Luminescence, circular dichroism and in silico studies of binding interaction of synthesized naphthylchalcone derivatives with bovine serum albumin

Article abstract of DOI:10.1002/bio.3319

Chalcones possess various biological properties, for example, antimicrobial, anti-inflammatory, analgesic, antimalarial, anticancer, antiprotozoal and antitubercular activity. In this study, naphthylchalcone derivatives were synthesized and characterized

Full text of DOI:10.1002/bio.3319

Received: 8 August 2016
DOI: 10.1002/bio.3319
Revised: 7 December 2016
Accepted: 22 February 2017
R E S E A R C H A R T I C L E
Luminescence, circular dichroism and in silico studies of binding
interaction of synthesized naphthylchalcone derivatives with
bovine serum albumin
Sharda Pasricha1 Deepti Sharma2 Himanshu Ojha2
Pragya Gahlot1
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Mallika Pathak3 Mitra Basu2 Raman Chawla2 Sugandha Singhal2 Anju Singh4
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Rajeev Goel2 Shrikant Kukreti4 Shefali Shukla1
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1 Department of Chemistry, Sri Venkateswara
Abstract
College, University of Delhi, Delhi, India
2 Division of CBRN Defence, Institute of
Nuclear Medicine and Allied Sciences, Delhi,
India
Chalcones possess various biological properties, for example, antimicrobial, anti‐inflammatory,
analgesic, antimalarial, anticancer, antiprotozoal and antitubercular activity. In this study,
naphthylchalcone derivatives were synthesized and characterized using 1H NMR 13C NMR, Fou-
rier transform infrared and mass techniques. Yields for all derivatives were found to be >90%.
Protein–drug interactions influence the absorption, distribution, metabolism and excretion
(ADME) properties of a drug. Therefore, to establish whether the synthesized naphthylchalcone
derivatives can be used as drugs, their binding interaction toward a serum protein (bovine serum
albumin) was investigated using fluorescence, circular dichroism and molecular docking tech-
niques under physiological conditions. Fluorescence quenching of the protein in the presence
of naphthylchalcone derivatives, and other derived parameters such as association constants,
number of binding sites and static quenching involving confirmed non‐covalent binding interac-
tions in the protein–ligand complex were observed. Circular dichroism clearly showed changes
in the secondary structure of the protein in the presence of naphthylchalcones, indicating binding
between the derivatives and the serum protein. Molecular modelling further confirmed the bind-
ing mode of naphthylchalcone derivatives in bovine serum albumin. A site‐specific molecular
docking study of naphthylchalcone derivatives with serum albumin showed that binding took
place primarily in the aromatic low helix and then in subdomain II. The dominance of hydrophobic,
hydrophilic and hydrogen bonding was clearly visible and was responsible for stabilization of the
complex.
3 Department of Chemistry, University of
Delhi, Delhi, India
4 Nucleic Acid Research Laboratory,
Department of Chemistry, University of Delhi,
Delhi, India
Correspondence
Himanshu Ojha, Scientist D, Division of CBRN
Defence, Institute of Nuclear Medicine and
Allied Sciences, Timarpur, Delhi 110054, India.
Email: himanshu.drdo@gmail.com
Funding Information
Defence Research and Development Research
KEYWORDS
bovine serum albumin, circular dichroism, docking, luminescence, quenching
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1
INTRODUCTION
isoniazid are used in the treatment of tuberculosis. However, Mycobac-
terium tuberculi has developed resistance to these drugs over time;
According to the World Health Organization Tuberculosis Report
2015, in 2014, there were 9.6 million cases of tuberculosis worldwide,
23% of which were in India. Currently, streptomycin, rifampicin and
therefore, there is an urgent need to search for new potential antitu-
bercular agents.[1]
Chalcones are well‐known intermediates in the synthesis of vari-
ous heterocyclic compounds. The chalcones have been reported to
possess various biological properties, such as antimicrobial, anti‐
inflammatory, analgesic, vasorelaxant, antimalarial, anticancer,
antiprotozoal, antioxidant, antihyperglycaemic, inhibition of chemical
mediator release (e.g. interleukin‐5, leukotriene B4, tyrosinase and
aldose reductase), and antitubercular activities.[2]
Abbreviations used: ADME, Absorption, distribution, metabolism and excretion;
Arg, Arginine; Asp, Aspartic acid; BSA, Bovine serum albumin; 2D, Two‐
dimensional; CD, Circular dichroism; Cys, Cysteine; Glu, Glutamic acid; FTIR,
Fourier transform infrared; Leu, Leucine; Lys, Lysine; NMR, Nuclear magnetic
resonance spectroscopy; Phe, Phenylalanine; Pro, Proline; Trp, Tryptophan;
UV, Ultraviolet; Val, Valine; Vis, Visible
Luminescence. 2017;1–11.
wileyonlinelibrary.com/journal/bio
Copyright © 2017 John Wiley & Sons, Ltd.
1

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PASRICHA ET AL.
