NaCl was increased, the magnitude of binding constant decreased sharply. This suggests for the existence

lP

of electrostatic interaction in addition to the hydrophobic interaction [Datta et al., 2013], as

na

shown in Table 2. Therefore, it can be deduced that both the electrostatic and hydrophobic interactions are responsible for the stability of the BSA-memantine complex.

ur

The contribution of electrostatic forces in the interaction of memantine with BSA was examined

Jo

using the following relation [Rahman et al., 2018]: Na+] =

(8)

where Z is the apparent charge on the ligand, and

is the fraction of [Na+] bound to the

negatively charged amino acids of BSA. The slope of the plot of log value of zeta potential (

), and it was to be 0.68 (Figure 7).

21

and log [Na+] gave the

vs log [Na+] for the effect of ionic strength on binding of memantine on BSA.

re

Figure 7 Plot of log

-p

ro

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R2a

S.Db

3.85*10 3

0.9996

0.264

1.31*10 3

0.9720

0.3135

ur

NaCl (0-200 mM).

1.35*10 3

0.9920

0.3583

8.01

0.9089

0.5946

5.74

0.9666

0.5786

Kb

0 50 µM

na

Concentration of NaCl

200 mM a

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200 µM 50 mM

lP

Table2 The binding constants for the BSA- memantine system with increasing concentrations of

R2 is the correlation coefficient. b SD is the standard deviation. The results shown are the means

SD of

three experiments.

3.8 Effect of metal ions The binding of drugs with serum albumin is influenced by the metal ions, especially the divalent cations present in blood plasma. Hence, the effect of metal ions (Ca2+, Mg2+, Zn2+, Cu2+, Fe3+) on the binding of memantine with BSA was assessed. The metal ions were also used in different

22

Journal Pre-proof possible combinations. Table S1 shows the effect of combinations of metal ions on the BSAmemantine binding. It is interesting to observe that the binding constant was increased four times in the presence of a combination of Ca2+ + Mg2+ + Zn2+. The binding constant was also increased in the presence of Ca2+ alone and combination of Ca2+ + Mg2+ + Zn2+ + Fe3+. This increase in binding constant was because of conformational changes in BSA induced by the metal ions, favouring the complex formed between BSA and memantine [Li, D., 2017]. This will

of

increase the storage time resulting in the slow release of the drug from BSA, thus decreasing the

ro

therapeutic effects of memantine [Li, D., 2017]. The binding constant of memantine was

-p

decreased in the presence of other metal ions like Fe3+, Cu2+, and other combinations. This

re

decrease is possibly due to the following reasons: (1) the competition between the binding of metal ions and memantine to BSA, at the same binding site, thereby, weakening the memantine

lP

interaction with BSA and (2) difficulty in the binding of memantine due to metal ion-induced

na

conformational changes in BSA [Rahman et al., 2018]. From the pharmacokinetic point of view, the decrease in binding constant may result in quick clearance of memantine from the body.

ur

Therefore, higher doses of memantine are needed to achieve the desired therapeutic effects [Li,

Jo

D., 2017]. Inside the body, metal ions do not affect the binding independently but have a cumulative effect. Hence, it is inferred that the dose of memantine should be changed according to supplements given to patients containing either Ca2+ or combination of Ca2+ + Mg2+ + Zn2+ and Ca2+ + Mg2+ + Zn2+ + Fe3+.

23

Journal Pre-proof 3.9 Competitive site marker displacement assay and continuous variation analysis (Job’s Plot) The two central regions where a ligand preferably bind to BSA are the cavities in subdomains IIA and IIIA, also referred to as sites I and II, respectively [Thoppil et al., 2008]. These sites consist of hydrophobic amino acid residues, for example presence of Trp213 in sub-domain IIA.

of

In some cases, the sub-domain IB (site III) can also bind ligands, which has Trp-134 at its binding site. To identify the correct binding site of memantine, the specific site marker

ro

experiment was performed using compounds that are known to bind to these sites of BSA

-p

specifically. From previous studies, it is known that warfarin binds to site I (subdomain IIA),

re

ibuprofen binds to site II (subdomain IIIA), and bilirubin or digitoxin binds to site III

lP

(subdomain IB) [Ali and Al-Lohedan, 2017].

Fluorescence spectroscopy of the BSA-memantine complex was performed with

na

increasing concentrations of the displacement probes, i.e., warfarin, ibuprofen, and bilirubin.

ur

Change in the fluorescence intensity was recorded to get knowledge about the specific binding site of memantine. The percentage displacement of memantine by the probes was calculated

Jo

using the following, according to the method described earlier [Sudlow et al., 1976]:

where

and

(9)

represent the fluorescence of the memantine-BSA complex with and without the

probe, respectively [Sudlow et al., 1976]. Figure 8a shows the percentage displacement of memantine bound to BSA on the addition of probes. A noticeable displacement of memantine was observed by bilirubin (78%) than that observed in the case of warfarin (53%) and ibuprofen (12%). This suggests that memantine binds to BSA at site III, i.e., sub-domain IB.

