Synthesis, characterization and bioactivity studies of novel 1,3,4-oxadiazole small molecule that targets basic phospholipase A2 from Vipera russelli

Article abstract of DOI:10.1007/s11010-016-2888-6

Secretory phospholipase A₂ (sPLA₂) is a key enzyme participating in the inflammatory cascade followed by the action of cyclooxygenase-2 and lipoxygenases. Therefore, inhibitors of sPLA₂ could be used as potent anti-inflammatory agents to treat the early phase of inflammation. In this study, we have prepared the fenoprofen and ibuprofen analogs containing 1,3,4-oxadiazole nucleus and tested against Vipera russelli venom's basic sPLA₂ (VRV-PL-VIIIa). Among the tested ligands 5(a–t),2-(2-chlorophenyl)-5-(1-(4-phenoxyphenyl) ethyl)-1,3,4-oxadiazole (5m) inhibited the catalytic activity of VRV-PL-VIIIa with an IC₅₀ value of 11.52?μM. Biophysical studies revealed that the 5m quenches the intrinsic fluorescence of VRV-PL-VIIIa, in a concentration dependent manner. Also, the compound 5m affected VRV-PL-VIIIa conformation, which was observed by circular dichroism spectra that recorded the prominent shift in the α-helix peak and the random coil formation of VRV-PL-VIIIa. Further, molecular docking analysis revealed that the compound 5m possess strong hydrophobic interactions at catalytic triad region of the VRV-PL-VIIIa. Evident to in vitro and in silico studies, 5m strongly inhibited the hemolysis of red blood cells. Our in vivo pharmacological studies revealed that the compound 5m inhibited the edematogenic activity of VRV-PL-VIIIa in?mouse foot pad. Additionally, the 5m inhibited VRV-PL-VIIIa-induced myotoxicity and lung hemorrhage in mice. Overall, our ADMET results depicted that 5m possess better druggable property. Thus, this study explored the new fenoprofen and ibuprofen analog 5m as the lead-structure that serves as an anti-inflammatory agent.

Full text of DOI:10.1007/s11010-016-2888-6

Mol Cell Biochem
DOI 10.1007/s11010-016-2888-6
Synthesis, characterization and bioactivity studies of novel 1,3,4-
oxadiazole small molecule that targets basic phospholipase A2
from Vipera russelli
1
2
3
1
Vivek Hamse Kameshwar
•
Kumar J. R.
•
Babu S. Priya
•
S. Nanjunda Swamy
Received: 4 July 2016 / Accepted: 15 November 2016
Ó Springer Science+Business Media New York 2016
Abstract Secretory phospholipase A (sPLA ) is a key
2
blood cells. Our in vivo pharmacological studies revealed
that the compound 5m inhibited the edematogenic activity
of VRV-PL-VIIIa in mouse foot pad. Additionally, the 5m
inhibited VRV-PL-VIIIa-induced myotoxicity and lung
hemorrhage in mice. Overall, our ADMET results depicted
that 5m possess better druggable property. Thus, this study
explored the new fenoprofen and ibuprofen analog 5m as
the lead-structure that serves as an anti-inflammatory agent.
2
enzyme participating in the inflammatory cascade followed
by the action of cyclooxygenase-2 and lipoxygenases.
Therefore, inhibitors of sPLA could be used as potent anti-
2
inflammatory agents to treat the early phase of inflamma-
tion. In this study, we have prepared the fenoprofen and
ibuprofen analogs containing 1,3,4-oxadiazole nucleus and
tested against Vipera russelli venom’s basic sPLA (VRV-
2
PL-VIIIa). Among the tested ligands 5(a–t),2-(2-chlor-
ophenyl)-5-(1-(4-phenoxyphenyl) ethyl)-1,3,4-oxadiazole
Keywords 1,3,4-Oxadiazole Á VRV-PL-VIIIa Á Anti-
inflammation Á NSAIDs Á ANS
(
5m) inhibited the catalytic activity of VRV-PL-VIIIa with
an IC50 value of 11.52 lM. Biophysical studies revealed
that the 5m quenches the intrinsic fluorescence of VRV-
PL-VIIIa, in a concentration dependent manner. Also, the
compound 5m affected VRV-PL-VIIIa conformation,
which was observed by circular dichroism spectra that
recorded the prominent shift in the a-helix peak and the
random coil formation of VRV-PL-VIIIa. Further, molec-
ular docking analysis revealed that the compound 5m
possess strong hydrophobic interactions at catalytic triad
region of the VRV-PL-VIIIa. Evident to in vitro and in
silico studies, 5m strongly inhibited the hemolysis of red
Introduction
Venom of venomous snakes acts as natural advance killer
machines to kill its prey, among which V. russelli is one of
the most dangerous species found in the Indian subconti-
nent and other parts of Asia, causing high morbidity and
mortality rate among the big four venomous snakes [1].
Annual snakebite is a majorly neglected medical emer-
gency reported in modern India representing about 45,900
annual deaths affecting to a very great degree in rural areas
(
97%). It is found in order as Uttar Pradesh [ Andhra
Electronic supplementary material The online version of this
article (doi:10.1007/s11010-016-2888-6) contains supplementary
material, which is available to authorized users.
Pradesh [ Bihar. Sex ratio of the victim bitten by snake
were more common in males (59%) than females (41%)
which contributing 25% at ages of 15–29 years during the
monsoon months of June–September. Hence, an effective
treatment involving both education and antivenom provi-
sion would reduce snakebite deaths globally [2].
&
S. Nanjunda Swamy
nanju_chem@yahoo.com; nanjuchem@gmail.com
1
2
3
Department of Biotechnology, JSS Science and Technology
University, Mysuru 570006, India
Among the big four family, Viperidae family snake
venom possess sPLA2 enzyme belonging to group II,
whose enzymatic activity is critical with the presence of
amino acid residue of His48 and Asp49 (VRV-PL-VIIIa-
Department of Biochemistry, Faculty of Life Sciences, JSS
University, Mysuru, India
Department of Studies in Chemistry, University of Mysore,
Manasagangotri, Mysuru 570006, India
venom of Russelli viper phospholipase A VIIIa peak).
2
123