Various chalcones with naphthalene, pyrrole, piperizine,
and hexane (≥ 99%) were purchased from Sigma Aldrich (USA). Potas-
sium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phos-
phate (K2HPO4) and silica precoated alumina plates (200 μm thick)
were purchased from E‐Merck (Germany) and labelled actual mass
fraction purities were >99%. HCl was from Thermo Fischer Scientific
(India). The water used to prepare solutions was 18 MΩ MQ grade
derived from a Millipore water system (model Elix 3, Millipore Corp.,
USA). BSA protein stock solution was prepared in 10 mM phosphate
buffer (pH 7.4) and the protein solution was dialyzed overnight using
dialysis cellulose membrane against the same phosphate‐buffered
saline at 277 K. Its concentration was determined using UV/vis absorp-
tion measurement, using the extinction coefficient of ε280nm
6.60 ꢀ 0.02. Stock solutions (1 mM) of all naphthylchalcone deriva-
tives were prepared in methanol.
benzoazepine, morpholine and similar heterocyclic rings have been
reported to have antitubercular activity. Natural‐derived chalcone
licochalcone 1, obtained from Glycyrrhiza inflate, has shown antibacte-
rial activity against M. tuberculosis, M. avium and M. bovis.[3] Lipophilic
chalcones and their conformationally restricted styrenylchromanones
have strong antitubercular potential.[4] Some triazole methyl chalcones
are known to show >80% inhibition of recombinant M. aurum bacte-
ria.[5] Currently, a new series of naphthylchalcones have been reported
as inhibitors of M. tuberculosis, targeting protein tyrosine phosphatases
as more effective antitubercular agents.[6] Based on Scheme 1, some
naphthylchalcones were synthesized and characterized using nuclear
magnetic resonance (1H NMR and 13C NMR), Fourier transform infra-
red (FTIR) and mass techniques.
Serum albumin is the most abundant protein in the circulatory sys-
tem and also the most widely studied. It has a wide range of physiolog-
ical functions involving the binding, transport and deposition of many
endogenous and exogenous ligands present in blood. Bovine serum
albumin (BSA) has 583 amino acid residues in a single polypeptide
chain cross‐linked with 17 disulfide bonds. It has three homologous
domains (I–III), each comprising of two subdomains (A and B).[7] The
two tryptophan (Trp) residues of BSA contribute significantly to its
intrinsic fluorescence; Trp134 is located in domain IB and Trp212 is
buried in a hydrophobic pocket in domain IIA.[8]
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2.2
Synthesis of naphthylchalcone derivatives
A series of four chalcones (Table 1).were synthesized by Aldol
condensation between acetophenone (1.0 mM) and 1‐naphthaldehyde
or 2‐naphthaldehyde (1.0 mM) in methanol and KOH (50% w/v) at
room temperature with magnetic agitation for 24 h; the volume of
KOH varied according to the reaction. On completion of the reaction,
the reaction mixture was cooled on ice and then acidified with 10%
HCl. The crude product was obtained by vacuum filtration and
recrystallized from alcohol (Figure 1).[4] The purity of the sample was
analysed using thin layer chromatography using 200‐μm thick Merck
silica precoated alumina plates, with solvent systems of different
polarities. The desired products were obtained with an average yield
of >90% after purification. Their structures were established using
FTIR, NMR and mass spectrometry. 1H NMR spectra showed that only
E‐chalcones were obtained. Other spectral data for the synthesized
compounds are presented in Figures S1–S3. The general structure of
naphthylchalcones is shown in Figure 2 and their substitution patterns
are given in Table 1.
It is well accepted that many drugs bind to serum albumin, and
their bioavailability and efficacy depend on their binding ability.[9]
Hence, the interaction between ligand and serum albumin is important
in understanding drug bioavailability and efficacy. In this study, the
interaction between naphthylchalcone derivatives and BSA was inves-
tigated using fluorescence spectroscopic techniques, circular dichroism
(CD) and molecular docking under physiological conditions. Fluores-
cence quenching is often used to monitor molecular interactions and
to study the mechanism of binding between drugs and plasma proteins
because of its high sensitivity, reproducibility and relative ease of
use.[10] The binding constants and number of binding sites were deter-
mined from the double logarithm equation. Molecular docking
revealed the nature of the binding sites, as well as the prevalence of
different types of interactions involved in binding. Furthermore, an
absorption, distribution, metabolism and excretion (ADME) study was
performed to confirm the suitability of naphthyl derivatives for use
as drugs by analysing their physiochemical properties and applying
Lipinski's rule of five.
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2.3
Fluorescence measurements
Fluorescence steady‐state measurements were performed on a spec-
trofluorimeter (FS920, Edinburgh Instruments, UK) with a standard
3.5 ml quartz cell. The experiment was conducted at 298 K. The exci-
tation source was a xenon lamp (450 W) and the sample chamber was
equipped with a Peltier Accessory. Scanning parameters for all mea-
surements were optimized with a slit width of 3.0 nm for excitation
and emission, a dwell time of 0.2 s and a wavelength step of 0.5 nm.
