Unravelling the binding mechanism and protein stability of human serum albumin while interacting with nefopam analogues: a biophysical and insilico approach

Article abstract of DOI:10.1080/07391102.2016.1216895

In this study, molecular binding affinity was investigated for Nefopam analogues (NFs), a functionalized benzoxazocine, with human serum albumin (HSA), a major transport protein in the blood. Its binding affinity and concomitant changes in its conformation, binding site and simulations were also studied. Fluorescence data revealed that the fluorescence quenching of HSA upon binding of NFs analogues is based on a static mechanism. The three analogues of NFs binding constants (K_A) are in the order of NF3?>?NF2?>?NF1 with values of 1.53?±.057?×?10⁴, 2.16?±.071?×?10⁴ and 3.6?±.102?×?10⁵?M^?1, respectively. Concurrently, thermodynamic parameters indicate that the binding process was spontaneous, and the complexes were stabilized mostly by hydrophobic interactions, except for NF2 has one hydrogen bond stabilizes it along with hydrophobic interactions. Circular dichroism (CD) studies revealed that there is a decrease in α-helix with an increase in β-sheets and random coils signifying partial unfolding of the protein upon binding of NFs, which might be due to the formation of NFs-HSA complexes. Further, molecular docking studies showed that NF1, NF2 and NF3 bound to subdomains IIIA, IB and IIA through hydrophobic interactions. However, NF1 have additionally formed a single hydrogen bond with LYS 413. Furthermore, molecular simulations unveiled that NFs binding was in support with the structural perturbation observed in CD, which is evident from the root mean square deviation and R_g fluctuations. We hope our insights will provide ample scope for engineering new drugs based on the resemblances with NFs for enhanced efficacy with HSA.

Full text of DOI:10.1080/07391102.2016.1216895

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20
Unravelling the Binding Mechanism and
Protein Stability of Human Serum Albumin
while Interacting with Nefopam Analogues: A
Biophysical and Insilco approach
Mahesh Gokara, Vidadala V. Narayana, Vineet Sadarangani, Shatabdi Roy
Chowdhury, Sreelaxmi Varkala, Dhevalapally B. Ramachary & Rajagopal
Subramanyam
To cite this article: Mahesh Gokara, Vidadala V. Narayana, Vineet Sadarangani, Shatabdi
Roy Chowdhury, Sreelaxmi Varkala, Dhevalapally B. Ramachary & Rajagopal Subramanyam
(2016): Unravelling the Binding Mechanism and Protein Stability of Human Serum Albumin
while Interacting with Nefopam Analogues: A Biophysical and Insilco approach, Journal of
Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2016.1216895
To link to this article: http://dx.doi.org/10.1080/07391102.2016.1216895
Accepted author version posted online: 25
Jul 2016.
Published online: 25 Jul 2016.
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Date: 25 July 2016, At: 17:37

Publisher: Taylor & Francis
Journal: Journal of Biomolecular Structure and Dynamics
DOI: http://dx.doi.org/10.1080/07391102.2016.1216895
Unravelling the Binding Mechanism and Protein Stability of Human
Serum Albumin while Interacting with Nefopam Analogues: A
Biophysical and Insilco approach
Mahesh Gokara†, Vidadala V. Narayana‡, Vineet Sadarangani†, Shatabdi Roy Chowdhury†,
Sreelaxmi Varkala†, Dhevalapally B.Ramachary‡, Rajagopal Subramanyam§*
† Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad
500046, India
‡School of Chemistry, University of Hyderabad, Hyderabad 500046, India
§Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad
500046, India
*Corresponding author
Rajagopal Subramanyam
Department of Plant Sciences
School of Life Sciences
University of Hyderabad 500 046 India
Tel: +91-40-23134572
Fax: +91-40-23010120
Email: srgsl@uohyd.ernet
1

Abstract
In this study, molecular binding affinity was investigated for Nefopam analogues (NFs), a
functionalized benzoxazocine, with human serum albumin (HSA), a major transport protein in
the blood. Its binding affinity and concomitant changes in its conformation, binding site and
simulations were also studied. Fluorescence data revealed that the fluorescence quenching of
HSA upon binding of NFs analogues is based on a static mechanism. The three analogues of NFs
binding constants (KA) are in the order of NF3>NF2>NF1 with values of 1.53 0.057 × 104, 2.16
0.071 ×104 and 3.6 0.102 ×105 M-1, respectively. Concurrently, thermodynamic parameters
indicate that the binding process was spontaneous, and the complexes were stabilized mostly by
hydrophobic interactions, except for NF2 has one hydrogen bond stabilizes it along with
hydrophobic interactions. Circular dichroism studies revealed that there is a decrease in α-helix
with an increase in β-sheets and random coils signifying partial unfolding of the protein upon
binding of NFs, which might be due to the formation of NFs-HSA complexes. Further, molecular
docking studies showed that NF1, NF2 and NF3 bound to subdomain IIIA, IB and IIA through
hydrophobic interactions. However, NF1 have additionally formed a single hydrogen bond with
LYS 413. Furthermore, molecular simulations unveiled that NF’s binding was in support with
the structural perturbation observed in circular dichroism, which is evident from the RMSD and
Rg fluctuations. We hope our insights will provide ample scope for engineering new drugs based
on the resemblances with NFs for enhanced efficacy with HSA.
Keywords: human serum albumin, binding studies, nefopam, protein stability, docking studies
2