Mol Cell Biochem
Since Gly30 and Asp49 are among the highly conserved
2-chlorobenzoic acid, 2,3-dichlorobenzoic acid, 8-anilino-1-
naphthalenesulfonic acid (ANS), 1,2-dimyristoyl-sn-glycero-
3-phosphocholine (DMPC), Sephadex G-75, Sephadex G-50,
and CM-Sephadex C-25 were purchased from Sigma–
Aldrich, India. 1-myristoyl-2-hydroxy-sn-glycero-3-phospho-
choline (LPC) was purchased from Avanti Polar Lipids.
HPLC grade Methanol and analytical grade HCl were
obtained from Fisher Scientific. Tris (hydroxymethyl) amino-
methane, fatty acid-free bovine serum albumin (BSA) fraction
V, Sodium hydroxide, Silica gel (60–120 mesh), DMSO, pre-
coated TLC plates, Acetonitrile, Ethyl acetate, Calcium chlo-
ride dihydrate, and Sodium chloride were purchased from
Merck. Triton X-100 was obtained from HiMedia Laborato-
ries. Sodium acetate, glycine, and ethylene glycol bis (b-ami-
residues in PLA enzymes residing at catalytic triad. Car-
2
bonyl group of Asp49 forms a hydrogen bond with the
2
?
2?
Ca ion which is very crucial for the Ca ion to interact
with the substrate for catalysis [3, 4].The pathophysiology
of basic VRV-PL-VIIIa is that, it exerts its local intense
toxicity effects and systemic effects [5]. sPLA is a primary
2
upstream enzyme, which cleaves fatty acids at the sn-2
position of glycerol, the backbone of phospholipids
releasing free fatty acid and lysophospholipid [6, 7], which
are substrates for downstream enzymes like cyclooxyge-
nase (COX-2), lipoxygenase (LOXs), and lysophospholipid
acyl-CoA acyltransferase (LAT). The reaction has partic-
ular importance when the fatty acid released from the sn-2
position is arachidonic acid, which is further oxidatively
metabolized by downstream enzymes like COX-2 and
LOX enzymes, resulting in the release of proinflammatory
mediators like prostaglandins, thromboxanes, and leuko-
trienes [8–10]. Specific inhibitor which can block sPLA2
could have significant anti-inflammatory property, which
blocks the substrate for COX-2 and LOXs.
1
noethyl ether) N, N -tetraacetic acid (EGTA) were purchased
from SRL Chemicals. Milli-Q water was used throughout the
experiments. All chemicals were of analytical grade, and sol-
vents were of HPLC grade. Nanodrop ND3300 Fluorospec-
trometer from Thermo Scientific was used to record the
fluorescence. This study was carried out in accordance with
Animal ethical committee of the JSS Medical College,
Mysuru, India. The protocol was approved by the Institutional
Animal Ethics Committee (IAEC) on the Ethics of Animal
Experiments. (Registration no 261/CPSCE, dated October 16
Oct, 2000; Sanctioned letter no JSSMC/PR/IAEC/24/2091/
2013-14, dated July 29, 2013). Healthy Swiss albino male mice
weighing 20–25 g were obtained from Central animal house
facility, JSS Medical College, Mysuru. All dissection was
performed under sodium pentobarbital anesthesia, followed by
euthanasia (cervical dislocation) to minimize suffering.
Currently, Nonsteroidal
anti-inflammatory
drugs
(NSAIDs) are used to treat acute as well as chronic inflam-
matory conditions targeting COX-2. Continuous use of
NSAIDs results in an unacceptable risk like Gastrointestinal-
adverse drug reactions (GI-ADRs), associated with the renal
and cardiovascular system failure, due to the presence of a
free carboxylic acid group of NSAIDs (fenoprofen and
ibuprofen) [11]. Previously, findings suggested that cycliza-
tion of the free carboxylic group of aroyl propionic acid
resulted in significant decrease of ulcerogenic activity [12].
The presence of bulky ester group in these ligands was
responsible for the inhibition of venom PLA . In medicinal
2
General procedure for the synthesis of 2,5
disubstituted 1,3,4-oxadiazole derivatives 5(a–t)
chemistry, the 1,3,4-oxadiazole-based heterocyclic ring was
used as replacements for ester bonds [13]. Also, oxadiazole, a
5
-membered heterocyclic ring, was used for designing potent
Aromatic acids 1(a–d) were refluxed for 3–4 h with 6.5 ml
of ethanol containing 5–6 drops of concentrated H SO .
bioactive entities [13, 14]; Henceforth, we focused to prepare
anti-inflammatory drugs like fenoprofen and ibuprofen ana-
logs, which contain 1,3,4-oxadiazole ring by replacing the
2
4
Progress of reaction was monitored and confirmed by TLC
(hexane:ethyl acetate:7:3), the reaction mass was extracted
with dichloromethane thrice. The solvent was evaporated
under vacuum, to obtain aryl benzoates. Resultant aryl
benzoates 2(a–d) were added dropwise to 6–8 ml of
ethanol containing 100% NH NH .H O (6.5 ml), and the
terminal-free carboxylic group to target sPLA of V. russelli.
2
Further, this is the first study to report the anti-PLA activity
2
of the newly synthesized ligands containing 1,3,4-oxadiazole
elucidating better correlation between CD data and Kabsch–
Sander’s secondary structural predication along with in silico
studies validating our findings.
2
2
2
reaction mass was refluxed for 8–10 h. The solvent was
evaporated under vacuum completely. Resultant residual
mass was stirred with 5–6 ml of water at 5–8 °C and fil-
tered. The pure crystalline compounds were obtained by
recrystallizing it in ethyl acetate to yield aryl hydrazides
Materials and methods
3(a–d). The reaction mixture containing aryl hydrazides
Lyophilized powder of V. russelli venom was purchased from
Irula Co-operative Society Ltd., Chennai, India. Fenoprofen
calcium, ibuprofen, 2-methylbenzoic acid, 2,3-dimethylben-
zoic acid, 4-methylbenzoic acid, 2,5-dimethylbenzoic acid,
3(a–d) (0.002 mol) and aromatic acids 4(a–f) (0.001 mol)
were dissolved in 8 ml of phosphorous oxychloride and
refluxed for 4–6 h under nitrogen. The reaction mixture
was cooled to room temperature and quenched using
1
23

