Naproxen intercalates with DNA and causes

photocleavage through ROS generation

Mohammed A. Husain1, Zahid Yaseen2, Sayeed U. Rehman1, Tarique Sarwar1 and
Mohammad Tabish1
1 Department of Biochemistry, Faculty of Life Sciences, A.M. University, Aligarh, Uttar Pradesh, India
2 Department of Chemistry, Faculty of Sciences, A.M. University, Aligarh, Uttar Pradesh, India

Keywords Naproxen is an important non-steroidal anti-inflammatory drug with many

DNA binding; docking; intercalation;
naproxen; ROS
Correspondence
M. Tabish, Department of Biochemistry,
Faculty of Life Sciences, A.M. University,
Aligarh, Uttar Pradesh 202002, India
Tel: +91 9634780818
E-mail: tabish.bcmlab@gmail.com
(Received 8 July 2013, revised 17
September 2013, accepted 30 September
2013)
doi:10.1111/febs.12558
pharmacological and biological properties. In this study, we have attempted
to ascertain the mode of action and the mechanism of binding of naproxen
to DNA. We have also demonstrated that, upon irradiation with white
light, naproxen generates reactive oxygen species, causing DNA cleavage.
Generation of reactive oxygen species from photo-irradiated naproxen as
determined spectrophotometrically was found to lead to nicking of plasmid
DNA as analyzed by agarose gel electrophoresis. Without photo-irradia-
tion, naproxen binds to DNA and forms drug–DNA complexes as revealed
by spectroscopic techniques. Several experiments such as determination of
the effect of urea, iodide-induced quenching and a competitive binding
assay with ethidium bromide showed that naproxen binds to DNA primar-
ily in an intercalative manner. These observations were further supported
by CD analysis, viscosity measurements and molecular docking. Using
DNA as a template, fluorescence resonance energy transfer between nap-
roxen and ethidium bromide was also observed, further strengthening the
evidence for intercalative binding of naproxen with DNA.

Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are
widely used for the treatment of rheumatic and
arthritic diseases [1]. Naproxen [2-(6-methoxynaphtha-
len-2-yl) propanoic acid, Fig. 1A] belongs to this class,
and its pharmacological activity primarily comprises
inhibition of cyclo-oxygenase (COX). A general hall-
mark of most NSAIDs is the generation of reactive
oxygen species (ROS) in the presence of light, and
naproxen is a well-known photosensitizer that is able
to generate ROS upon illumination [2].
Among the NSAIDs, compounds containing aryl-
propionic acid (naproxen) in their chemical structure
behave as photosensitizers and produce biomolecule
modifications that are thought to be responsible for the
occurrence of photo-induced effects [3,4]. Drug–DNA
photoreaction results in undesired effects, and DNA
molecules are susceptible to damage caused by the
absorption of light by photosensitizing molecules [5,6].
It is well-established that ROS react with nuclear
DNA, resulting in breakage of the DNA strand. The
DNA strand breakage was found to be facilitated by a
non-covalent drug–DNA interaction, induced by both
electron and energy transfer to DNA, as recently sug-
gested for some NSAIDs [7,8]. In this context, the pri-
mary event is absorption of photons of the appropriate
wavelength, which allows the chromophore to reach an
excited state. The excitation energy is then transferred
to oxygen molecules, generating ROS such as superox-
ide and hydroxyl radicals [9,10]. These generated ROS
result in local oxidative stress, causing breakage in

Abbreviations
ctDNA, calf thymus DNA; EtBr, ethidium bromide; FRET, Fo €rster resonance energy transfer; NBT, nitroblue tetrazolium;
NSAIDs, non-steroidal anti-inflammatory drug; ROS, reactive oxygen species.

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Naproxen–DNA interaction
A B
Fig. 1. (A) Structure of the naproxen (+ isomer) sodium. (B) Agarose gel electrophoresis of EtBr-stained pBR322 DNA after treatment with
naproxen. Lane A is the ‘control’, which contains only plasmid DNA. The concentrations of naproxen in lanes B–F were 100, 150, 200, 250
and 300 lM, respectively. The arrows labelled ‘OC’, ‘L’ and ‘SC’ indicate the relaxed, linear and supercoiled forms of plasmid DNA.
genomic DNA and oxidative modifications in proteins
and lipids within cell membranes [11]. In order to avoid
the undesirable effects of photoreactive compounds,
efforts have been made to establish a model system for
estimation of photosensitive potential through analyti-
cal and biochemical methods [12].