1. Introduction
Studies on the interaction between drugs and human serum albumin (HSA) have been a
subject of interest in therapeutic drug monitoring. It has a significant contribution to
understand the physicochemical mechanism, several biological and behavioral activities
of drugs transported in human body (Kanakis, Tarantilis, Polissiou, Diamantoglou &
Tajmir-Riahi 2006; Sarkar, Paul & Mukherjea 2013). HSA is the most abundant protein in
blood serum, it accounts for about 60% of the total plasma proteins corresponding to a
concentration of 42 mg/mL, and provides about 80% of the colloidal osmotic pressure of blood
(Peters Jr 1995). It is the major soluble depot and transport protein, capable of binding,
transporting, and delivering an extraordinarily diverse range of endogenous and exogenous
compounds like bilirubin, fatty acids, thyroxine, and hemin (Tabachnick 1964; Beaven, D'Albis
& Gratzer 1973; Curry, Brick & Franks 1999; Bhattacharya, Grnne & Curry 2000; Zunszain,
Ghuman, Komatsu, Tsuchida & Curry 2003; Simard, Zunszain, Hamilton & Curry 2006). HSA
also binds to exogenous drug molecules like warfarin, digitoxin, ibuprofen, and quinidine with
binding constants of 3.3 × 105, 4 × 105, 2.6 × 106, and 1.6 × 103 M−1, respectively (Varshney et
al. 2010). It also maintains normal osmolality in plasma as well as in interstitial fluid and
exhibits antioxidant and enzymatic functions (Peters Jr 1995).
HSA has only one tryptophan residue, Trp-214 in the IIA subdomain (Carter et al. 1989;
Lakowicz 2006). This protein also contains two principal drug binding sites, Site-I and Site-II.
Site-I is located within the hydrophobic cavity of subdomain IIA which is capable of binding
mostly neutral, bulky heterocyclic compounds by strong hydrophobic interactions, whereas Site-
II is within the IIIA subdomain and binds to many aromatic carboxylic acids by dipole–dipole,
3

van der Waals and hydrogen bonding interactions (Sudlow, Birkett & Wade 1975; Sudlow,
Birkett & Wade 1976; Varshney et al. 2010).
Drug interaction with HSA generally enhances the distribution and bioavailability of the
drug depending on the specific pharmacokinetic properties of the drug molecules.
Additionally, because of its abundance, HSA play a significant role in the pharmacokinetic
behavior of a variety of drugs, including drug half-life in the blood stream, regulating drug
efficacy, decreasing drug toxicity and improving drug targeting specificity (Sleep, Cameron
& Evans 2013). Therefore, a clear and quantitative information on the nature of a given drug–
plasma protein interaction should provide a firm basis for its rational use in clinical practice even
if it is not the only factor predictive of serum concentrations of a free drug (Kurono, Fujii,
Murata, Fujitani & Negoro 2006; Shang et al. 2014). Recently, we have reported interaction of
biologically significant molecules like trimethoxyflavone, asiatic acid, chitosan oligomers, 7-
hydroxycoumarin derivatives, piperine, L-dopa and lupeol and its derivatives with HSA (Gokara,
Sudhamalla, Amooru & Subramanyam 2010; Gokara, Malavath, Kalangi, Reddana &
Subramanyam 2014; Yeggoni et al. 2014; Yeggoni & Subramanyam 2014; Gokara, Kimavath,
Podile & Subramanyam 2015; Kallubai, Rachamallu, Yeggoni & Subramanyam 2015; Yeggoni,
Rachamallu, Kallubai & Subramanyam 2015).
Nefopam is a centrally acting non-opioid analgesic benzoxazocine and has been used to treat
mild to moderate postoperative pain since the mid-1970s (Heel, Brogden, Pakes, Speight &
Avery 1980; Kapfer, Alfonsi, Guignard, Sessler & Chauvin 2005; Beloeil, Eurin, Thevenin,
Benhamou & Mazoit 2007). In clinical practice, the administration of nefopam has been reported
to reduce the use of opioid analgesics, which reduces the prevalence of Postoperative nausea and
vomiting (Lee, Kim & Cheong 2013). It is reported that the nefopam plasma half-life is 3-5 h;
4

with its peak plasma concentrations reaching after 15 – 20 min through intravenous injection.
Owing to a first-pass metabolism, oral bioavailability is only 40%. Nefopam undergoes extensive
hepatic biotransformation to desmethylnefopam (which seems to be biologically active) and N-
oxide-nefopam (Aymard et al. 2003). It is reported that nefopam binding with protein is ~75%,
and the major route of elimination (87%) is renal whereas a small part (8%) is excreted in the
faeces. Ninety-five per cent of an initial dose is excreted within five days and 5% as unchanged
substance (Heel et al. 1980). Nefopam is considered to be safe and well tolerated. Reported
adverse effects are mostly minor and includes drowsiness, nausea and vomiting, and sweating
(Mimoz et al. 2001; Du Manoir et al. 2003; Kapfer, Alfonsi, Guignard, Sessler & Chauvin 2005).
Unlike non-steroidal anti-inflammatory drugs, nefopam has no effect on platelet function and in
contrast to opioids, this drug does not seem to increase the risk of respiratory depression (Gasser &
Bellville 1975).
In this study, we used synthesized NFs-analogues which are functionalized benzoxazocines
(Ramachary, Narayana, Prasad & Ramakumar 2009). Recent studies showed that functionalized
benzoxazocines are pharmaceutically acceptable salts that might act as analgesic agents and are
used for the treatment of emesis, depression, posttraumatic stress disorders, attention deficit
disorders, obsessive compulsive disorders, sexual dysfunction and as centrally acting skeletal
muscle relaxants (Stillings et al. 1985; Ohnmacht et al. 2004; Seto, Tanioka, Ikeda & Izawa
2005; Seto, Tanioka, Ikeda & Izawa 2005; Seto, Tanioka, Ikeda & Izawa 2005). Since, nefopam
has very important role in pain relief, their products are expensive. It is for this purpose we have
attempted to synthesize nefopam analogues which may mimic similar function as original
nefopam. To understand the pharmacokinetics with HSA, we have investigated the binding,
including quenching mechanisms, binding energies, the number of binding sites, and secondary
5