Mol Cell Biochem
crushed ice. On neutralization of the contents, resultant
mass was washed with 20% sodium bicarbonate solution
followed by brine wash. Resultant residual product was
dried, and recrystallization was done using ethyl acetate to
give 5(a–t) as shown in Table 1. Spectral properties were
consistent with their assigned structures.
substrate stock (1 mM DMPC in methanol containing
2 mM Triton X-100 in Milli-Q water) was added and
incubated for 5 min at 37 °C. Activity was initiated by
adding 10 ng of sPLA alone or pre-incubated with dif-
2
ferent concentrations of 5(a–t) ranging from 0 to 100 lM
for 5 min at 37 °C. Reaction mixtures were incubated for
45 min at 37 °C. 50 ll of quenching solution was added at
a final concentration of 2 mM NaN , 50 lM ANS, and
3
Purification of sPLA (VRV-PL-VIIIa) from V.
2
russelli venom
50 mM EGTA, vortexed for 30 s, and incubated for 5 min
at RT. 2 ll of this solution was pipetted to measure RFU in
a Nanodrop ND3300 Ver 2.8 using an excitation UV-LED
(370 ± 10 nm) and emission was recorded at 480 nm in
dark condition. Enzyme activity was calculated by
Eq. (A.1), where DRFU is the change in RFU of test (with
sPLA ) with respect to control (without sPLA ) in the
sPLA from V. russelli venom was purified to homogeneity as
2
reported earlier [5]. Protein was estimated byLowry’s method
[15] . Briefly, V. russelli venom (100 mg) was fractionated on
pre-equilibrated Sephadex G-75 column (1.5 9 140 cm)
using 50 mM phosphate buffer pH 7.0. The protein was
resolved into majorthree peaks. The secondpeak, constituting
2
2
presence of inhibitor(s). The resultant RFU was compared
with the standard LPC curve to determine the sPLA2
activity in the presence of inhibitor [16].
about 30% of the total protein, showed major sPLA activity.
2
This sPLA peak fraction was lyophilized and further sub-
2
jected to pre-equilibrated CM-Sephadex C-25 column
(
1.5 9 40 cm) chromatography. The fractions were eluted
stepwise using phosphate buffers of varied ionic strength
50–200 mM) and pH (7.0–8.0). They were resolved into two
Intrinsic fluorescence spectra of sPLA -5m adduct
2
(
Quenching studies of deeply buried aromatic amino acid
residues at active site were performed in the presence of
fractions labeled as V and VIII, respectively. The above eluted
two fractions were similar to V and VIII protein profiles as
reported earlier [5]. The lyophilized fraction VIII was next
subjected to Sephadex G-50 column (1.0 9 40 cm) chro-
matographyandelutedusing50 mMphosphate bufferpH7.0,
sPLA with and without inhibitor (5m) after excitation at
2
280 nm. 1 ml of reaction mixture containing 30 lg of sPLA2,
10 mM CaCl , 20 mM Tris–HCl buffer, pH 7.5, with dif-
2
ferent concentrations of inhibitor 5m (0–100 lM) was incu-
bated at 37 °C for 10 min. Fluorescence emission spectra
(395–500 nm) of sPLA alone and mixture of sPLA -5m
and the obtained peak was checked for sPLA activity.
2
Homogeneity was checked by SDS-PAGE and RP-HPLC as
reported earlier [5] (data not shown).
2
2
were recorded. Tryptophan was used as standard to correct
non-specific quenching of enzyme with an inhibitor.
In vitro inhibition of VRV-PL-VIIIa by 2,5
disubstituted 1,3,4-oxadiazole derivatives 5(a–t)
Circular dichroism studies
sPLA2 activity was assayed according to the reported
Binding study of 5m with sPLA was carried out by UV-
2
method [16]. Briefly, a 50 ll buffer containing 50 mM
CD spectrum in the absence and presence of 5m at 5 lM
concentration, using Jasco J715 spectropolarimeter.
Tris–HCl buffer pH 7.5, 10 mM CaCl , and 100 lM
2
Table 1 List of 2,5
disubstituted 1, 3, 4-oxadiazole
derivatives 5(a–t)
Ligands
R
R
1
Ligands
R
R
1
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
2-Cl C
6
H
5
4-Cl C
4-Cl C
4-Cl C
4-Cl C
6
6
6
6
H
H
H
H
5
5
5
5
(4a)
(4a)
(4a)
(4a)
5k
5l
2-CH
3
C
6
H
5
2,3-CH
2,3-CH
3
3
C
C
6
H
5
5
(4d)
(4d)
2,3-Cl
2-CH
2,3-CH
2-Cl C
2,3-Cl
2-CH
2
C
6
H
4
2,3-CH
3
C
6
H
5
4
4
6
H
3
C
6
H
5
5m
5n
5o
5p
5q
5r
5s
2-Cl C
6
H
5
Fenoprofen (4e)
Fenoprofen (4e)
Fenoprofen (4e)
Fenoprofen (4e)
Ibuprofen (4f)
Ibuprofen (4f)
Ibuprofen (4f)
Ibuprofen (4f)
3
C
6
H
4
2,3- Cl
2
C
6
H
4
H
6 5
2,5-CH
2,5-CH
2,5-CH
2,5-CH
3
3
3
3
C
C
C
C
6
H
6
H
6
H
6
H
4
4
4
4
(4b)
(4b)
(4b)
(4b)
2-CH
3 6 5
C H
2
C
6
H
4
3 6
2,3-CH C H
g
h
i
3
C
6
H
5
2-Cl C
6
H
5
2,3-CH
2-CH
2,3-CH
3
C
6
H
4
4
2,3- Cl
2
C
6
H
4
3
C
6
H
5
2-CH
2-CH
3
3
C
6
H
H
5
(4c)
2-CH
3 6 5
C H
j
3
C
6
H
C
6
5
(4c)
5t
3 6
2,3-CH C H
123