The protein concentration was kept at 7.5 μM and the ligand concen-
trations were varied from 0 to 32.25 mM for titration. The excitation
wavelength was set at 295 nm to selectively excite the Trp residues
of BSA. The emission spectra were recorded over a wavelength range
of 310–450 nm at a scan rate of 100 nm min−1 in the absence and
presence of naphthylchalcone derivatives.
This work will further help in the design and synthesis of new
improved chalcone‐based derivatives with better inhibitory and phar-
macological actions.
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2
EXPERIMENTAL
Materials
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2.1
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2.4
CD measurements
BSA (Fraction V, ~ 99%; protease free and essentially γ‐globulin free),
acetophenone (≥ 98% purity), 1‐naphthaldehyde (≥ 95% purity), 2‐
naphthaldehyde (≥ 98% purity), methanol (≥ 99.9% purity), KOH (≥
85% purity), ferric chloride (≥ 99.9% pure), diethyl ether (≥ 99.5% pure)
Alterations in the secondary and tertiary structure of the protein in the
presence of BSA were studied on a CD spectropolarimeter (J815, Jasco,
Japan) equipped with a Peltier Accessory. The spectropolarimeter was

PASRICHA ET AL.
3
TABLE 1 Substitution patterns of the naphthylchalcone derivatives
Compound
IUPAC name
R1
R2
R3
R4
R5
1a
1c
1d
2a
1‐(2,5‐dimethoxy‐phenyl)‐3‐naphthalen‐1‐yl‐propenone
1‐(3‐bromo‐phenyl)‐3‐naphthalene‐1‐yl‐propenone
1‐(2_hydroxy‐phenyl)‐3‐naphthalene‐1‐yl‐propenone
1‐(2,5‐dimethoxy‐phenyl)‐3‐naphthalene‐2‐yl‐propenone
OCH3
H
H
H
H
H
H
H
H
H
OCH3
H
Br
H
H
H
H
H
OH
OCH3
The molecular structure of naphthylchalcone derivatives in the ground
state was optimized using Becke's three‐parameter exchange
functional (B3), which is a linear combination of Hartree Fock, local
and gradient‐corrected exchange terms. The B3 hybrid functional
was used with the LYP correction (B3LYP), which is the correlation
functional of Lee, Yang and Parr. The basis set used for calculations
was the Pople's polarization 6‐311G ++(d,p) basis.[11]
The protein structure was subjected to refinement using the pro-
tein preparation wizard of the Schrödinger suite in which hydrogen
bonds and missing side chains in the protein structure were optimized.
Co‐crystallized water compounds beyond 5 Å of the active site were
removed and hydrogen atoms were added to the structure by applying
an all‐atom force field. Protein structure was further refined using a
restrained molecular mechanics calculation with the OPLS_2005 force
field to remove potential steric clashes. The energy minimization of the
structure was performed until the average root mean square deviation
of the non‐hydrogen atoms reached 0.3.
FIGURE 1 Synthesis of naphthylchalcones (Scheme 1)
FIGURE 2 General structure of naphthylchalcone
The receptor grid was generated by using a receptor grid genera-
tion program. After import of optimized structure of ligands and gener-
ation of the grid on the prepared protein, the docking calculations were
performed.
sufficiently purged with 99.9% dry nitrogen before measurement. For
CD measurements, the protein concentration and path lengths used
were 1 μM and 0.1 cm, respectively. The spectra were collected at a
scan rate of 50 nm min−1, and a response time of 1 s. Each spectrum
was baseline corrected, and the final plot was taken as an average
of three accumulated plots in the range of 200–300 nm. The con-
centration of all naphthylchalcone derivatives was kept at 40 μM.
Molar ellipticity [θ] was calculated from the observed ellipticity Θ
as given in equation 1:
Glide provides three different level of docking precision (HTVS,
high‐throughput virtual screening; SP, standard precision; and XP,
extra precision). In this study, the XP mode of the docking program
was selected for docking of different ligands with the target
protein.[12,13]
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2.6
ADME screening
The QikProp program from Schrödinger Maestro 9.2[14] was used to
obtain the ADME properties of the analogues. It predicts both physi-
cally significant descriptors and pharmaceutically relevant properties.
The program was processed in the normal mode, and 44 proper-
ties were predicted for the seven naphthylchalcone derivatives. These
properties consisted of principal descriptors and physiochemical prop-
erties with detailed analysis of the predicted octanol/water partition
coefficient (QlogPo/w), octanol/gas partition coefficient (QPlogPoct),
water/gas partition coefficient (QlogPw), polarizability in cubic Å
(QPolrz), % human absorption in the intestines (QP%), brain/blood
partition coefficient (QPlogBB), IC50 value for blockage of HERG K+
channels (logHERG), skin permeability (QPlogKp), binding to human
serum albumin (QPlogKhsa), apparent Caco‐2 cell permeability in
mm/s (QPPCaco), and apparent MDCK cell permeability in mm/s
(QPPMDCK). Caco‐2 cells are a model for the gut–blood barrier,
whereas MDCK cells are considered to be good mimics for the
blood–brain barrier. It also evaluates the acceptability of the analogues
based on Lipinski's rule of five,[15] which is essential for rational drug
½Θꢁ ¼ 100 x ðθ=c×lÞ
(1)
where c is the concentration of the protein in μM and l is the path
length of the cell (cm). Changes in [Θ] of each protein sample were
measured at 298 K.