Interaction studies of drugs with DNA are useful
for understanding of the reaction mechanism as well
as to provide guidance for the application and design
of new drugs. Binding studies of drugs with DNA
have been performed in order to develop design princi-
ples for targeting of specific DNA sequences in order
to control gene expression [13]. Important modes of
drug–DNA interactions included (a) electrostatic inter-
actions between the negatively charged phosphate
groups of DNA and ionic portion of the molecules,
(b) binding of molecules to the major or minor
grooves, involving van der Waals interactions, and (c)
intercalation of the molecules with the base pairs of
nucleic acid [14,15]. Intercalation describes the forma-
tion of a ‘molecular sandwich’ between an incoming
planar or flat molecule and two layers of stacked
nitrogenous bases. This is the most effective mode of
interaction for drugs targeting DNA. There are a large
number of molecules that interact with DNA, and the
factors responsible for such interactions are quite com-
plex [16].
The availability of the genome sequence, the well-
studied three-dimensional structure of DNA, and the
predictability of accessible chemical and functional
groups make DNA as attractive drug target. However,
the number of known drugs that target DNA is still
very limited compared to the drugs that target proteins
[17]. We wished to investigate the interaction of nap-
roxen with DNA in detail. The ultimate objective was
to determine the mode of action and mechanism of
binding of naproxen with DNA in the presence and
absence of light. Moreover, the photosensitizing action
of naproxen in formation of strand breaks in the dou-
ble-stranded supercoiled pBR322 plasmid was also
investigated. Our results clearly demonstrate that
6570
M. A. Husain et al.
naproxen binds to DNA primarily in an intercalative
manner. Such interaction is also supported by the
results of biophysical experiments such as determina-
tion of the effect of urea, iodide-induced quenching
and displacement of ethidium bromide (EtBr). Our
results also suggest that F€ orster resonance energy
transfer (FRET) takes place between naproxen and
EtBr in the presence of DNA, providing an opportu-
nity to use such systems in optoelectronic devices and
for detection of DNA.
Results and Discussion
Interaction of photo-irradiated naproxen with
DNA: ROS generation assay
In order to demonstrate the photocleavage activity of
naproxen, a reaction mixture containing naproxen and
plasmid DNA pBR322 was irradiated for 2 h at
37 °C. Significant DNA damage was caused by nap-
roxen after irradiation with white light. Exposure of
plasmid pBR322 to increasing concentrations of nap-
roxen in the presence of light caused conformational
changes from a supercoiled to an open circular form.
As shown in Fig. 1B, the increase in the intensity of
the band representing the open circular form and
appearance of a DNA band corresponding to linear
DNA indicates plasmid DNA strand break activity.
Naproxen did not promote DNA strand breaks in the
absence of irradiation [6]. It is well documented that
ROS such as singlet oxygen and superoxide act
as major toxic mediators in drug-induced phototoxic
cascades [18].
There are at least three direct mechanisms by which
photo-excited molecules can damage DNA. First, pho-
to induced molecules cause formation of photo-ad-
ducts by damaging DNA directly through covalent
binding. Second, an excited molecule may transfer the
excitation energy to DNA, leading to production of a
pyrimidine dimer. Last, DNA damage in the form of
oxidative modification of guanine may occur due to
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M. A. Husain et al. Naproxen–DNA interaction

abstraction of an electron or a hydrogen atom by a
photo-excited chromophore. In addition to these direct
mechanisms, there are at least two indirect mechanisms
of DNA damage by excited photoreactive chemicals,
which include (a) reactive oxygen-mediated DNA dam-
age, and (b) production of reactive decomposition
products [19]. In a nitroblue tetrazolium (NBT) assay,
naproxen produced superoxide anions upon induction
with white light; these superoxide anions reduce NBT
via a one-electron transfer reaction, producing par-
tially reduced monoformazan (NBT+) as a stable
intermediate. Formation of monoformazan was
recorded spectrophotometrically at 560 nm (Fig. 2A).
In addition, increasing concentrations of naproxen in
the presence of white light lead to a progressive
increase in the formation of hydroxyl radicals
(Fig. 2B). It has been reported that, among oxygen
radicals, hydroxyl radicals are the main activated oxy-
gen species involved in DNA cleavage [6]. Therefore,
activated species such as superoxide and hydroxyl radi-
cals formed during the photolysis of naproxen result in
DNA strand breaks, leading to structural changes.