Mol Cell Biochem
Briefly, 1 ml reaction mixture containing 0.2 mg/ml con-
force field. Using PROPKA, pH was fixed and optimized to
7.5. Non-hydrogen atoms were minimized by restrained
centration of sPLA , 10 mM Tris–HCl buffer pH 7.5,
2
˚
minimization to default RMSD to 0.3 A. The docking
1
0 mM CaCl , and with/without 5m was added and the
2
spectrum was recorded at RT between 195 and 260 nm
using quartz cuvette with path length of 1 cm. The band-
width and response time were 2 nm and 2 s, respectively.
The protein spectrum was corrected by subtracting a blank
solution containing 10 mM Tris–HCl buffer (pH 7.5) and
study of selected target and ligands 5(a–t) was done by
using Maestro 9.3, docking module Glide XP (extra pre-
cision). Receptor grid generation of VRV-PL-VIIIa was
defined as Asp47, His48, and Gly30 to prepare a grid box
˚
˚
˚
of 30 A x 30 A x 30 A size to dock ligands. Docking
precision and ligand sampling were fixed to flexible mode.
Twenty numbers of posses per ligands were chosen for
accurate docking with the target. The resultant output file
was visualized on maestro; further, the docking calculation
was analyzed and validated by comparing with the standard
inhibitor. Additionally, molecular dynamics (MD) simula-
tions were performed after the docking simulations to
confirm the stability of the ligand in the proposed site with
the Desmond program inbuilt OPLS 2005. VRV-PL-VIIIa-
5m complex was positioned in an orthorhombic cell soaked
with a pre-equilibrated box of explicit solvent including
1
0 mM CaCl . The scale on the CD spectra was normal-
2
ized to mean residue ellipticity (MRE), which was mea-
2
-1
sured in degrees cm d mol
where H is raw ellipticity measured in machine units
millidegrees). The mean residue weight (MRW) for the
residue using Eq. (B.1),
(
peptide bond was calculated from Eq. (B.2), where M is the
molecular mass of the polypeptide chain (in Da); N is the
number of amino acids present in the target protein; l the is
path length in cm; and [P] is the protein concentration in
mg/ml [17]. The MRE was plotted against the corre-
sponding wavelength. Secondary structure calculations of
CD spectra were determined by using K2D3 server [18].
10 mM CaCl salt with single-point charge (SPC) water
2
˚
˚
˚
model in a cubic box with 10 A 9 10 A 9 10 A dimen-
sion. Overlapping solvent molecules were removed, and an
appropriate number of counter ions were added to maintain
charge neutrality and energy minimization for both SPC
water models and the protein before subjecting the system
to MD simulation runs [20].
Inhibitor-binding analysis using ANS
as fluorescence probe
The 50 ll reaction mixture containing 100 lM substrate
and different concentrations of inhibitor 5m (0–100 lM) in
5
0 mM Tris–HCl buffer, pH 7.4, and 10 mM CaCl was
2
incubated for 10 min at 37 °C. Fifty microliters of
quenching solution was prepared and added as mentioned
earlier and vortexed for 30 s and incubated for 5 min at
RT. Two microliters of this solution was pipetted to mea-
sure RFU. Emission coefficient (EC) was measured as
the slope of the plot of fluorescence intensity as a function
of ANS along with DMPC complex in the presence or
absence of 5m: EC =Em /Em , where Em and Em are the
Inhibition of indirect hemolytic activity by 2,5
disubstituted 1,3,4-oxadiazole derivatives
Indirect hemolytic activity was assayed according to the
method of Boman and Kalletta [21]. Briefly, packed human
erythrocytes, egg yolk, and phosphate-buffered saline pH
7.5 were mixed in the ratio of 1:1:8 (v/v). 1 ml of this
suspension was incubated separately for 60 min at 37 °C
t
b
t
b
ECs of ANS in the ternary (DMPC-5m-ANS) and in the
binary (DMPC-ANS) complexes, respectively.
with 8 lg of sPLA alone or pre-incubated with different
2
concentrations of inhibitor 5m ranging from 0 to 100 lM
for 5 min at RT. Reaction was stopped by adding 9 ml of
ice-cold PBS with pH 7.5; reaction mixtures were cen-
trifuged at 4 °C for 10 min at 15009g. The resultant % of
hemoglobin released in supernatant was measured spec-
trophotometrically at 540 nm.
In silico molecular docking studies
The structural drawing and geometry cleaning of the 2,5
disubstituted 1,3,4-oxadiazole derivatives 5(a–t) were
performed in Maestro 9.3 and then subjected to other
parameters, namely, energy minimization by using OPLS
Neutralization of edema-inducing activity
of sPLA2
2
005 force field. The crystal structure of a VRV-PL-VIIIa
from V. russelli, complex with 2-hydroxy-4-aminobenzoic
˚
acid (PDB ID:1SXK, 1.2 A resolution) [19], was retrieved
The procedure of Yamakawa et al. [22], as modified by
Vishwanath et al., on groups of four animals each [23] was
followed. Swiss albino male mice weighing 20–25 g were
through Brookhaven protein data bank (PDB). Crystal
structure was imported and refined by a multistep process
through the protein preparation wizard of Maestro 9.3,
which includes energy minimization using OPLS-2005
injected with 8 lg of sPLA (VRV-PL-VIIIa) with and
2
without 5m (0–200 lM) in a total volume of 30 ll saline
1
23