structural fluctuations of HSA using spectroscopic methods. Further molecular docking and
molecular simulation were also used to gain insight into the binding modes and stability of the
NF’s-HSA complexes.
2. Materials and Methods
2.1 Synthesis of Nefopam analogues
Nefopam analogues, 1) 6-(phenyl)-7-methyl-9 ethyl 5, 6-dihydro-2H- benzo[b] [1, 4] oxazocine-
8-carboxylic acid ethyl ester Nefopam analogue 1 (NF1); 2) 9-(4-chloro phenyl)-6-(4-hydroxy
phenyl)-7-methyl-5, 6-dihydro-2H-benzo[b] [1, 4] oxazocine-8-carboxylic acid ethyl ester
Nefopam analogue 2 (NF2); 3) 6-(4-hydroxy phenyl)-7-methyl-9-propyl-5, 6-dihydro-2H
benzo[b] [1, 4] oxazocine-8-carboxylic acid ethyl ester Nefopam analogue 3 (NF3) were
synthesised according to our previous method (Ramachary et al. 2009).
General Experimental Procedures for the Synthesis of Nefopam Analogues (NF1, NF2 and NF3)
from corresponding Hagemann’s esters involves the following three-steps.
Piperidine/K2CO3-Catalyzed Three-Component Enamine Amination/Iso-Aromatization/O-
Allylation Reactions in One-Pot: In an ordinary glass vial equipped with a magnetic stirring
bar, to 1.0 mmol of the Hagemann’s esters was added 2.0 mL of solvent, and then the catalyst
piperidine (0.05 mmol, 4.95 μL) was added and the reaction mixture was stirred at 25 °C for the
0.5 h; then 0.5 mmol of nitrosobenzene was added in one-portion and the reaction mixture was
stirred at 25 °C for 1 h. To the reaction mixture, allyl bromide (181.5 mg, 1.5 mmol) and K2CO3
(345.5 mg, 2.5 mmol) were added and stirring continued at RT for 24 h. The crude reaction
mixture was worked up with aqueous NH4Cl solution, and the aqueous layer was extracted with
dichloromethane (2 x 20 mL). The combined organic layers were dried (Na2SO4), filtered and
concentrated. Pure one-pot enamine amination/iso-aromatization/O-allylation products were
obtained by column chromatography (silica gel, a mixture of hexane/ethyl acetate).
N-Allylation: At 0 °C, to a suspension of sodium hydride (14.4 mg, 0.6 mmol, 3.0 equiv.), in
DMF (2 ml, 0.1 M) were added monoene amine (0.2 mmol). After being stirred for 5 min, allyl
bromide (48.39 mg, 0.4 mmol, 2.0 equiv.) is added to the reaction mixture at 0 °C, and then the
reaction mixture was stirred for 3 to 5 h from 0 °C to RT. The reaction mixture poured into
6

saturated NH4Cl solution and extracted with DCM (2 x 15 mL). The combined DCM extracts
were washed with brine, dried over Na2SO4, and evaporated to dryness. Purification of the
residue by column chromatography (silica gel, mixture of hexane/ethyl acetate) gave diene
amine product.
RCM Reaction: A 10 ml oven-dried round bottom flask equipped with a stir bar was charged
with diene amine (0.1 mmol), CH2Cl2 (2 ml, 0.05 M) and first generation Grubb’s catalyst (4.11
mg, 0.005 mmol, five mol-%). The reaction mixture was stirred under N2 at room temperature
for 8 to 12 h. Solvent CH2Cl2 were distilled off at ambient pressure and the nefopam analogues
(NF1, NF2 and NF3) were purified by column chromatography (silica gel, a mixture of
hexane/ethyl acetate).
2.2 Sample preparation.
Fat-free HSA (purchased from Sigma-Aldrich) was dissolved in a physiological aqueous solution
of 0.1M phosphate buffer pH 7.2 to the final concentration of 1.5mM protein according to the
previous procedure (Subramanyam, Gollapudi, Bonigala, Chinnaboina & Amooru 2009). The
purity of HSA was confirmed by SDS gel and mass spectrometry (data not shown). The exact
concentration of HSA was determined spectrophotometrically using molar extinction coefficient
of 35700 M−1 cm−1 in a Perkin-Elmer lambda 35 (Chatterjee, Pal, Dey, Chatterjee & Chakrabarti
2011). Synthesized NF’s were prepared in 20:80 ethanol: distilled water mixture. From our
previous work, a solution containing 20% ethanol showed no effect on HSA secondary structure
(Subramanyam et al. 2009; Subramanyam et al. 2009). The optimum physiological pH for HSA
was set to be 7.2 as it has the maximum absorption and fluorescence at this pH (Subramanyam et
al. 2009; Subramanyam et al. 2009). Thus, for all the experiments, we have used 0 . 1 M
phosphate buffer at pH 7.2 as a physiological buffer. The optimum incubation time for binding of
NF1, NF2, and NF3 with HSA was found to be 15 min as assessed by fluorescence emission
spectroscopy. Thus, we followed the same incubation time for all the experiments.
7