24

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-p

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Figure 8 (a) Effect of site marker probe bilirubin, ibuprofen and warfarin (0-12 μM) on the fluorescence of BSA-memantine complex (1:7), and (b) Job’s Plot for change in fluorescence intensity of BSA vs mole fraction.

re

Fluorescence spectroscopy was also implemented to assess the binding stoichiometry between

lP

memantine and BSA using continuous variation analysis (Job’s Plot). Upon the addition of memantine, a decrease in fluorescence intensity of BSA was observed at 337 nm. The difference

na

in the emission of BSA alone and BSA-memantine complex versus the molar ratio of BSA was

ur

plotted. The point of inflection provided the mole fraction of BSA bound to memantine in the complex and the value of inflection was found at a 0.8 mole fraction (Figure 8b). The binding

Jo

stoichiometry of BSA: memantine was found to be 1:4.

3.10 Molecular docking The mode of interaction and binding site of memantine with BSA was also studied by molecular docking. Among all the clusters, the most populated and lowest energy conformational cluster was selected and analysed for binding orientation. It was observed that memantine binds near site IB of BSA as shown in Figure 9a. Memantine binds to BSA through hydrogen bonds with

25

Journal Pre-proof Glu-186 and Thr-190 (Figure 9b and 9c). This suggests that the electrostatic interaction is involved in the stability of memantine-BSA interaction. The free energy of reaction was found to be

6.67 kcal/mol, showing similarity with the value obtained through fluorescence

spectroscopy-based experiments. The values obtained through in silico and in vitro experiments shows slight discrepancy. This discrepancy may have arisen due to the fact that unlike in vitro experiments, in silico experiments were performed in the absence of water molecules and with a

Jo

ur

na

lP

re

-p

ro

of

rigid protein structure.

Figure 9(a) Relative binding site of memantine (green) and bilirubin (blue), (b) Amino acid residues near binding site of memantine, and (c) 2-D plot of the interacting amino acid residues of BSA with memantine.

3.11 Molecular dynamics simulation

26

Journal Pre-proof MD simulation studies were conducted to gain insight into the stability of the BSA-memantine complex. Structures of the complex with the lowest docking energy were taken as initial conformations for MD simulation. Initially, the root-mean-square deviations (RMSDs) of the starting backbone structure of BSA in the presence and absence of memantine and memantine alone were calculated to investigate the stability of the complex as described earlier [Cui et al., 2015]. Figure 10a shows the RMSD value of BSA in the absence of memantine, which shows

of

that BSA reaches equilibrium with an average value of 0.50 nm. In the presence of drugs,

ro

fluctuation in the BSA backbone has been observed, which might be due to slight alterations in

-p

the local conformation of the BSA in the presence of the drug. The RMSD of memantine

re

remains smooth during the simulation time.

lP

To further verify the stability of the complex, the physicochemical parameters such as total energy and potential energy were also calculated (Figure 10b). The smooth curve of total energy

na

and potential energy shows that the system reaches equilibrium and remains stable during the

ur

simulation period [Liao et al., 2014].

VMD software.

Jo

Meanwhile, the evolution of the conformation of the complex over time was also displayed using

To further reveal the dynamic interaction of memantine with BSA, the different snapshot conformations of BSA-memantine complex were captured at 85 ns, 90 ns, 95 ns, and 100 ns and analysed. It was observed that memantine remains in the same position throughout the simulation after 50 ns. Figure 10c shows four snapshots representing the 2D and 3D conformation of BSAmemantine complex. The memantine is mostly surrounded with the same residue at a different time interval, indicating the best binding pose of memantine.

27

Journal Pre-proof From Figure 10c, it is also observed that during the drug-protein binding, hydrophobic interaction plays an essential role in the binding process. A list of interacting amino acid residues involved in the interaction with memantine at various time intervals is given in Table S2. The conformational changes in BSA at four snapshots were also calculated by using the DSSP method in GROMACS and are listed in Table S3. No significant difference in the secondary

of

structure of BSA is observed during every 100 frames as it reaches equilibrium states. During the simulation, as the memantine reaches this binding site, nearby residues change their

ro

conformation to form a stable complex system. From Figure 10c, if we compare the structure of

-p

BSA at all the four-time interval (i.e. 85 ns, 90 ns, 95 ns, and 100 ns), it is observed that at 90 ns

re

Lys439, and 95 ns Trp213 are the only different residues involved in the interaction whereas all

lP

other residues are same at all the four-time interval, further confirming the binding site of memantine. All these residues interact with memantine through hydrophobic interactions to

na

create a more stable complex. Therefore, we conclude that hydrophobic forces between the

ur

memantine and hydrophobic pocket of BSA play a significant role in the binding process [Ross

Jo

and Subramanian, 1981].

28

na

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-p

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Journal Pre-proof

Jo

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Figure 10(a) The RMSD of protein backbone black line (absence of memantine), red line (presence of memantine) and memantine (green line) during the simulation period, (b) the potential energy (red line) and the total energy (black line) of the BSA-memantine complex during simulation, and (c) binding modes of memantine with BSA at (A) 85 ns, (B) 90 ns, (C) 95 ns, (D) 100 ns during MD simulation. Hydrophobic residues in 2D plot are shown with curve red line. In 3D plot memantine carbons are shown in yellow colour and protein residue carbons are shown in magenta colour.