Mol Cell Biochem
into the intraplantar surface of the right hind footpad. Left
footpad received 30 ll of saline and served as vehicle.
After 45 min, the mice were sacrificed by giving anesthesia
before injecting intraperitoneally(test). After 4 h of
administration of respective controls and test samples, mice
were anesthetized by administering pentobarbitone (30 mg/
kg, i.p) and sacrificed by cervical dislocation. The lung
tissue was dissected, processed, and visualized as men-
tioned above.
(
pentobarbitone, 30 mg/kg) through i.p, and both hind
limbs were removed at the ankle joint and weighed indi-
vidually after cervical dislocation. The increase in weight
due to edema is expressed as Eq. (C.1) Percentage increase
of sham-injected control compared to the un-injected limb
was found to be 175 ± 4%.
Statistical analysis
Results were presented as the mean ± SEM. Two-way
ANOVA followed by Dunnett’s multiple comparisons test
and Sigmoidal four parametric logical (4PL) fit was com-
Myotoxicity
Ò
Myotoxicity was determined as reported earlier in groups
of four animals each [24].Briefly, half the LD50 value [5]
puted and analyzed using the GraphPad Prism V.6.05
(GraphPad Software, Inc., USA). Statistical significance
(
5.3 mg/kg body weight) of VRV-PL-VIIIa in 100 ll sal-
was
represented
as
*P \ 0.05,
***P \ 0.001,
ine was injected intramuscularly into the right thigh of
positive control grouped mice (n = 4). The groups are
receiving 100 ll saline (vehicle) or only 5m in saline
served as negative control. For inhibition studies, the
****P \ 0.0001.
Results
sPLA was pre-incubated with the inhibitor in the ratio of
2
1
:5 (sPLA :5m, w/w) for 15 min before injecting intra-
2
Chemical synthesis of 1,3,4-oxadiazoles
muscularly (test). After 4 h of administration of respective
controls and test samples, mice were anesthetized by
administering pentobarbitone (30 mg/kg, i.p) and sacrificed
by cervical dislocation. The abdominal cavity was opened,
and blood was drawn by puncturing the aorta. The 1:20
diluted serum was estimated for cytosolic markers like
AST, LDH, and CK levels by Agappe diagnostic kits using
Toshiba TBA 40FR Accute analyzer. Activities were
expressed as units/l.
The title compounds were synthesized by reacting aryl
hydrazides 3(a–d) with various aromatic acids 4(a–f)
(0.001 mol) in the presence of the cyclizing agent phos-
phorous oxychloride by refluxing the mixture under nitro-
gen atmosphere (Scheme 1). The resultant residual product
was dried and recrystallized using ethyl acetate to obtain
title compounds 5(a–t) (Table 1). Spectral properties were
consistent with their assigned structures.
The thigh muscle tissue was dissected from the site of
injection and fixed in Bouin’s solution followed by
sequential dehydration by processing the tissue through
different grades of alcohol and chloroform:alcohol mixture.
The processed tissue was embedded in paraffin block and
cut into 3 lM–4 lM thin sections. The sections were
stained with hematoxylin and eosin for microscopic
observation. The sections were observed under a micro-
In vitro inhibitory effect of 1,3,4-oxadiazoles
on VRV-PL-VIIIa
The 1,3,4-oxadiazole ring underwent nucleophilic attack at
carbon, followed by ring cleavage and electrophilic attack
at nitrogen [25]. The series of 1,3,4-oxadiazoles 5(a–t)
were assessed for PLA inhibition studies, and the results
2
scope, and photographs were taken using Jenoptik camera
Ò
are tabulated in Table 2. The tested 2,5–disubstituted-
(
ProgResCapturePro ) attached to the microscope.
1,3,4-oxadiazoles 5(a–t) inhibited sPLA in dose-depen-
2
dent manner with an IC50 value ranging from 11.52 to
72 lM which are computed and analyzed using sigmoidal
Lung hemorrhage
4PL curve fit. Among the tested compounds, 5m showed
significant inhibition against VRV-PL-VIIIa with IC50
value of 11.52 lM (Fig. 1), when compared to other
structurally related molecules. Overall the result indicates
that dimethyl/mono chloro at ortho position in R is most
favorable for inhibition of VRV-PL-VIIIa in terms of
hydrophobicity (5j) and electronegative (5e), respectively
(Table 2). With this view, we derivatized fenoprofen with
1,3,4 oxadiazole nucleus where mono chloro/dimethyl
group was present at ortho position in case R of 5m to 5p.
Lung hemorrhage was determined as reported earlier in
groups of four animals each [24]. Half the LD50 value of
VRV-PL-VIIIa in 100 ll saline was injected intraperi-
toneally (i.p) into the positive control grouped mice
(n = 4). The groups received 100 ll saline (vehicle) or
only inhibitor 5m in saline served as negative control. For
inhibition studies, the sPLA was pre-incubated with the
2
inhibitor in the ratio of 1:5 (sPLA :5m, w/w) for 15 min
2
123