Interaction of naproxen with DNA without
irradiation: UV/vis spectroscopy
UV/vis absorption spectroscopy is used to explore the
structural changes and formation of biomacromole-
cules upon addition of small ligands. The effect of
naproxen on the UV absorption spectra is shown in
Fig. 3. The absorption spectra showed large hyper-
chromic shifts (increase in band intensity) upon
increasing the concentration of calf thymus DNA
(ctDNA), with a band centered at 230 nm, indicating
formation of adducts between ctDNA and naproxen.
However, no red shift was observed in the spectra of
the naproxen–DNA complex. As seen in Fig. 3, there
is no clear isosbestic point, which indicates that there
is more than one type of binding and/or that 1 : 1
drug:DNA stoichiometry is not maintained during the
process [20]. Therefore, we conclude that formation of
naproxen–DNA complexes does occur; however, these
experiments do not provide mechanistic details.
Steady-state fluorescence
In order to study the interaction between naproxen
and DNA, the steady-state fluorescence technique was
employed. The fluorescence emission spectra showed
the effect of ctDNA on the emission spectrum of nap-
roxen. Upon addition of ctDNA to naproxen solution,
we observed significant quenching of the fluorescence
intensity of naproxen (Fig. 4). The change in the emis-
sion spectra of naproxen due to the presence of
ctDNA indicated an interaction between naproxen and
ctDNA, causing fluorescence quenching of naproxen.
The quenching pattern observed is potentially due to
formation of a non-fluorescent complex of naproxen
with DNA. To rationalize the results of the fluores-
cence experiments, the ratio of the peak fluorescence
intensity in the presence and absence of ctDNA (F/F0)
was plotted as a function of DNA concentration for
naproxen. The inset in Fig. 4 shows that the fluore-
scence intensity decreases with an increasing concen-
tration of DNA, i.e. the binding interaction increases
gradually with subsequent addition of DNA.

A B

Fig. 2. (A) Concentration-dependent photo-generation of superoxide anions by naproxen, as measured by the reduction of NBT. Naproxen
was dissolved in 50 mM sodium phosphate buffer (pH 7.8) at the indicated concentrations, and then exposed to white light for 2 h, and the
absorbance was measured at 560 nm. (B) Concentration-dependent generation of hydroxyl radicals by photo-irradiated naproxen. Naproxen
was dissolved in 10 mM Tris/HCl (pH 7.2) at the indicated concentrations, and then exposed to white light for 1 h, and the absorbance was
measured at 532 nm. The increase in absorbance at 532 nm is due to the formation of a coloured complex between malondialdehyde and
thiobarbituric acid. Values are means
SD of three experiments. Asterisks indicate statistically significant differences compared with the
control (*P < 0.05).

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Naproxen–DNA interaction
Fig. 3. UV/vis absorption spectra for naproxen (20 lM) in the
presence of various concentrations of ctDNA in Tris/HCl buffer (pH
7.2). The arrow shows that the absorbance increases with
increasing amounts of ctDNA.
Determination of the binding mechanism of
naproxen and DNA
Iodide quenching
The fluorescence intensity of naproxen in the presence
or absence of DNA was analyzed using potassium
iodide (KI) as a quencher, as described previously [21],
and the correlation between the degree of accessibility
of each molecule to the quencher and steric bulk was
observed. Interaction of KI with the negatively charged
phosphate backbone of DNA was assumed to be repul-
sive in nature. As a consequence, an intercalatively
bound small molecule experiences little protection com-
pared with groove/surface binders in the presence of an
anionic quencher. Quenching of the fluorescence inten-
sity of naproxen by KI in the presence and absence of
DNA was calculated using the Stern–Volmer equation:
(F0/F) = 1 + KSV [Q]
where F0 and F are the fluorescence intensities in the
absence and presence of the quencher KI (Q), and KSV
is the Stern–Volmer quenching constant. KSV indicates
the accessibility of the bulky quencher (iodide) to the
fluorophore. The slopes of the plots of (F0/F – 1) ver-
sus [KI] yield the values of KSV (Fig. 5). The KSV values
for of naproxen in the presence of KI and the absence
and presence of DNA were 30.06 and 7.52 M, respec-
tively. Table 1 summarizes the calculated KSV from
Stern–Volmer plots. The value of KSV for naproxen
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M. A. Husain et al.