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2.5
Molecular docking
Molecular docking was performed by using Glide module suit
Schrödinger Maestro 9.2. Glide (Grid‐based ligand docking with ener-
getic) is designed to assist in the screening of potential ligands based
on binding mode and affinity for a given receptor.
The crystal structure of BSA (PDB ID: 4JK4) was obtained from
the PDB database (www.rcsb.org) and the ligand structures were
drawn using ChemDraw and geometry optimized using Gaussian
software.
Studies were carried out at the DFT level with Gauss View 5.0
molecular visualization program and Gaussian 2009 program package.

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PASRICHA ET AL.
design. Poor absorption or permeation is more likely when a ligand vio-
lates Lipinski's rule of five, i.e. has more than five hydrogen donors, the
molecular weight is >500, logP is >5 and the sum of N and O molecules
is >10.
Anal. calcd for C19H14O2 (274.31) calc. C: 83.19; H 5.1; O: 11.66;
found C: 83.78; H: 5.17.
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3.1.3
Compound 3: 1‐(2‐hydroxy‐phenyl)‐3‐napthalene‐
1‐yl‐propenone
Yellow solid, mp 106–108°C. 1H NMR (CDCl3, δ in ppm) 6.97 (dd, 1H,
J = 7.4 Hz, H5′), 7.06 (d, 1H, J = 8.4 Hz, H3′), 7.51–7.62 (m, 4H, H3,
H6, H7, H4′), 7.76 (d, 1H, J = 8.4 Hz, H3′), 7.90–7.99 (m, 4H, H6′,
H4, H5, H8), 8.29 (d, 1H, J = 8.4 Hz, H2), 8.79 (d, 1H, J = 15.2 Hz,
Hb), 12.88 (S, 1H, OH), IR νmax cm−1 1635 (C = O), 1576 (C = C),
1203, 1015 (C–O), 3047, 3451, 1351, 1435, 972, 1162, 760 (Ar)
(KBr).13C NMR (CDCl3, δ ppm) d 1183.93 (C3′), 119.17 (C5′), 122.99
(Ca), 123.67 (C3), 125.59 (C8), 125.68 (C1′), 126.67 (C3), 127.41
(C6), 129.07 (C4, C5), 130.00 (C9, C6′), 131.48 (C10), 136.74 (C1,
C4′), 142.66 (Cb), 163.93 (C2′), 194.12 (C = O). Anal. calcd for
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3
RESULTS AND DISCUSSION
Chemistry
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3.1
Purified naphthylchalcone derivatives were obtained in yields of >90%.
Their structures were identified using FTIR, NMR and elementary
analyses. Melting points were determined with a Microquimica MG
APF‐301 apparatus and are uncorrected. Infrared (IR) spectra were
recorded with a FT Perkin Elmer 16 PC spectrometer on KBr disks.
NMR (1H and 13C NMR) spectra were recorded on a Brucker Ac‐
200 F (200 MHz) with tetramethylsilane as an internal standard. Ele-
mentary analyses were obtained on a Perkin Elmer 2400. Percentages
of C and H were in agreement with the product formula (within ꢀ0.4%
of theoretical values). The purity of the synthesized substances was
analysed using thin‐layer chromatography with 200‐μm thick Merck
silica precoated aluminium plates with several solvent systems of dif-
ferent polarities. Compounds were visualized with UV light (254 nm),
using ferric chloride solution followed by heat as the developing agent,
and purified by recrystallization from diethyl ether and hexane. 1H
NMR spectra revealed that all the naphthylchalcone derivatives were
geometrically pure and configured E (JHa–Hb = 15–16 Hz).[16]
C19H13OBr (337.21); calc. C: 67.67; H: 3.88; O: 4.74; Br: 23.73; found
C: 67.58; H: 3.80.
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3.1.4
Compound 4: 2E‐1‐(2′,5′‐dimethoxyphenyl)‐3‐(2‐
napthyl)‐2‐propen‐1‐one
Light yellow solid, mp 120–121°C. 1H NMR (CDCl3, δ in ppm) d 3.83
(s, 3H, OCH3), 3.89 (s, 3H, m‐OCH3), 6.97 (d, 1H J = 8.0 Hz, H4′),
7.06 (d, J = 8.0 Hz, 1H, H3′), 7.23 (s, 1H, H6′), 7.51–7.53 (m, 2H, H6,
H7), 7.53 (d, 1H, J = 16.0 Hz, Ha), 7.75 (d, 1H, J = 8.0 Hz, H3), 7.81
(d, 1H, J = 16.0 Hz, HB), 7.83–7.88 (M, 3H, H4, H5, H8), 7.99 (S, 1H,
H1). IR νmax cm−1 1644 (C = O), 1570 (C = C), 1336, 1130 (C–O),
3012, 2946, 2837, 1508, 1227, 1005, 693(Ar) (KBr). 13C NMR (CDCl3,
δ ppm) 55.89 (OCH3), 56.56 (m‐OCH3), 113.42 (C3′), 109.77 (C6′),
114.40 (C4′), 123.76 (Ca), 119.17 (C1′) 132.66 (C10), 126.68 (C3),
133.36 (C9), 127.78 (C5), 130.51 (C4), 128.63 (C8), 127.07–127.23
(C6, C7), 134.27 (C2), 143.46 (Cb), 152.58–153.64 (C2′, C5′),
192.48(C = O). Anal. calcd for C21H18O3 (318.36); calc. C: 79.22; H: 5.69;
O: 15.07; found C: 79.14; H: 6.00.