Mol Cell Biochem
Scheme 1 Substituted aromatic
acids 1(a–d) are converted into
the corresponding esters 2(a–d).
These esters were converted
into the corresponding
N
N
O
O
O
R -COOH
EtOH/H SO4
1
R
2
NH -NH -H O
2
2
2
4(a-f)
O
5(a-t)
R
OH
(a-d)
Reflux
R
OEt
R
NHNH2 POCl Reflux
3
R1
2
(a-d)
3(a-d)
1
hydrazides 3(a–d). Aryl
hydrazides 3(a–d) cyclized with
aromatic acids from 4(a–f) were
coupled in the presence of
phosphorous oxychloride under
reflux, to give respective 2,5–
disubstituted 1, 3, 4-oxadiazoles
derivatives 5(a–t) as mentioned
in Table 1
Where R
Where R1
4a
Cl
H C
CH3
3
1a
4d
CH3
CH3
Cl Cl
CH3
1
b
c
H C
3
4
b
c
1
O
H C
e†
3
4
CH3
4
CH3 CH3
1
d
CH3
CH3
f†
CH3
4
Table 2 In vitro and in silico
anti-inflammatory activity of
Ligands
In vitro assay
In silico analysis
2
4
,5 disubstituted 1, 3,
-oxadiazole derivatives
4PL fit IC50 (lM)
Glide Emodel
(kcal/mol)
XP
G-score
Glide energy
(kcal/mol)
Glide ligand efficiency
(
Scheme 1)
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
41.58
44.41
49.68
29.45
27.22
61.12
52.26
72.18
31.40
23.75
60.18
70.14
11.52
35.04
30.45
57.92
41.33
32.78
32.70
25.84
2.0
-29.44
-36.98
-28.10
-37.99
-33.16
-34.15
-35.40
-33.79
-37.53
-44.01
-36.96
-30.88
-44.87
-33.58
-32.44
-30.61
-43.74
-33.06
-36.71
-35.60
-40.54
-3.41
-3.38
-3.07
-3.87
-3.89
-22.34
-26.01
-21.22
-22.74
-20.33
-30.05
-27.26
-27.67
-24.15
-34.71
-27.76
-25.55
-34.80
-25.57
-24.56
-23.71
-30.59
-0.81
-0.78
-0.74
-0.89
-0.90
-0.04
-0.72
0.79
-0.15
-2.90
3.15
g
h
i
-3.83
-4.21
-2.64
1.89
-0.88
-0.98
-0.66
0.47
j
k
l
m
n
o
p
q
r
s
-6.40
-3.47
-3.85
-2.86
-3.46
-3.62
-3.63
-4.09
-1.86
-1.03
-0.87
-0.90
-0.72
-0.80
-0.84
-0.84
-0.95
-0.46
-25.95
-23.07
-20.06
-32.20
t
Quercetin
Intrinsic fluorescence spectra of sPLA -5m adduct
2
Fig. 2a. Addition of 5m quenched the intrinsic fluorescence
spectra of sPLA enzyme in a dose-dependent manner. The
2
Intrinsic fluorescence of purified VRV-PL-VIIIa (sPLA2)
with lead molecule 5m alone gives the maximum intensity
corresponding to tryptophan amino acid [26] as showed in
RFU of VRV-PL-VIIIa decreases as the inhibitor concen-
tration increased (Fig. 2b).
1
23

Mol Cell Biochem
Circular dichroism studies
Far UV-CD spectra of VRV-PL-VIIIa enzyme with/without
5m were evaluated for major characteristic changes initiated
between 195 and 222 nm. In the presence of 5 lM con-
centration of 5m (&half IC50 value) [26], it was found that
enzyme peak deviate steeply downward compared to the
native structure. From 208 nm to 202 nm a large steep
negative peak was observed confirming the quenching of a-
helix in the presence of 5m wherein active site amino acid of
VRV-PL-VIIIa resides from 39 to 52 position in the helix.
Inhibitor-binding analysis using ANS
as a fluorescence probe
Fig. 1 Dose response curves showing the effect of 5m on VRV-PL-
VIIIa. IC50 was calculated using sigmoidal four parameter logistic fit
ANS being a strong anion binds predominantly to cationic
groups of choline head of DMPC through ion pair
(
4PL) plot. Inset percentage of sPLA
2
activity in the presence of 5m
(
0–100 lM). All data values represent mean ± SEM (n = 6)
Fig. 2 a Intrinsic fluorescence spectral interaction of VRV-PL-VIIIa
with 5m. Fluorescence spectra were recorded after excitation at
480 nm. All data values represent mean ± SEM (n = 6). c Inhibitor-
binding analysis using ANS as a fluorescence probe: Em /Em , where
t b
2
80 nm. Emission intensity was recorded from 395 to 500 nm.
Emt and Emb are the ECs of ANS in the ternary (DMPC-5m-ANS)
and in the binary (DMPC-ANS) complexes, respectively. All data
values represent mean ± SEM (n = 6)
Different concentrations of 5m ranging from 0 to 100 lM, respec-
tively, were recorded. b Effect of fluorescence intensity of ANS-
DMPC in the presence of 5m. Emission intensity was recorded at
123