Fig. 4. Fluorescence emission spectra for naproxen (20 lM) in the
presence of various concentrations of ctDNA. The excitation
wavelength was 230 nm and the emission wavelengths were 300–
500 nm. The inset shows the variation in the respective
fluorescence intensities with DNA concentrations. The arrow shows
that the intensity decreases upon increasing the concentration of
ctDNA. Values are means
SD of three experiments.
Table 1. Variation of KSV values for naproxen in the absence and
presence of ctDNA environments. Values are the means
SD of
four experiments.
Drug KSV (M) Ra SDb
Buffer 30.06
0.24 0.99874 0.7238
Drug–DNA complex 7.52
0.20 0.99921 0.7830
a
R is the correlation coefficient.
b
SD is standard deviation of naproxen and KI, naproxen, DNA and KI.
is low in the presence of DNA compared to its
absence, clearly indicating intercalative binding.
Effect of urea
Chemical agents such as urea, guanidinium chloride
(1)
etc. are often used to denature biomacromolecules such
as DNA and protein, resulting in release of interacting
drug molecules by destabilizing the interaction between
the DNA and the drug [22,23], leading to a change in
the fluorescence behaviour of the drug molecule. Urea
is used extensively as denaturing agent in biochemistry,
destabilizing the double-stranded DNA double helix
[24]. As evident from Fig. 6, there is a progressive
increase in the fluorescence intensity of naproxen by
addition of urea to naproxen-bound ctDNA. The
extent of intercalation of naproxen as a function of
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M. A. Husain et al.
Fig. 5. Stern–Volmer plot for fluorescence quenching of naproxen
(50 lM) by KI in the absence and presence of ctDNA (13.5 lM) in
Tris/HCl buffer (pH 7.2). The concentration of KI was varied from 0
to 72 mM. Values are means
SD of three experiments.
Fig. 6. Fluorescence spectra of naproxen in the presence of urea.
The fluorescence intensity increases with addition of urea. The
inset shows the variation of the fluorescence intensity of ctDNA-
bound naproxen as a function of urea concentration. Values are
means
SD of three experiments. Asterisks indicate statistically
significant differences compared with the control (*P < 0.05).
urea concentration, given by the ratio of its fluores-
cence intensities in the presence and absence of urea
(F/F0), is shown in Fig. 6. This confirmed that urea is
able to release naproxen from the DNA strands. This
also provides further evidence that the binding mode of
naproxen with ctDNA is intercalative.
EtBr displacement assay
It is well known that enhancement of fluorescence
emission of EtBr occurs as a result of intercalation
FEBS Journal 280 (2013) 6569–6580 ª 2013 FEBS
Naproxen–DNA interaction
between DNA base pairs [25,26]. In order to examine
the ability of naproxen to displace EtBr from interca-
lated complexes between EtBr and DNA, an EtBr dis-
placement assay was performed. As shown in Fig. 7,
emission of intense fluorescence from EtBr was
observed in the presence of DNA due to its strong
intercalation with DNA base pairs. However, addition
of naproxen induced a progressive decrease in the
DNA-induced EtBr emission, possibly due to displace-
ment of EtBr by naproxen. The observed quenching of
the EtBr fluorescence by addition of naproxen suggests
that naproxen probably binds to ctDNA in an interca-
lative manner.
Viscosity measurement
Viscosity measurements of drug–DNA complexes pro-
vide reliable evidence for the intercalative mode of
binding because the length of DNA changes upon
intercalation with the drug, affecting the viscosity [27,
28]. In the case of classical intercalation binding, a
ligand lengthens the DNA helix due to separation of
base pairs, resulting in increased DNA viscosity
[27,28]. On the other hand, if the drug is a groove
binder or interacts electrostatistically with DNA, the
viscosity of solution does not change significantly
[29–31]. In Fig. 8, a plot of (g/go)1/3 versus the ratio of
the naproxen concentration to the DNA concentration
Fig. 7. Fluorescence titration of the ctDNA–EtBr complex with
naproxen. The fluorescence intensity decreases with addition of
naproxen. The inset shows the intensity observed at 590 nm
versus the concentration of naproxen. The excitation wavelength
was 475 nm.
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Naproxen–DNA interaction
Fig. 8. Effect of increasing the concentration of naproxen on the
viscosity of ctDNA. The concentration of DNA was kept constant
(0.1 mML1) and concentration of the complex was varied. Values
are means
SD of three experiments. Asterisks indicate
statistically significant differences compared with the control
(*P < 0.05).
gives a measure of the viscosity changes. As seen in
Fig. 8, with continuing addition of naproxen to DNA
solution, the viscosity of the solution increases gradu-
ally. This is explained by the fact that naproxen inter-
calates between the DNA base pairs. This observation
confirms the above results suggesting that naproxen
intercalates with DNA.