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3.1.1
Compound 1: (2E)‐1‐(2′,5′‐dimethoxyphenyl)‐3‐(1‐
napthyl)‐2‐propen‐1‐one
Yellow solid, mp 89–90°C. 1H NMR (CDCl3, δ in ppm): 3.85 (s, 3H,
OCH3), 3.77 (s, 3H, OCH3), 7.14 (d, 1H J = 8.2 Hz, H4′), 7.15 (dd,
J = 8 Hz, 1H, H4), 7.55–7.65 (m, 5H, H3, H5, H6, H7, H8), 7.99 (d,
1H, J = 7.2 Hz, H3′), 8.01 (d, 1H, J = 16 Hz, Hα), 8.33 (s, 1H, H6′),
8.04 (d, 1H, J = 8.0 Hz, H2), 8.19 (d, 1H, J = 16.0 Hz, Hβ). IR νmax cm−1
1657 (C = O), 1588 (C = C), 1223, 1043 (C–O), 2947, 1493, 977, 807,
787 (KBr) .13C NMR (CDCl3, δ ppm): 56.12 (OCH3), 56.72 (OCH3),
113.60 (C3′), 114.72 (C6′), 119.71 (C4′), 123.85 (Ca), 125.40 (C1′)
125.72 (C10), 126.45 (C3), 127.06 (C9), 128.96 (C5), 129.69 (C4),
129.86 (C8), 130.72 (C6), 132.01 (C2), 132.85 (C7), 133.96 (C1),
140.20 (Cb), 152.97 (C5′), 153.90 (C2′), 192.45 (C = O). Anal. calcd
for C21H18O3 (318.36) calc. C: 79.22; H 5.69; O: 15.07; found C:
79.20; H: 6.10.
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3.2
Fluorescence spectral measurements
For macromolecules, intrinsic fluorescence gives information about
their structure and dynamics. Fluorescence quenching is the decrease
in the fluorescence intensity of a fluorophore due to ground state com-
plex formation, excited state reaction, energy transfer, collision
quenching and molecular rearrangements.[17] Figure 3 shows the typi-
cal fluorescence band for BSA around 336 nm.[17] On subsequent addi-
tion of the naphthylchalcone derivatives, a significant decrease in the
emission intensity of BSA was observed. A red shift in the wavelength
maxima of 4 nm was observed for naphthylchalcone derivative 2a,
whereas in the case of naphthylchalcone derivatives 1a, 1c and 1d,
blue shifts in the wavelength maxima of 11, 6 and 4, respectively, were
observed. The red shift suggested an increase in the polarity of the
microenvironment around the Trp residues after binding of ligands
with BSA. It was probably due to loss of the compact structure of
hydrophobic subdomain IIA.[18–22] Therefore, it can be inferred that
the binding interaction between naphthylchalcone derivative 2a and
BSA resulted in enhancement of the hydrophilicity around theTrp res-
idues of BSA. The blue shift suggested that theTrp residue is buried in
the hydrophobic environment of the BSA protein, which signifies that
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3.1.2
yl‐propenone
Compound 2: 1‐(3‐bromo‐phenyl)‐3‐napthalene‐1‐
Yellow solid, mp 89–90°C. 1H NMR (CDCl3, δ in ppm) 6.97 (dd, 1H,
J = 7.4 Hz, H5′), 7.06 (d, 1H, J = 8.4 Hz, H3′), 7.57–7.68 (m, 4H, H3,
H6, H7, H4′), 8.01 (d, 1H, J = 8.4 Hz, H3′), 7.84–7.99 (m, 4H, H6′,
H4, H5, H8), 8.99 (d, 1H, J = 8.4 Hz, H2), 8.79 (d, 1H, J = 15.2 Hz,
Hb), 12.88 (S, 1H, OH), 13C NMR (CDCl3, δ ppm): 1183.93 (C3′),
119.17 (C5′), 122.99 (Ca), 123.67 (C3), 125.59 (C8), 125.68 (C1′),
126.67 (C3), 127.41 (C6), 129.07 (C4, C5), 130.00 (C9, C6′), 131.48
(C10), 136.74 (C1, C4′), 142.66 (Cb), 163.93 (C2′), 194.12 (C = O).

PASRICHA ET AL.