Mol Cell Biochem
Fig. 3 a Effect of 5m on
secondary structure of VRV-PL-
VIIIa. Analysis of far UV-CD
spectra for VRV-PL-VIIIa in
association with 5 lM of 5m.
Spectra for the VRV-PL-VIIIa
alone or VRV-PL-VIIIa ? 5m
are shown. The spectra shown
are unsmoothed and corrected
only by subtraction of buffer
blanks. The scale on the CD
spectra was normalized to MRE.
b PDB Kabsch and Sander’s
secondary structure prediction
of VRV-PL-VIIIa to elucidate
accurate bind mode of 5m
formation, imparting hydrophobicity resulting in an
increase in fluorescence [27]. When these interactions are
interrupted, fluorescence of ANS-DMPC decreases. As the
concentration of 5m (0–100 lM) is increased, the fluo-
rescence of ANS-DMPC adducts decreases marginally and
remains constant at 100 lM of 5m. This clearly suggests
that 5m is competing weakly with ANS binding to DMPC,
although the nitrogen atoms present in the 1,3,4 oxadiazole
ring at the 2nd and 3rd position of 5m lacks amphiphilicity
catalysis by forming hydrogen bonding between Asp99 and
Asp49 via water molecule as a part of catalytic network.
Thermodynamically, His48 contributes 30 kJ/mol to a total
catalytic energy of 40 kJ/mol [29, 30]. It is known that any
structural perturbations in His48 of N-terminal a-helix of
PLA play an important role in both membrane binding of
2
the enzyme and substrate binding to the catalytic site that
reduces the binding affinity of the enzyme–substrate
complex [31]. All the ligands showed interaction with
Trp31 which is close to the active site residing at random
coil. Depletion in a-helical and random coil clearly sug-
gests that 5m binds with His48 (a-helix), which resides
near random coil (Fig. 4a, b); thus, this authenticates the
CD data with respect to Kabsch and Sander’s secondary
structure prediction of VRV-PL-VIIIa (Figs. 3b, 4a). Our
MD simulation clearly reveals that all the ligands embed-
ded in the substrate-binding pocket completely blocks the
access of the substrate, near the catalytic site (data not
shown).
(
Fig. 2b). Thus, it concludes that 5m compete very weakly
with choline-binding space resulting in perturbed substrate-
binding site. Results are evidenced as reported earlier [16].
Further, our results show that EC of 5m (1.05) is com-
paratively less than chlorphentermine (4.24) [28] which
induces drug-induced phospholipidosis.
In silico interaction analysis of 5(a–t) against VRV-
PL-VIIIa
Molecular docking and molecular dynamics simulations
revealed that GII PLA has a defined conserved active site
2
In situ inhibition of indirect hemolytic activity
of VRV-PL-VIIIa by 5(a–t)
within
a
hydrophobic channel lined by invariant
hydrophobic residues. The active site residues His48,
Asp49, Tyr52, and Asp99 are directly connected to the
channel [3, 29]. His48 was known to play a major role in
In situ indirect hemolytic activity of VRV-PL-VIIIa, in
presence of compound 5m was evaluated using egg yolk
1
23

Mol Cell Biochem
Fig. 4 In silico analysis of
VRV-PL-VIIIa with 5m.
Docking pose of 5m with VRV-
PL-VIIIa showing molecular
interaction before molecular
dynamic simulation (a). Binding
geometry of 5m (wired model)
with hydrophobic core residing
at active site of VRV-PL-VIIIa
after MD simulations (b)
Fig. 6 Neutralization of edema-inducing activity of VRV-PL-VIIIa
by 5m. All data values represent mean ± SEM (n = 4)
Fig. 5 Neutralization of indirect hemolytic activity of VRV-PL-VIIIa
by 5m. All data values represent mean ± SEM (n = 6)
123

Mol Cell Biochem
Myotoxicity
VRV-PL-VIIIa is known to possess potent myotoxicity
32]. We evaluated the efficacy of 5m in neutralizing the
myotoxicity of VRV-PL-VIIIa in the ratio of 1:5
sPLA :5m). Intramuscular injection of VRV-PL-VIIIa
[
(
2
causes degeneration of muscle fibers resulting in infiltra-
tion of polymorphonuclear leukocytes that rupture blood
vessels in mice (Fig. 8; M2), when compared to normal
healthy controls (Fig. 8; M1) [3, 7]. The mice injected with
VRV-PL-VIIIa:5m (1:5 w/w) mixture showed decreased
levels of CK, LDH, and AST activities (Fig. 7a); this is
also associated with decrease in myolysis and infiltration of
polymorphonuclear leukocytes in skeletal muscle fibers
with intact musculature (Fig. 8; M3) and it is comparable
with normal healthy control. 99% of confidence interval
(Dunnett’s test) shows the band of significance, when
compared with the treated group (Fig. 7b).
Lung hemorrhage
Fig. 7 Effect of 5m on serum AST, (inset) CK, and LDH activities in
mice which received saline (vehicle), VRV-PL-VIIIa (positive
control), 5m (negative control), and treated group VRV-PL-
VIIIa ? 5m (1:5, w/w) intramuscular (a). Dunnett’s test with 99%
confidence intervals revealing the band of significance of 5m with
reduced cytosolic and myotoxicity markers when compared to control
represented in colored dotted line (b). All data values represent
mean ± SEM (n = 4). *P \ 0.05, ***P \ 0.001, ****P \ 0.0001
Intraperitoneal injection of VRV-PL-VIIIa is known to
induce hemolysis and lung damage resulting in rupture of
blood vessels in mice [5, 26]. When a group of mice were
injected (i.p.) with half of the LD50 dose of sPLA , lung
2
damage occurred, which can be visualized by hemolysis
and was confirmed by histopathological studies (Fig. 8).
Microscopic section of mice injected with saline (control)
showed lung parenchyma with normal histology (L1).
Positive control mice that received VRV-PL-VIIIa alone,
showed lung tissue with areas of interstitial, alveolar
hemorrhage associated with intense rupture of blood ves-
sels and intense infiltration of polymorphonuclear leuko-
and packed erythrocytes as substrate. VRV-PL-VIIIa alone
exhibited 96 ± 2.0% hemolysis of erythrocyte (positive
control) when compared with pure water (100% lysis).
VRV-PL-VIIIa incubated with 5m (0–100 lM), respec-
tively, for 60 min at 37 °C, inhibited hemolysis in a dose-
dependent manner (Fig. 5).
cytes (L2). The mice injected with sPLA with 5m (1:5
2
w/w) showed lung parenchyma with decrease in areas of
hemorrhage and rupture of blood vessels with intact red
blood cells (L3), which is comparable with normal healthy
control.
5
m showed comparatively significant dose-dependent
inhibition, induced by VRV-PL-VIIIa compared to positive
control.
In vivo neutralization of edema-inducing activity
of VRV-PL-VIIIa by 5m
Discussion
1,3,4-Oxadiazole is an important class of heterocycles,
The PLA from snake venoms, when injected into the
2
with a whole array of therapeutic values like analgesic,
bactericidal, antifungal, anticonvulsant, psychotropic, plant
growth regulating, and mono amino oxidase inhibition.
1,3,4-Oxadiazole exerts dual COX/LOX inhibitors consti-
tuting a valuable alternative to classical NSAIDs and
selective COX-2 inhibitors for the treatment of inflamma-
tory diseases. But COX and LOX operate downstream
inflammatory cascade [15]. So, we attempted to block the
mouse footpad, induced edema in the ratio of more than
1
75 ± 4 when compared to saline-injected mice. Since
sPLA s are edematous in vivo, we examined the effect of
2
5
m on the edema-inducing activity of VRV-PL-VIIIa at
different concentrations ranging from 0 to 100 lM. 5m
inhibited VRV-PL-VIIIa in a concentration-dependent
manner (inset of Fig. 6). 5m significantly inhibited edema-
inducing activity of VRV-PL-VIIIa (Fig. 6).
upstream main enzyme sPLA , which drives inflammatory
2
1
23