Circular dichroism (CD) studies
CD spectroscopy is a very sensitive technique that has
been used extensively for analysis of changes that occur
in the secondary structure of polypeptides, proteins and
DNA under the influence of interacting ligands [32,33].
Non-covalent drug–DNA interactions affect the structure
of DNA, and hence alter the CD spectral behaviour.
CD spectroscopy was used to obtain further information
on the binding of naproxen to DNA [34,35]. The CD
spectrum of DNA in the absence and presence of nap-
roxen is shown in Fig. 9. In the far-UV wavelength
range (200–300 nm), the intrinsic CD profile of DNA is
characterized by a positive peak at ~ 276 nm and a neg-
ative peak at ~ 247 nm, which represent base pair stack-
ing and the right-handed B-form of DNA, respectively
[36,37]. As shown in Fig. 9, there is a decrease in the
ellipticity of DNA with increasing concentration of
naproxen. As reported previously [14], the decrease in
the peak ellipticity at ~ 276 nm may be explained on the
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M. A. Husain et al.
Fig. 9. CD spectra of ctDNA (30 lM) in 10 mM Tris/HCl (pH 7.2)
with varying concentrations of naproxen. The numbers 1–3 indicate
the spectra at 0, 30 and 60 lM of naproxen, respectively. Each
spectrum was obtained at 25 °C with a 10 mm path length cell.
basis of disruption of the stacked nitrogenous bases due
to intercalation of naproxen with DNA in order to
optimize the binding interaction. Accommodation of the
intercalated naproxen within a particular base pair
results in adjustment of the relative orientation of bases.
The change in the peak position at 247 nm occurs due
to the change in the hydration layer of DNA, which in
turn affects the helicity of DNA. Minor groove binders
do not perturb the CD profile of DNA significantly.
Hence, the CD analysis confirms our finding that
naproxen binds to DNA in an intercalative manner.
€ rster resonance energy transfer (FRET)
Fo
FRET is a phenomenon that is observed when the emis-
sion spectrum of the donor molecules (D) overlaps with
the excitation spectrum of the acceptor molecules (A).
For FRET to be operational, the donor and acceptor
molecules must be in close proximity to one another
(1–10 nm), the donor and acceptor transition dipole
moment must be correctly oriented, and donor must
have a high quantum yield [38]. The emission spectrum
of DNA is not very pronounced, although it can
increase the fluorescence intensity of EtBr significantly.
In order to magnify the FRET, we used the naproxen–
DNA complex as an energy donor and EtBr as an
energy acceptor. As shown in Fig. 10A, the emission
spectrum of the donor drug–DNA complex overlaps
with the absorption spectrum of the acceptor EtBr. The
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M. A. Husain et al.
A emission spectra of naproxen in the presence of increas-
B
C the lifetime of the excited state), /D is the fluorescence
Fig. 10. (A) Spectral overlap between the normalized absorption
spectrum of EtBr and the emission spectrum of naproxen in the
presence of DNA. (B) Emission spectrum of naproxen in the
presence of EtBr. (C) Changes in the emission spectrum of
naproxen (20 lM) in the presence of DNA (20 lM) with addition of
EtBr. The concentration of EtBr was 0–7 lM.
FEBS Journal 280 (2013) 6569–6580 ª 2013 FEBS
Naproxen–DNA interaction
ing concentrations of EtBr do not show any appreciable
change, indicating limited interaction between EtBr and
naproxen (Fig. 10B), but the fluorescence emission of
EtBr increases in the presence of DNA (Fig. 10C).
These factors make EtBr a suitable candidate to study
FRET between naproxen and DNA. In order to under-
stand the relative interactions of naproxen and EtBr
with DNA, we monitored the emission intensity upon
addition of EtBr, and observed a significant decrease in
the excimer emission intensity. However, we also
observed the appearance of a new peak at 600 nm
(Fig. 10C) with addition of EtBr. The appearance of
emission from EtBr at 600 nm (while exciting the solu-
tion at 230 nm, at which EtBr has minimum absorption)
may be expected due to FRET between the excimer of
naproxen and EtBr in the presence of DNA.