2.3 Fluorescence spectroscopy studies
.
A quantitative analysis of the potential interaction between NF1, NF2, and NF3 with
HSA was performed by LS-55 Spectrofluorimeter (PerkinElmer, USA), with 1.0 cm quartz
o
cells. Fluorescence emission spectra were recorded at 25 C with a wavelength range of 300–450
nm and excited at 285nm. The bandwidth was fixed to 5.0 nm for both excitation and emission.
o
-6
x10 M, and the
The sample temperature was maintained at 25 C. HSA concentration was 1
concentrations of NFs were 1 to 9μM in 0.1M phosphate buffer with pH 7.2 (physiological
pH). Three independent experiments were performed and each time identical spectra were
obtained. All protein fluorescence spectra were corrected by subtraction of buffer. Furthermore,
the spectra were corrected to avoid inner filter effect by using the below equation.
The intensity of fluorescence observed (Fobs) was corrected for inner filter effect (For) (Kirby
1971; Lakowicz 2006)
Fcor = Fobs10 (Aꢀꢁꢂ + Aꢀꢃꢄ)/2 [1]
Where, For is the corrected fluorescence intensity, Aexc and Aemi represent the absorbance at the
fluorescence excitation (285 nm) and emission wavelengths (360 nm) for HSA and (340 nm) for
AGP. Fobs are the observed fluorescence (Lakowicz 2006). The corrected values allow
evaluating fluorescence quenching only due to the interaction of the drug with albumin (van de
Weert & Stella 2011). The detailed procedure was given in our previously published articles and
also can be referred these articles for further details (Subramanyam et al. 2009; Subramanyam et
al. 2009; Gokara et al. 2015).
8

2.4 Displacement experiments with Site I (Phenylbutazone) and II (Ibuprofen) and
lidocaine Markers.
For the competitive phenylbutazone/ibuprofen/Lidocaine HSA experiment, the
concentrations of HSA and the site probe were maintained at a constant concentration of 1 μM,
whereas nefopam analogues were titrated with increasing concentrations of NF-1, 2, and three
from 1 μM -9 μM. The excitation wavelength for phenylbutazone, ibuprofen and Lidocaine
complexes with HSA was 285 nm (Yeggoni et al. 2014).
2.5 Circular Dichroism
Circular dichroism (CD) measurements were made on a Jasco J-810 spectropolarimeter
(JAPAN) using a quartz cell with a path length of 0.2 cm cell in a nitrogen atmosphere. The
spectra were recorded in the range of 190 to 300 nm, and the scan rate is 50 nm/min with a
response time of 4 s. All the parameters were followed according to our previous procedures
(Subramanyam et al. 2009; Subramanyam et al. 2009). The induced ellipticity of the protein
alone was defined as the ellipticity of the drug-HSA mixture minus the ellipticity of drug alone at
-6
the same wavelength. The concentration of HSA was 0.001mM (1x10 M) and the
concentrations of NF1, NF2 and NF3 were 0.001, 0.005, and 0.009mM, respectively. Each time
three scans were averaged and likewise three independent spectra were measured with identical
spectra. The relative contents of secondary structure; α-helix, β-pleated sheet, β-turn, and random
coils of HSA were calculated by using CDNN 2.1 software S Table 1). All the Spectra were
corrected for baseline with respective blanks for HSA control and NF’s-HSA complexes.
2.6 Molecular docking
Since the experimental evidence showed NF’s binding to HSA, we were interested in pinpointing
the probable binding mechanism and important interactions in atomic details in the binding site
via molecular docking. AutoDock program (Version AutoDock 4.2.3) was used to identify the
9

potential ligand binding sites (Morris et al. 2009). Lamarckian genetic algorithm (LGA)
implemented in AutoDock was applied to estimate the possible conformations of the ligand–
protein complex. HSA crystal structure used in the docking studies was obtained from the RCSB
Protein Data Bank (PDB ID: 1AO6). The receptor (HSA) and NF’S were pretreated. For the
ligand preparation, 3D structures of NF1, NF2, and NF3 were built from their 2D structures, and
their geometry was optimized using discovery studio 3.5 software according to our previous
work (Yeggoni et al. 2014; Gokara et al. 2015). For the recognition of all binding sites in HSA,
docking was performed by setting the grid box size at 126 Å 126 Å 126 Å, which was followed
by blind docking (Hetenyi & van der Spoel 2006). All the parameters were inserted at their
default settings. Among 30 conformations, the conformer with the lowest binding free energy
was also matched with experimental data for further analysis as reported earlier (Sudhamalla,
Gokara, Ahalawat, Amooru & Subramanyam 2010; Malleda, Ahalawat, Gokara &
Subramanyam 2012). Further to elucidate the affinity of nefopham analogues over other
analogues we have carried out docking with nefopham-aliphatic (NF-R1), Nefopham-bromide
(NF-R2), nefopham-cholride (NF-R3) (for structures see Figure S2.). These three analogues
where docked with the three binding sites i.e., IB, IIA and IIIA. The result with the lowest
docking energy analysis in cluster rank 1 was used for further analysis. For visualization of the
docked conformations, Pymol v 1.5 was used. All the parameters that are mentioned above were
according to our previous work (Sudhamalla et al. 2010; Yeggoni et al. 2014; Gokara et al.
2015).
2.7 Molecular dynamic simulations (MD Simulation)
The structure of NF’s-HSA complexes obtained from the docking procedure was used as starting
point for the MD simulation. A 10 ns MD simulation was carried out using GROMOS96 force-
10