The methods of MM-PBSA were used to analyse and calculate the forces involved in the binding process [Siddiqui et al., 2019]. MM-PBSA was done from the last 10 ns of MD simulation. The MM-PBSA binding energy was calculated for the 100 snapshots of every 0.1 ns MD simulation, as shown in Table S4. Depending upon the chemical nature of memantine, different types of

29

Journal Pre-proof interaction ranging from hydrophobic, electrostatic, polar interactions are formed between memantine atoms and distinct residues of BSA. Each type of interaction either contributes positively or negatively to the overall binding energy (BE). Polar solvation energy (PSE) impaired binding of memantine while van der Waals (vdW), solvent accessible surface area energy (SASA), and electrostatic forces (Elec) favored the binding process as shown in Figure 11a. Electrostatic contribution towards the binding process is very small (-0.196 kcal/mol) and

of

almost negligible as there is no hydrogen bond formation taking place. Solvent accessible surface

12.299 ± 3 kcal/mol.

Jo

ur

na

lP

re

-p

binding energy of the memantine-BSA complex was

ro

area (SASA) interaction energy contribution is also minimal. The total average calculated the

30

Journal Pre-proof Figure 11(a) Energy plot showing the contribution of various types of interaction to the overall binding energy, (b) polar (blue bar) and apolar (red bar) energy contributions of the key residues to the total binding energy (green bar). Negative values are favourable and positive values are unfavourable for binding process, (c) Ramachandran plot of BSA, and (d) Ramachandran plot of BSA-memantine complex.

Table 3

and

angles for BSA alone and BSA-memantine complex phi angle (

psi angle(

-71.04

-39.34

BSA-memantine complex

-79.38

-26.15

re

-p

ro

of

BSA alone

From the ligand binding pose, the binding free energy of amino acid residues key to the binding

lP

process can be calculated. The polar, apolar and total binding energy contributions of 8 key

na

residues were plotted in Figure 11b and the data are given in Table S5. Residues Arg194, Trp213, Arg217, Lys439, Pro446, and Cys447 contribute positively towards the entire binding

ur

process (green bar in Figure 11b), whereas residues Glu443 and Asp450 contribute negatively

Jo

towards the entire binding process as polar energy (blue bar in Figure 11b) contribution overwhelmed the apolar contribution. The conformational change in BSA alone and BSA simulated with memantine (Figure 11c and 11d) was calculated in terms of dihedral angles from the Ramachandran plot as shown in Figures 11c and 11d, respectively. The dihedral angles for BSA alone and the BSA-memantine complex is summarised in Table 3.

31

Journal Pre-proof 4. Conclusions In this study, various spectroscopic and in silico techniques were employed to assess memantine and BSA interaction. Steady-state fluorescence data revealed the complex formation between memantine and BSA. The binding constant was found to be 3.85*103 M-1. The synchronous fluorescence and 3D fluorescence spectroscopy confirmed the conformational changes in BSA. Further, the circular dichroism (CD) indicated changes in the BSA secondary structure resulting

of

in the reduction of the α-helical content on interaction with memantine. Additionally, the binding

ro

strength was increased 4 times in the presence of Ca2+ + Mg2+ + Zn2+. With the help of site

-p

markers like bilirubin, warfarin, and ibuprofen, memantine was found to bind at site IB of BSA.

re

Docking studies also corroborated the binding of memantine at site IB. The CD-based thermal unfolding profile indicated the stabilization of BSA in the presence of memantine. RMSD plot

lP

reflects the changes in the local conformation of BSA in the presence of memantine. The primary

na

binding forces involved in the stable complex formation was found to be hydrogen bonding, van

Jo

ur

der Waals forces and hydrophobic interactions.

Conflict of Interest

The authors declare that there is no conflict of interest in this work.

Acknowledgements We are thankful to the CSIR, New Delhi for providing SRF to FA, and DST-INSPIRE SRF to SS. We are also thankful to the UGC-DRS-SAP, DST-FIST and DST-PURSE for generous funding to the Department of Biochemistry, Faculty of Life Sciences and BRAF at C-DAC, Pune for providing supercomputer facility to carry out MD simulation.

32

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Author statement

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Faisal Ameen: Writing- Original draft preparation, Conceptualization, Methodology, Formal

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analysis, Software, Validation Sharmin Siddiqui: Writing - Review & Editing, Software, Formal analysis Ishrat Jahan: Software Shahid M. Nayeem: Software Sayeed ur Rehman:

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Writing - Review & Editing and Mohammad Tabish: Supervision

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Graphical abstract

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of ro -p re lP na ur Jo

    

Highlights Memantine interacts with BSA near tryptophan residue in sub-domain IB. Memantine causes conformation changes around tryptophan residues in BSA. Memantine increases α-helical content as well as the thermal stability of BSA. Ca2+ + Mg2+ + Zn2+ increased the binding strength of memantine and BSA. The stabilizing forces are van der Waal’s, hydrophobic and hydrogen bonding.

41