According to F€ orster’s theory [39], the efficiency of
FRET depends on the inverse sixth power of the dis-
tance between the two components:
EFRET = [1 + (RDA/Ro)6]1 (2)
The term Ro is known as the F€ orster distance, and
represents the distance at which the efficiency of trans-
fer is 50%. The F€ orster distance [39] may be calculated
as [39]
Ro = 8.8 9 1025 k2 /D N4 J (3)
where j is the orientation factor (value 2/3; the value
2
is valid when both components are freely rotating and
may be considered to be isotropically oriented during
quantum yield of the donor in the absence of FRET,
N is the refractive index of the medium (assumed to be
equal to 1.3) [40], and J represents the spectral overlap
of the emission spectrum of the donor with the
absorption spectrum of the acceptor, and is given as
R
ðkÞeðkÞk4 dk
JðkÞ ¼ R (4)
FðkÞdk
where F(k) is the fluorescence intensity of the donor in
the wavelength range k to k + dk and is dimensionless,
and e(k) is the extinction coefficient (in M1cm1) of the
acceptor at k. EFRET may also be estimated using the
fluorescence emission intensity both in the absence and
presence of acceptor by using the following equation:
EFRET = 1 – (F/Fo) (5)
On solving the above equations (in the presence of
DNA), we calculated the spectral overlap, the F€
orster
distance, the distance between the energy donor and
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Naproxen–DNA interaction
acceptor and the FRET efficiency, and these were found
to be 3.2 9 1015, 1.92 nm, 2.11 nm and 35%, respec-
tively. Hence, the value of RDA is close to that of Ro, i.e.
the F€ orster distance or critical distance at which the
energy transfer is 50%, indicating that the energy trans-
fer from naproxen–DNA to EtBr occurs with high prob-
ability. As the UV energy absorbed by naproxen may be
efficiently transferred to the intercalated EtBr in the
presence of DNA, this strongly suggests that naproxen
binds to DNA in intercalating mode. In the case of
groove binders, no FRET is observed because of the
greater distance between donor and acceptor molecules
and the fact that orientation of the dipoles is inadequate
for the energy transfer.
Molecular docking
Molecular docking is a useful tool for obtaining infor-
mation about the structural features of ligand–receptor
complexes and the binding affinity of a ligand with its
receptor [41–43]. Docking studies were therefore per-
formed in an attempt to ascertain the type and amount
of interaction between a double-stranded DNA dode-
camer [d(CGCGAATTCGCG)2, PDB ID 1BNA] and
naproxen. The drug was made flexible to attain different
conformations in order to predict the best-fit orienta-
tion, and the best energy-docked structure was analyzed.
As evident from Fig. 11, naproxen binds to DNA by
intercalating within the nucleotide base pairs of DNA.
Furthermore, there is possibility of hydrogen bonding as
the oxygen-bearing groups of naproxen are in proximity
A B
C
Fig. 11. (A, B) Molecular docked structure of naproxen complexed
with DNA. (C) The possibility of hydrogen bonding to adjacent
deoxyadenosines (DA4 and DA6 of strand A) of the dodecamer
duplex of sequence (CGCGAATTCGCG)2 (PDB ID 1BNA).
6576
M. A. Husain et al.
to the deoxyadenosines (DA4 and DA6 of strand A) of
the dodecamer (Fig. 11C). The resulting relative binding
energy of the docked DNA–naproxen complex was
found to be ~ 170.11 kJM. Despite the electrostatic
repulsion between naproxen and DNA (which bear the
same charge), the large negative value of binding energy
indicates a high binding potential of the naproxen with
DNA. Hence, there is agreement between the results of
spectral techniques and molecular modeling, providing
important information about the mode and mechanism
of interaction of naproxen with DNA.
In conclusion, the present study clearly demonstrates
the action of naproxen in the presence and absence of
light based on studies on ROS generation as well as
DNA binding by naproxen. These results may be very
useful from the medical point of view as they show
that naproxen has photochemical and/or photobiologi-
cal properties in vitro. Our study also established the
binding mode of naproxen with DNA. The results of
the detailed studies performed here are in total agree-
ment with interaction of naproxen with DNA by an
intercalative mode of binding. The observation of
FRET between naproxen and EtBr in the presence of
DNA suggests the possibility of using such a system
for efficient DNA drug design.