field through the GROMACS4 software (Ver. 4.5, www.gromacs.org) (Berendsen, van der Spoel
& van Drunen 1995; van Gunsteren et al. 1996; Van Gunsteren, Daura & Mark 1998). The
topology parameters of HSA were created by using Gromacs program. The topology parameters
of NF1, NF2, and NF3 were built by the Dundee PRODRG2.5 server (Schuttelkopf & van
Aalten 2004). Then the complex was immersed in a cubic box (7.335×6.135×8.119nm) of
extended simple point charge water molecules (Berendsen, Postma, Van Gunsteren & Hermans
1981). All the parameters that are mentioned above were according to our previous work
(Sudhamalla et al. 2010; Yeggoni et al. 2014; Gokara et al. 2015). The MD simulation and
results analysis were performed on OSCAR Linux cluster with 16 nodes (dual xeon processor) at
Bioinformatics Facility, School of Life Sciences, and University of Hyderabad.
3. Results and discussion
To study the binding mechanism of Nefopam analogous with HSA, the fluorescence emission
spectra is an important tool shown in Figure 1. When the excitation wavelength is set at 285 nm,
HSA shows a strong emission band around 360 nm. The continuous addition of NFs, the
fluorescence maxima of HSA decreased gradually, which reveals NF’s quenched the intrinsic
fluorescence of HSA. Dynamic and static quenching mainly causes fluorescence quenching.
Thus, dynamic and static quenching can be distinguished by temperature and viscosity, or by the
difference in their fluorescence lifetime (Lakowicz 2006). Dynamic quenching is a process in
which the fluorophore and the quencher come into contact during the transient existence of the
excited state. Static quenching refers to fluorophore (HSA)-quencher (NF’s) complex formation.
To verify whether the quenching is static or dynamic in NF-HSA complexes, bimolecular
quenching constant were calculated from the slope of F0/F vs. [Q] (S-Figure 1), which are found
11

to be linear for NFs- HSA complexes indicating that the quenching is mainly static. The Kq was
estimated according to the Stern-Volmer equation:
ꢅꢆ
[ ]
= 1 + ꢇꢈꢉꢊ ꢋ = 1 + ꢌꢍ[ꢋ]
[2]
ꢅ
Where F and F are the fluorescence intensities in the absence and presence of quencher, [Q]
o
is the quencher concentration, and K is the Stern-Volmer quenching constant (K ), which can
D
q
be written as K =k t ; where k is the bimolecular quenching rate constant, and t is the
D q 0
q
0
lifetime of the fluorophore in the absence of quencher, lifetime of fluorophore for HSA been 5.6
ns (Tayeh, Rungassamy & Albani 2009).
The rate constant (Kq) of NF1, NF2 and NF3 were 5.5 × 1013, 5.4 × 1013 and 4 × 1013 M-1 S-
1, respectively. In general, the collisional quenching constant for various types of quencher with
biopolymer is 2.0 × 1010 L/mol/s (Zhang, Que, Pan & Guo 2008). From the analysis, it is
apparent that bimolecular quenching constants are much greater than this value, which implies
that there is non-fluorescent complex formation between the NFs and HSA. The static quenching
may be the main mechanism of the fluorescence quenching of HSA by NFs under our
experiment conditions
.
For static quenching, the binding constant (Ks) and the number of binding sites (n) can be
obtained from modified Stern–Volmer regression curves based on the following equation (Zsila,
Bikádi & Simonyi 2003; Min et al. 2004).


[3]
log F
0
− F ÷ F = log K
s
+ n×log Q
[ ]
(
)


Where n is the slope (i.e. the number of binding sites), Ks is the binding constant and [Q] is
the quencher concentration. The results showed that the number of Nefopam molecules binding
12

to HSA was about 0.87, 0.96 and 0.9 for NF1, NF2 and NF3 respectively, which indicates that
the binding of NFs was approximately one to one ratio for HSA (Figure 1, inserts). From the
intercept of the plot, the binding constants of NF1, NF2 and NF3 with HSA was calculated from
the intercept as 1.53 0.057 × 104, 2.16 0.071 ×104 and 3.6 0.102 ×105 M-1 for NF1, NF2 and
NF3, respectively (Figure 1), which indicates strong binding of these analogues with HSA. The
order of the binding constants for these analogues are NF3>NF2>NF1. The different binding
constants between these three analogues are due to the variation in the functional groups. This is
concurrence with our recent report which was synthesized molecules of different coumarin
derivatives with the various functional group attached behaved the similar kind of binding
(Archit et al. 2013; Yeggoni et al. 2014). Further, we have discussed about the role of functional
groups in docking results.
Site-specific marker analysis of HSA with Nefopam analogues.
Displacement experiments were employed with constant one μM concentration of HSA and for
the site-specific probes. The intensity of the HSA-site probe fluorescence was recorded with the
increasing concentrations of NF’s. Remarkably, fluorescence quenching of NF’-HSA complexes
was observed with titration of different concentrations of NF-1, NF-2, and NF-3 with respective
site markers, i.e., Ibuprofen, lidocaine, and phenylbutazone. The binding constant values
obtained for NF’s with HSA were found to be more than the HSA-site markers, which indicates
that there is competition between NF’s and site-specific markers (Table 1). Additionally, to make
sure that whether the NF’s have more than one binding site, we performed fluorescence with site
markers in different combinations; which showed insignificant fluorescence quenching/no
fluorescence quenching. With this experimental evidence, we can firmly assure that three NF’s
have only one specific binding site.
13