5
FIGURE 3 Fluorescence emission spectra of BSA [7.5 μM] in the presence of naphthylchalcone derivatives (i) 1a, (ii) 1c, (iii) 1d and (iv) 2a. The
concentration of each naphthylchalcone derivatives varied from (a) 0, (b) 6.62, (c) 13.15, (d) 19.6, (e) 25.97 and (f) 32.25 μM
binding of the ligands creates a more apolar environment around the
Trp residue.[23–25] Some previous studies have reported the binding
of chalcones with serum proteins, but most have reported a blue shift
in the wavelength maxima in the fluorescence emission spectra of the
serum protein.[26,27] The shift in the wavelength maxima of BSA in the
presence of naphthylchalcone derivatives suggests their binding to BSA.
Fluorescence quenching was further analysed using the Stern‐
Volmer equation:
where, F0 and F are the fluorescence intensities in the presence and
absence of quencher, respectively, KSV is the Stern‐Volmer quenching
constant and [Q] is the concentration of quencher.
The Stern‐Volmer plots for all four derivatives are shown in
Figure 4 and the obtained KSV values are given in Table 2.
Molecular contact between the fluorophore and quencher is nec-
essary in both static and dynamic quenching. In dynamic quenching,
quencher must interact with the fluorophore during the excited state
lifetime and no permanent change occurs during this type of
quenching. But, in static quenching, a non‐fluorescent complex is
formed between fluorophore and a quencher on contact. [17]
F0=F ¼ 1 þ KSV½Qꢁ
(2)
FIGURE 4 Stern–Volmer plots F0/F versus [Q] for the binding of BSA with naphthylchalcone derivatives, (a) 2a, (b) 1c, (c) 1a and (d) 2a

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PASRICHA ET AL.
TABLE 2 Association constant (K, n) and stern–Volmer quenching
constant (KSV) values obtained for binding of naphthylchalcone deriv-
atives 1a, 1b, 1c and 2a for bovine serum albumin using fluorescence
quenching method
1010 M−1 s−1, hence it confirms that the quenching is essentially static
in nature.[25,28]
Complex
KSV (104) M−1
K (106) M−1
N
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3.3
Analysis of binding equilibria
2a
1c
1a
1d
8.02
5.63
4.23
1.62
1.61
0.82
0.26
0.79
1.07
1.335
0.952
1.419
When small molecules bind independently to a set of equivalent sites
on a macromolecule, equilibrium between the free and bound mole-
cules is established. The association constant (K) and number of bind-
ing sites (n) for the naphthylchalcone derivative–BSA interaction
were determined according to Feng et al.[29] using equation 5,
In order to establish whether the quenching mechanism is
dynamic or static, the bimolecular quenching rate constant value was
obtained from equation 3.
logðF0–FÞ=F ¼ logK þ n log½Qꢁ
(5)
F0=F ¼ 1 þ KSV½Qꢁ ¼ 1 þ kqτ0½Qꢁ
(3)
where K and n are the association constant and the number of binding
sites, respectively. A linear plot between log(F0 – F)/F and log[complex]
was observed for all four derivatives (Figure 5).
where, kq is the second‐order bimolecular quenching rate constant, τ0
is the lifetime of excited Trp residues in BSA in the absence of
quencher and KSV is the Stern‐Volmer constant. The τ0 value for Trp
in proteins is usually around 10−9 s.[17] Putting the values of KSV and
τ0 for the Trp residue in equation 3, the value of kq obtained was in
order of 1013 M−1 s−1. The value of kq depends on collision between
the fluorophore and quencher and its probability depends on their rate
of diffusion (D), size and concentration. It can be shown that
The binding parameters obtained from the plot of log(F0 – F)/F0
against log[Q] for each of the naphthylchalcone derivatives binding
with BSA are given in Table 2.
The values of n are ~1 for all the naphthylchalcone derivatives,
which emphasizes that there is only one equivalent site in BSA for
the binding of naphthylchalcone derivatives. The magnitude of the
association constant, K (106) suggests a role for non‐covalent binding
interactions in the complexation of BSA with naphthylchalcone deriv-
atives.[30,31] Similarly, Naik and Nandibewoor[32] reported the binding
of BSA with chalcone using the fluorescence quenching method. The
reported values of n and K for binding of chalcone with BSA were of
the same order of magnitude as the values reported here. Therefore,
thorough analysis of the fluorescence data clearly suggest the binding
of naphthylchalcones with BSA.
kq ¼ 4πaDNa×103
(4)
where, D is the sum of the diffusion coefficients of quencher and
fluorophore, a is the sum of molecular radii and Na is the Avogadro's
number. The upper limit of kq for a diffusion‐controlled process is
1010 M−1 s−1 [17]
In the current study, the value of kq was 1000 to
.
FIGURE 5 The plot of log(F0–F)/F0 against log[Q] to determine the value of association constant and number of binding site of BSA with
naphthylchalcone derivatives (a) 2a, (b) 1c, (c) 1a and (d) 1d respectively

PASRICHA ET AL.