Experimental procedures
Materials
Naproxen and calf thymus DNA were purchased from
Sigma-Aldrich (St Louis, MO). Ethidium bromide was pur-
chased from Himedia (Mumbai, India). Plasmid pBR322
DNA was purified as described previously [44]. All other
chemicals and solvents were of reagent grade and used with-
out purification.
Calf thymus DNA preparation
ctDNA was dissolved in 0.1 M Tris/HCl buffer (pH 7.2) at
room temperature with occasional stirring to ensure forma-
tion of a homogeneous solution. The purity of the DNA
solution was determined by measuring the absorbance ratio
A260 nm/A280 nm. As this ratio was in the range 1.8–1.9, no
further purification was necessary. The concentrations of
DNA solutions were determined by using a mean extinc-
tion coefficient value of 6600 M1cm1 for a single nucleo-
tide at 260 nm [45].
Plasmid nicking assay
To examine the generation of nicks in double-stranded
DNA by ROS generated by naproxen, a plasmid nicking
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M. A. Husain et al.
assay was performed. The reaction mixture contained
0.5 lg pBR322 plasmid DNA, the desired concentration of
naproxen, and 10 mM Tris/HCl (pH 7.5) to a final volume
of 25 lL in all tubes. Irradiation was performed with white
light for 2 h at 37 °C. After incubation, 5 lL of 6X track-
ing dye solution containing 40 mM EDTA, 0.05% brom-
ophenol blue and 50% glycerol was added, and the
reaction mixture was subjected to electrophoresis using a
1% w/v agarose gel. The gel was stained with EtBr and
viewed and photographed on a UV transilluminator
(Wealtec Corporation, Model MD-25, Sparks, Nevada,
USA).
Irradiation procedure
Irradiations were performed using a fluorescent lamp at a
distance of 10 cm. The irradiation test was performed at
37 °C using an irradiance of 38.6 W/m2 as measured using
a Lasermate coherent power meter (Coherent Inc., Santa
Clara, CA, USA), and there was no measurable change in
the temperature of the solution after irradiation for 2 h.
Superoxide generation assay
In order to study the superoxide generation by naproxen,
the NBT reduction assay was performed essentially as
described previously [46]. The assay mixture contained
50 mM sodium phosphate buffer (pH 7.8), 0.3 mM NBT,
0.1 mM EDTA and 0.06% Triton X-100 in a total volume
of 3.0 mL. The reaction was started by addition of increas-
ing concentrations of naproxen (0–200 lM). After mixing,
the absorbance was recorded at 560 nm against a blank
that did not contain naproxen.
Hydroxyl radical assay
The generation of hydroxyl radicals was measured as
described previously [47]. ctDNA (300 lg) dissolved in
10 mM Tris/HCl (pH 7.2) was used as a substrate with
increasing concentrations of naproxen (0–300 lM), and vol-
ume of the reaction mixture was adjusted to 3 mL by add-
ing 10 mM Tris/HCl (pH 7.2). Incubation was performed
for 60 min at 37 °C in the presence of white light. Forma-
tion of hydroxyl radicals generates malondialdehyde from
deoxyribose, which then reacts with thiobarbituric acid to
give a coloured complex that was assayed by recording the
absorbance at 532 nm.
UV spectroscopic method
The UV spectrum was recorded using a Beckman DU40
spectrophotometer (Beckman Coulter Inc., Fullerton, CA,
USA) with 1 9 1 cm quartz cuvettes. The UV spectra of
naproxen and the DNA–naproxen complex were recorded
in the wavelength range 200–250 nm. The experiment was
FEBS Journal 280 (2013) 6569–6580 ª 2013 FEBS
Naproxen–DNA interaction
performed in the presence of a fixed concentration of nap-
roxen (20 lM) in a total volume of 3 mL, and titrated using
varying concentrations of DNA (0–12 lM). DNA solutions
of the same concentrations without naproxen were used as
blanks to observe the UV spectra specific to the naproxen–
DNA complex.
Fluorescence measurements
All fluorescence emission spectra were recorded on a Shima-
dzu 5000 spectrofluorometer (Kyoto, Japan) equipped with
a xenon flash lamp using 1.0 cm quartz cells. Fluorescence
emission spectra of naproxen–DNA, naproxen–DNA with
KI and naproxen–DNA with urea were recorded at 300–
500 nm upon excitation at 230 nm, with the widths of both
the excitation and the emission slits set to 10.0 nm. All
reaction mixtures were prepared in 10 mM Tris/HCl (pH
7.2).