3.1 Free energy calculations
Fundamentally, there are four main types of non-covalent interactions involves the interactions
of ligands with proteins, including hydrogen bonding, van der Waals forces, hydrophobic and
electrostatic interactions (Qi et al. 2008). The signs and magnitudes of the thermodynamic
parameters for protein reactions can be accounted to identify the main forces contributing to
ligand–protein stability. Free energy can depict the type of interactions involved in the formation
of NF1, NF2 and NF3 and HSA-Nfs complexes. The free energy (ΔG0) of the binding was
calculated from the following equation:
ΔG° = −RT ln K
[4]
Where, ΔG is free energy, K is the binding constant at the corresponding temperature, which
can be obtained from fluorescence data, R is the gas constant and T is absolute temperature.
The standard free energies were calculated to be approximately -5.48, -5.7 and -7.50
kcal/mol for NF1, NF2 and NF3 respectively at 25 oC. These results indicate that HSA-NF3
complex is more stable than the HSA-NF2 and HSA-NF1. Docking experiments revealed that
the mode of binding between NF1, NF2 and NF3 and HSA were located in three different
domains. Even though the backbone of these NFs is the same but the variation in the binding is
due to the differences in the functional groups. The small change in the functional group would
obvious change of the acceptability of the drug and their binding. Our recent studies showed
such kind of interactions played an important role in the binding of biologically significant drugs
in the formation of HSA-Drug complexes (Gokara et al. 2014; Yeggoni et al. 2014; Gokara et al.
2015; Yeggoni et al. 2015).
14

3.2 Secondary Structure analysis: Studied by Circular Dichroism
To decipher the secondary structural changes of HSA caused by its interaction with NF’s, we
have carried out CD experiment of HSA in the presence of different concentrations of NF’s. CD
spectroscopy is one of the best tools to monitor the structure, conformation, and stability of
proteins in solution. It can provide us with a very good estimation of the fraction of the residues
in the protein structures which are involved in α -helical, β -sheet, turn, and random coil and
possibly their inter-conversions. The CD spectrum of native HSA in far-UV region has two
negative bands in the region of 208 and 222 nm (Li et al. 2005; Yeggoni et al. 2014), which is
characteristic of α-helix. When various concentrations of NF1, NF2 and NF3 were added to free
HSA the intensity at 208 and 222 nm decreased in a concentration-dependent manner (Figure 2).
The decrease of α-helix, increasing order of β-helix and random coils follows as NF3>NF2>NF1
(Figure 2, Figure 3 and S Table 1). Conformational analysis revealed that α-helix of HSA while
binding with three analogues were decreased, while consequent increase (in %) in the β-sheets
and random coils, NF1: α-helix; 57.2 2.5 to 47 2.5: β-sheets; 25 0.8 to 30.8 1: Random Coil;
17.8 1.0 to 22.2 1.2. NF2: α-helix; 57.2 2.5 to 41.7 1.82: β-sheets; 25 0.9 to 32.4 1.02:
Random coil; 17.9 1.8 to 25.9 2.6.NF3: α-helix; 57.1 2.5 to 36.8 1.6: β-sheets; 25 0.88 to
34.1 1.07: Random coil; 17.9 1.82 to 29.1 2.94. The decrease in the α-helix accompanied with
the increase in the β-sheets and random coils indicated the partial unfolding of HSA with NFs
analogues, suggesting the formation of NFs-HSA complexes. The difference in the
conformational changes of NF’s is attributed to their structural complexity, which is in the order
of NF3>NF2>NF1 and this might be the reason for alteration of protein conformation. This is
very well supported by fluorescence studies where the order of binding affinity is
(NF3>NF2>NF1) suggesting that with an increase in binding affinity leads to the decrease in the
15

secondary structure of the protein. The differences in α-helical contents can be attributed to the
different structural arrangements of the protein in solid state and in aqueous solution. Similar
conformational transitions from α-helix to β-sheet structure were observed for protein unfolding
(Li et al. 2005). Thus, upon binding of NFs to HSA, the secondary structure appears to be
partially unfolded due to change in microenvironment of the protein. Similar studies were
reported by our group that upon binding of different drug molecules to HSA lead to the change
in protein conformation (Sudhamalla et al. 2010; Yeggoni et al. 2014; Gokara et al. 2015;
Yeggoni et al. 2015).
3.3 Molecular Docking Studies
Molecular docking was employed to understand the interaction NF’s with specific binding sites
on HSA. The binding location of NF’s with HSA molecule is of crucial importance. Since the
knowledge of the binding location within the protein environment of a biologically active drug
molecule is a crucial prerequisite for understanding the efficacy and also its function as a
therapeutic agent. The docking results indicated that among the NF’s, NF1 is bound to the
Sudlow site 2 i.e., IIIA (Figure 4A, 4B and 4C) surrounded by Val 409, Arg 410, Thr 540, Leu
544, Glu 542, Leu 529, Lys 545, Ala 406, Asn 405; NF2 is bound to IB (Figure 4D, 4E and 4F)
Met 125, Pro 118, Arg 117, Val 116, Phe 134, Leu 182, Leu 115, Tyr 161, Lys 137, Tyr 138, Glu
141, Ile 142, His 146 and NF3 is bound to Sudlow site 1, i.e., IIA (Figure 4G, 4H and 4I) Glu
208, Arg 209, Lys 212, Ala 213, Val 216, Asp 324, Val 325, Phe 228, Leu 327, Gly 328, Glu
354, Leu 331, Ala 350. It is observed that among the three analogues only NF1 has two hydrogen
bonds (Figure 4B) with Lys 413 with the bond length of 2.1 Å and 2.6 Å, whereas NF2 and NF3
are stabilized completely by hydrophobic interactions (Figure 4E and Figure 4H). Furthermore,
16