7
FIGURE 6 a) Circular dichroism spectra of BSA (1 μM) in the presence of 40 μM each with naphthylchalcone derivatives (1a, 1c, 1d and 2a); b)
Circular dichroism spectra of 40 μM naphthylchalcone derivatives (1a, 1c, 1d and 2a)
TABLE 3 GlideScore and other detailed parameters values of the naphthylchalcone compounds (2a, 1a, 1c and 1d) at the three different binding
sites
Minimization converged
Docking
score
Glide
evdw
Glide
energy
Glide
einternal
Glide
emodel
XP
HBond
Compound
Site
OPLS‐2005
Aromatic low helix (115–184)
2a
1c
1a
1d
0.0169
0.0207
0.0119
0.0003
–7.3829
–9.0279
–8.2146
–8.9169
–1.4822
–2.6214
–1.5175
0.9171
10.8891
9.2283
15.0231
5.3734
–37.1077
–46.3070
–48.1499
–38.7695
–0.5361
–0.5083
–0.6242
–0.7
–0.3076
–0.3761
–0.3422
–0.4246
Sudlow I (198–582)
Sudlow II (307–582)
2a
1c
1a
1d
0.0169
0.0207
0.0119
0.0003
–5.1437
–4.4882
–3.9796
–3.9071
–0.2479
1.7591
1.9437
–7.0765
4.0687
3.0826
10.8729
1.1343
–49.6995
–32.3191
–33.1240
–49.3145
0
–0.2143
–0.1870
–0.1658
–0.1860
0
–0.2770
–0.48
2a
1c
1a
1d
0.0169
0.0207
0.0119
0.0003
–3.2807
–3.998
–1.0148
–3.2669
–3.0492
–7.3464
0.5395
7.0975
0.2172
3.3683
–31.8181
–43.0629
–39.9841
–37.0088
–0.2495
–0.5360
–0.8813
–1.372
–0.1366
–0.1666
–0.1511
–0.0797
–3.6280
–1.6744
FIGURE 7 Representative 2D (a) and 3D (b) docked model of Naphthylchalcone derivative (2a) with BSA at sudlow I site

8
PASRICHA ET AL.
|
of naphthylchalcone derivatives to BSA results in a decrease in the
magnitude of double minimum, reflecting conformational changes
3.4
CD studies
The CD spectra of free BSA and in the presence of 40 μM of each
naphthylchalcone derivative were recorded over a wavelength range
of 200–250 nm (Figure 6a). None of the synthesized derivatives show
any CD signal over this wavelength range (Figure 6b). It is known that,
the α helices of protein show a strong double minimum at 209 and
220 nm.[33,34] The two negative peaks at 209 and 220 nm originate
in n ! π* transitions of the amide groups in the peptide bond and
reflect the characteristic signal of the α‐helical conformation of the
protein.[35,36] A negative band near 222 nm is observed due to the
strong hydrogen‐bonding environment of the α‐conformation. The
intensity of this double minimum reflects the amount of helicity of
BSA and indicates that BSA contains >50% helical structure. Addition
leading to
a decrease in α‐helical content. The addition of
naphthylchalcone derivatives leads to a decrease in the α‐helical con-
tent of BSA from 48% to ~20%. A similar percentage decrease in the
α‐helical content of serum proteins in the presence of chalcones was
reported by Naik and Nandibewoor[32] and He et al.[26] The decrease
in the α‐helical content of BSA clearly showed that the binding of
naphthylchalcones induces changes in the secondary structure of
BSA. The mechanism of α‐helix unfolding may be related to gradual
opening of the BSA molecule with ligands, followed by interactions
between the hydrophobic sites of the protein and hydrophobic
groups of ligands, resulting in subsequent changes in the protein
FIGURE 8 Representative 2D (a) and 3D (b) docked model of Naphthylchalcone derivative (2a) with BSA at sudlow II site
FIGURE 9 Representative 2D (a) and 3D (b) docked model of Naphthylchalcone derivative (2a) with BSA at aromatic low helix

PASRICHA ET AL.
9
FIGURE 10 Correlation of association constant determined using fluorescence quenching study with docking score values for sites a) Sudlow I, b)
Aromatic low helix and c) Sudlow I
Table 3 shows that all four ligands have diverse GlideScores rang-
ing from −7.3829 to −9.0279 (aromatic low helix), −3.9071 to −5.1437
(Sudlow I) and −1.6744 to −3.998 (Sudlow II) in the different binding
sites. Two‐dimensional (2D) interaction maps suggested that in the
docking site of BSA there was dominance of hydrophobic, as well as
hydrogen binding, interactions. It has been reported and our previous
studies indicate that both hydrophobic and hydrogen binding interac-
tions play pivotal roles in the complexation of ligands with
proteins.[24,25,28,38]
The docking scores suggested that the compounds bind to the
Sudlow I site most effectively followed by Sudlow II. Compound 1c
showed the highest binding score of −9.0279 for aromatic low helix
via various hydrophobic interactions with Cys513, Val554, Cys558
and Val551; ionic interactions with Asp555, Lys563, Lys556, Asp562
FIGURE 11 Correlation of association constant (K) versus logKhsa for
given in the Supporting Information.