Naproxen–DNA interaction
The fluorescence emission spectra were recorded by keeping
the concentration of naproxen constant (20 lM) and vary-
ing the DNA concentration from 0–5.75 lM in a total vol-
ume of 3 mL containing 10 mM Tris/HCl (pH 7.2).
Potassium iodide (KI) quenching method
Potassium iodide quenching was performed in the presence
and absence of DNA. Naproxen (50 lM) was dissolved into
a 3 mL reaction mixture containing 10 mM Tris/HCl (pH
7.2), and emission spectra were recorded for varying con-
centrations of KI (0–72 mM). In another experiment, fixed
concentrations of naproxen (50 lM) and ctDNA (13.5 lM)
were used, and KI was added subsequently from 0–72 mM.
The volume of the reaction mixture was made up to 3 mL
by adding 10 mM Tris/HCl (pH 7.2).
Effect of urea
This was performed by using fixed concentrations of nap-
roxen (50 lM) and ctDNA (13.5 lM) in a 3 mL reaction mix-
ture containing 10 mM Tris/HCl (pH 7.2). Emission spectra
were recorded for varying concentrations of urea (0–3.6 M).
Ethidium bromide displacement assay
The EtBr displacement assay was performed as described
previously [48]. ctDNA (2.4 9 104 M) was dissolved in
10 mM Tris/HCl (pH 7.2), and EtBr was added to a final
concentration of 2 lM. The concentration of naproxen was
varied from 0–264 lM. The solution was excited at a
wavelength of 475 nm, and emission was recorded at a
wavelength of 500–700 nm.
6577
Naproxen–DNA interaction
Viscosity measurement
To further elucidate the binding mode of naproxen, viscos-
ity measurements were performed by keeping the DNA
concentration constant (0.1 mML1) [49] and varying the
concentration of the complexes. Viscosity measurements
were performed using an Ubbelohde viscometer (Canon,
Model-9721-K56, Coleparmer, USA) suspended vertically
in a thermostat at 25 °C (accuracy
0.1 °C). The flow
time was measured using a digital stopwatch, and each
sample was tested three times to obtain a mean calculated
time. The data are presented as g/g0 versus the ratio of the
DNA concentration to the naproxen concentration, where
g and g0 are the viscosity of naproxen in the presence and
absence of DNA, respectively [50].
CD studies
In order to study the structural changes in DNA due to the
presence of naproxen, CD spectra were analyzed. CD spec-
tra were recorded in the range of 220–320 nm for ctDNA
dissolved in 10 mM Tris/HCl (pH 7.2) in the absence and
presence of various concentrations of naproxen (0–60 lM).
Spectra were recorded on a CIRASCAN spectrophotometer
(Applied Photophysics, Leatherhead, UK) equipped with a
Peltier temperature controller. All CD spectra were collected
with a scan speed of 200 nm/min and a spectral band width
of 10 nm. Each spectrum was the average of four scans.
Molecular docking
An interactive molecular graphics program, Hex 6.3 [51]
was used to understand the naproxen–DNA interactions.
The structure of the B-DNA dodecamer d(CGCG
AATTCGCG)2 (PDB ID 1BNA) was downloaded from
the Protein Data Bank (http://www.rcsb.org./pdb). The
strucuture-data file (SDF) of naproxen was obtained from
http://www.drugbank.ca/drugs/DB00788 and converted
into PDB format using Avagadro’s 1.01 [52]. Hex 6.3 per-
forms docking using spherical polar Fourier correlations,
and requires the ligand and the receptor to be input in
PDB format. The parameters used for docking include: cor-
relation type, shape only; FFT mode, 3D; grid dimension,
0.6; receptor range, 180; ligand range, 180; twist range,
360; distance range, 40. Visualization of the docked pose
was performed using PyMol (DeLano Scientific, San
Carlos, CA, USA).
Statistical analysis
The results are expressed as means
SE of at least three
independent observations. Student’s t test was used to
examine statistically significant differences. Analysis of vari-
ance was performed by ANOVA. P values < 0.05 were
considered statistically significant.
6578
M. A. Husain et al.
Acknowledgements
The authors are thankful to University Grants Com-
mission (UGC) (New Delhi, India) for the award of a
University Grants Comission-Maulana Azad National
Fellowship and A.M. University (Aligarh, India) for
providing the necessary facilities. We also thank the
Advanced Instrumentation Research Facility, Jaw-
aharlal Nehru University (New Delhi, India) for per-
forming CD experiments.
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