the binding of NF2 with non-conventional site, i.e., Subdomain IB might lead to the exposure of
hydrophobic cavity of Subdomain IIA, which is in perfect agreement with the fluorescence of
NF2-HSA, which showed the red shift of NF2-HSA fluorescence. Although there are no studies
reported with nefopam, there are other studies which showed that non-opioid analgesics like
phenylbutazone, indomethacin bind strongly with HSA with binding constants of 7.0 × 105 and
1.4 × 106 M-1, respectively (Varshney et al. 2010). The calculated binding energies of NF1, NF2,
and NF3 are -5.66, -5.90, -7.57 Kcal M-1, respectively. These values are close and corroborate
with the experimental values suggesting that these analogues are strong enough to bind with
HSA, which is important for proper disposition and therapeutic action.
For further validation to understand the functional group role in binding, we performed
docking with HSA to NF-R1(CH2 )5, NF-R2 (Br) and NF-R3 (Cl). The analysis of docking results
(See S Table 2) showed that nefopham analogues are having higher affinity and free energies
compared with NF-R1, NF-R2 and NF-R3. Despite of binding within the same proximity, NF
with different R groups showed less binding, which clearly suggests that functional groups of
NF1, NF2 and NF3 plays a central role in forming stable complexes with HSA with more
binding and free energies. Thus, other than NFs analogous the additionally added R groups like
NF-R1(CH2 )5, NF-R2 (Br) and NF-R3 (Cl) are binding with different amino acids in same
binding pocket (See Figure S2 to 5). Hence, the docking results of NF-R1(CH2)5, NF-R2 (Br)
and NF-R3 (Cl) are having weaker binding when compare to the proposed NFs analogous,
suggested that NFs analogues have good binding activity.
17

3.4 Molecular dynamics simulations
The NF’s-HSA complexes were further analyzed by MD simulations to evaluate the reliability of
the docking results and the stability of the complexes. Complexes of NF analogues and HSA
were simulated for ten ns using the GROMACS MD package as discussed in materials and
methods.
The root mean square deviation (RMSD) of the trajectories from their initial structures was
used to examine whether the structure of the NFs- HSA complex remained stable or not under
the simulation conditions (Figure 5). As seen in Figure 5 during the MD simulations the RMSD
reached a plateau after the first 2.5 ns. After that the deviations in RMSD (nm) are nearly equal
for HSA alone and NFs-HSA complexes, with a subsequent increase up to the start of the Cα-
backbone of the HSA, and NFs-HSA analogues are stabilized at approximately 2.5 ns. Further, it
remained stable without any drastic fluctuations till the end of the simulation indicating that the
molecular system well behaved after that. Interestingly the fluctuations of RMSD are in perfect
agreement with the CD data. Order of fluctuations with respect to Cα-backbone of HSA are in
the order of NF2>NF3>NF1, compared to CD studies as it is in the order of NF3>NF2>NF1.
The difference might be due to the binding of NF2 in the different domain, i.e., IB; it is believed
that the drugs bind to subdomain IIA and IIIA. However, recently it was reported that IB is also
involved in the binding (Zsila 2013). The possible explanation can be drawn from the
fluorescence graph (see Figure 1B), where there is a red shift in the emission maximum of NF2-
HSA complexes. Indicating that binding of NF2 induced conformational changes globally (i.e.,
Cα-backbone) but not strong enough to induce a conformational change in secondary structural
elements (α-helix, β-sheets, and random coils). This is in perfect agreement with Rg data (Figure.
6B).
18

Rg gives the compactness/structural integrity of the HSA values against time, and it describes
the overall spread of the molecule (HSA). It is defined as the root-mean-square distance of the
collection of atoms from their common center of gravity. In the present MD studies, we
determined the Rg values of free HSA and NFs-HSA complexes as shown in Figure 6, in which
it is observed that the compactness of NF1-HSA is almost the same as HSA. Whereas with NF2-
HSA and NF3-HSA the peaks at around 4.5 – 6.5 ns are deviated about 1.5 0.25 Å with respect
to HSA.
To evaluate the NF’s binding site and its reliability with HSA, we analyzed root mean square
fluctuation (RMSF). These studies indicated that IIIA, IB, and IIA are rigid with respect to the
remaining domains of NFs-HSA complexes (Figure 7). This is consistent with docking studies
suggesting that NF1, NF2, and NF3 specifically bind with IIIA, IB and IIA, respectively.
As mentioned earlier that the differential binding could be various functional molecules present
in the NF’s. These MD simulations support that the HSA binding sites IIIA, IB and IIA
specifically interact with NFs through conformational changes which allow to modify several
molecular properties of HSA at different times (ns) or even at the same time without any
significant loss of local conformations of HSA. Previously we have reported similar kind of
work which showed MD simulations are consistent with the experimental data (Sudhamalla et al.
2010; Malleda et al. 2012; Gokara et al. 2014; Yeggoni et al. 2014; Gokara et al. 2015).
Furthermore, this supports the notion that the HSA protein is ligand promiscuous, as it shows
several binding sites at different time points within movements that conserve its 3D structure.
4. Conclusion
In conclusion, interaction and secondary structural changes of HSA with NF-analogues were
analyzed using fluorescence and CD spectroscopy, molecular docking and molecular dynamics
19