compound 1a, 1c, 1d and 2a
In Sudlow I (Figure 7). Asp118 forms a hydrogen bond via a back-
conformation.[34] The CD studies conclude that all four synthesized
bone with the carbonyl group of compounds. Other residues, Pro117,
Leu122, Phe133 and Leu115 showed hydrophobic interactions that
are common for all four ligands, whereas Glu125, Glu140, Lys116,
Lys136 and Arg185 showed hydrophilic interaction with ligands. The
2D docked model of all four naphthylchalcones derivatives with BSA
at Sudlow I site are given in the Supporting Information.
naphthylchalcone derivatives bind effectively with BSA.
|
3.5
Molecular docking
The binding affinity may be visualized further using a molecular
docking technique. In this study, receptor PDB ID: 4J4K was docked
with true structures of naphthylchalcone derivatives. The true struc-
tures of the synthesized compounds were obtained after geometry
optimization based on energy minimization using Gaussian 2009 under
solvent conditions. The three binding sites, aromatic low helix, Sudlow
I and Sudlow II, were selected in BSA PDB based on the binding pref-
erence of the aromatic compounds in BSA.[37] The extent of binding of
the ligands to the receptor protein was determined on the basis of
GlideScore values.
For Sudlow II (Figure 8). Lys116 forms hydrogen bonding via a
backbone with the carbonyl group of ligand 2a. Other residues,
Pro117, Pro516 and Leu515 showed hydrophobic interactions that
are common for all four ligands, whereas Gln 519 and Asp517 showed
hydrophilic interaction with the ligands. The 2D docked model of all
four naphthylchalcones derivatives with BSA at Sudlow II site are given
in the Supporting Information.
In aromatic low helix (Figure 9). Lys116 forms a hydrogen bond via
a backbone with the carbonyl group of ligand 2a. Other residues,
Cys513, Ala 560 and Ile 512 showed hydrophobic interactions that

10
PASRICHA ET AL.
TABLE 4 The absorption, distribution, metabolism and excretion (ADME) properties of the four naphthylchalcone derivatives
Naphthylchalcone derivative
logPo/W
logS
PCaco
logKhsa
logBB
% human oral absorption
1a
1c
1d
2a
6.007
5.323
5.214
6.191
–4.85
–5.52
–4.69
–5.15
3758.65
3341.60
1428.75
3516.28
0.502
0.699
0.611
0.535
–0.225
0.037
91
97
95
96
–0.556
–0.272
are common for all four ligands, whereas Asp517 and Lys520 show
hydrophilic interaction with the ligands. The 2D docked model of all
four naphthylchalcones derivatives with BSA at the aromatic low helix
site are given in the Supporting Information.
|
4
CONCLUSION
The characterization of synthesized derivatives confirmed that all the
naphthylchalcone derivatives obtained were E‐chalcones. Fluores-
cence quenching of the protein in the presence of ligands was static
Docking score values with experimentally determined association
constant values using fluorescence quenching data for all the three
sites are shown in Figure 10. In the case of the Sudlow I binding site,
the association constant values are linearly correlated with the docking
scores values. For Sudlow II, the association values are more and less
linearly correlated with the docking score, except for compound 2a.
However, in the case of aromatic low helix, the experimentally deter-
mined values show poor correlation with the docking scores. There-
fore, comparison of the association values with the docking score
confirmed the binding of naphthylchalcone compounds in Sudlow I
and II binding sites.
in nature, implying the formation of
a
complex between
naphthylchalcone derivatives and BSA. The conformational change in
the BSA on binding to naphthylchalcone derivatives was confirmed
by CD spectroscopy. Docking of naphthylchalcone derivatives with
BSA showed strong binding at the three binding sites in the protein.
The docking data showed that hydrogen bonding, hydrophobic and
hydrophilic interactions are the dominant binding forces.
ACKNOWLEDGMENTS
Authors are thankful Dr A. K. Singh, Director, Institute of Nuclear
Medicine & Allied Sciences, Defence Research & Development Organi-
zation for his support and thorough guidance. One of the authors Ms
Deepti Sharma thanks University Grants Commission for junior
research fellowship.
|
3.6
In silico ADME prediction
To extrapolate the serum albumin ligand binding affinity, it is useful to
assess the in silico ADME properties of the four naphthylchalcone
derivatives. In ADME screening, 44 parameters are used to predict
the suitability of the synthesized compounds for use as drugs. These
ADME parameters include: molecular descriptors and pharmaceutically
relevant properties like the partition coefficient (logPo/w) and water
solubility (logS), critical for estimation of the absorption and distribu-
tion of drugs within the body; blood–brain barrier permeability (logBB),
which is prerequisite for the entry of drugs into the brain; logKhsa,
which predicts binding to human serum albumin, (PCaco); a model for
the gut–blood barrier; the percentage of human oral absorption; and
Lipinski's rule of five. These properties calculated for four compounds
are given in Table 4.
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Luminescence, circular dichroism and in silico studies of binding
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