techniques. The results revealed that NF-analogues quenched HSA fluorescence via static
quenching. The affinity of NF analogues–HSA was diverse for three analogues which are in the
descending order as NF3>NF2>NF1. The negative values of ΔG indicated that the binding
processes were spontaneous; hydrophobic interactions were considered to be the main binding
force between NF analogues and HSA except NF1, which has one hydrogen bond.
Conformational analysis revealed that α-helix of HSA with three analogues decreased with
consequent increase in the β-sheets and random coils. The structural perturbation of HSA
indicates NF’s complexation has minimal effect on the secondary structure of HSA which is
further supported by molecular simulations, from which it can be presumed that the inherent
function of HSA wasn’t affected. Binding site analysis by docking study validated that among
NF analogues NF1, NF2 and NF3 bound to IIIA, IB, and IIA binding sites respectively.
Molecular simulations studies revealed that RMSD fluctuations remained the same after 2.5 ns
without any fluctuations for three analogues, whereas Rg indicated that compactness of NF1-
HSA is almost the same as HSA, whereas with NF2-HSA and NF3-HSA the peaks at around 4.5
– 6.5 ns deviate about 1.5 0.25 Å with respect to HSA. RMSF indicated that NF1, NF2, and
NF3 are specifically binding with IIIA, IB and IIA, respectively which reflects that the
compactness of HSA remains rigid after the binding. Hence, the interaction between HSA and
NF-analogues will be an important factor in our understanding of the pharmacokinetics and
pharmacological effects of NF analogues.
ACKNOWLEDGMENTS
We thank CIL, and BIF for CD and computational facility, University of Hyderabad, India. We thank
UPE-I and II, UGC, UGC-DSKPDF India for financial support. We dedicate this article for our coauthor
Mr. Chandramouli Malleda who passed away during the preparation of this manuscript.
20

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Scheme 1. Structure of Nefopam Analogues
1. 6-(phenyl)-7-methyl-9 ethyl 5, 6-dihydro-2H- benzo[b] [1, 4] oxazocine-8-carboxylic acid
ethyl ester Nefopam analogue 1 (NF1). 2. 9-(4-chloro phenyl)-6-(4-hydroxy phenyl)-7-methyl-5,
6-dihydro-2H-benzo[b] [1, 4] oxazocine-8-carboxylic acid ethyl ester Nefopam analogue 2
(NF2). (NF2). 3. 6-(4-hydroxy phenyl)-7-methyl-9-propyl-5, 6-dihydro-2H benzo[b] [1, 4]
oxazocine-8-carboxylic acid ethyl ester Nefopam analogue 3 (NF3).
Figure 1.Fluorescence emission spectra of Nefopam analogues with HSA in 0.1M phosphate
buffer pH 7.2, λex = 285 nm. λem = 362 nm. (A) Free HSA (0.001 mM) and free HSA with
different concentrations of NF1 (0.001-0.009 mM), (A1)Modified Stern-Volmer plot HSA-NF1
complex (B) Free HSA (0.001 mM) and free HSA with different concentrations of NF2 (0.001-
0.009 mM), (B2) Modified Stern-Volmer plot HSA-NF2 complex (C) Free HSA (0.001 mM)
and free HSA with different concentrations of NF3 (0.001-0.009 mM), (C1)Modified Stern-
Volmer plot HSA-NF3 complex
Figure 2.CD spectra of the free HSA and Nefopam analogues with HSA. (A) HSA and HSA-
NF1 complexes in aqueous solution with an HSA concentration of 0.001 mM and NF1
concentrations were 0.001, 0.005 and 0.009 mM (B) HSA and HSA-NF2 complexes in aqueous
solution with an HSA concentration of 0.001 mM and NF2 concentrations were 0.001, 0.005 and
0.009 mM. (C) HSA and HSA-NF3 complexes in aqueous solution with an HSA concentration
of 0.001 mM and NF3 concentrations were 0.001, 0.005 and 0.009 mM.
Figure 3.Spectra of Secondary Structure (Percentage %) free HSA and Nefopam analogues with
HSA; with an HSA concentration of 0.001 mM and NF’s concentrations were 0.001, 0.005 and
0.009 mM.
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Figure 4. Docking conformation of N1-HSA complex obtained from Autodock v 4.2. (A) N1
bound to IIIA domain on HSA (Protein represented in ribbon model and ligand represented in
stick model) (B) Stereo view of N1 bound to subdomain IIIA on HSA (prepared by using pymol
v 1.5) in which N1 is rendered as capped sticks and surrounding residues as lines (2 hydrogen
bonds with LYS 413) (C) The hydrophobic pocket of IIIA domain surrounding the N1. (D) N2
bound to IB domain on HSA (Protein represented in ribbon model and ligand represented in stick
model) (E) Stereo view of N2 bound to subdomain IB on HSA (prepared by using pymol v 1.5)
in which N2 is rendered as capped sticks and surrounding residues as lines (F) The hydrophobic
pocket of IB domain surrounding the N2.(G) N3 bound to IIA domain on HSA (Protein
represented in ribbon model and ligand represented in stick model) (H) Stereo view of N3 bound
to subdomain IIA on HSA (prepared by using pymol v 1.5) in which N3 is rendered as capped
sticks and surrounding residues as lines (I) The hydrophobic pocket of IIA domain surrounding
the N3.
Figure 5. RMSD vs Time (ns) plot of Nefopam analogues with has (A) Plot of RMSD of C–Cα–
N backbone vs simulation for solvated HSA and HSA + NF1 complexes during 10 ns. (B) Plot
of RMSD of C–Cα–N backbone vs simulation for solvated HSA and HSA + NF2 complexes
during 10 ns (C) Plot of RMSD of C–Cα–N backbone vs simulation for solvated HSA and HSA
+ NF3 complexes during 10 ns.
Figure 6. Rg vs Time (ns) Plot of Nefopam analogues with HSA (A) Rg values during 10ns of
MD simulation of HSA and NF1 complex. (B) Rg values during 10 ns of MD simulation of HSA
and NF2 complex. (C) Rg values during 10 ns of MD simulation of HSA and NF3 complex.
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Figure 7. RMSF vs Residue number over 10 (ns), Plot of Nefopam analogues with HSA (A)
RMSF values during 10ns of MD simulation of HSA and NF1 complex. (B) RMSF values during
10 ns of MD simulation of HSA and NF2 complex. (C) RMSF values during 10 ns of MD
simulation of HSA and NF3 complex.
29