Mol Cell Biochem
Fig. 8 Microphotographs (H 9 E 9 100) of lung and muscle tissue
histopathological studies: mice injected (i.m) with saline showed
normal skeleton muscle fiber (M1) and normal lung parenchyma (L1);
mice injected with VRV-PL-VIIIa showed skeleton muscle fiber with
area of rhabdomyolysis and intense polymorphonuclear infiltrate
polymorphonuclear infiltrate, and alveolar hemorrhage (L2); mice
injected with VRV-PL-VIIIa-5m (1:5, w/w) showed skeleton muscle
fiber with decreased polymorphonuclear infiltration (M3) and lung
showed decreasing interstitial hemorrhage associated with decreased
PMN leukocytes (L3)
(
M2), and lung parenchyma showed extensive interstitial, intense
cascade [33]. Hence, in our study, 2,5–disubstituted-1,3,4-
oxadiazoles 5(a–t) inhibited sPLA2 in dose-dependent
manner with an IC50 value ranging from 11.52 lM to
72 lM. Fenoprofen was derivatized with 1,3,4 oxadiazole
nucleus where chloro/methyl group present at ortho posi-
tion in case R of 5m to 5p showed comparative low
123

Mol Cell Biochem
Table 3 Predicted secondary structure percentages of 5m on VRV-
PL-VIIIa
it possesses chlorine at the ortho position and higher
hydrophobic moiety at R of fenoprofen (Fig. 1). Overall,
1
the result suggested that the change in R of 5(a–t) varies
1
VRV-PL-VIIIa
Native* (%)
% change in presence of 5m
treated*)
(
with binding affinity for VRV-PL-VIIIa with substrate.
This confirms that the ortho position is most vital for
a-helix
33.3
10.66
3.06
inhibition of sPLA (Table 2). When substrate or ligands
2
b-sheet
18.14
48.56
bind to active site of the target, it causes conformational
change resulting in a change in intrinsic fluorescence [34].
Random coil
13.72
*
Secondary structure contents were calculated using K2D3 server
Intrinsic fluorescence spectra of sPLA -5m adduct showed
2
quenching of fluorescence in a concentration dependent,
suggesting that 5m interacts with the active site of enzyme
(Fig. 2a) as reported earlier [26].
effective IC50 value, whereas in case of both ortho and
meta position it resulted in higher IC50 (5n, 5p). These
results suggest that electronegativity prevailing around the
One of the major hypotheses, which explains the strong
interaction of inhibitor (amphiphilic amine drugs) with
phospholipids, causes inappropriate substrate for phos-
pholipase to act on and thereby results in accumulation of
these adducts into lung tissue, which ultimately results in
phospholipidosis [28]. Phentermine and chlorphentermine
are structurally similar but pharmacologically they differ
mainly by two reasons. One is devoid of chlorine and
another is phentermine which does not cause phospho-
lipidosis, whereas chlorphentermine causes phospholipi-
dosis, because it contains amphiphilic chlorine molecule.
5m at ortho (chloro) position binds strongly with the active
site of VRV-PL-VIIIa.
However, in case of derivatizing ibuprofen with 1,3,4
oxadiazole nucleus 5(q–t), it demanded more electroneg-
ativity (5r) and hydrophobicity (5t) at R. These suggest
that the orientation of methyl group and chlorine molecule
plays an important role in interacting with amino acids at
the catalytic site for inhibiting VRV-PL-VIIIa. Notewor-
thy, these data validate that 5m is comparatively potent, as
Table 4 ADMET evaluation of
Name
Absorption, distribution, metabolism, excretion, and toxicity (ADME/tox)
2
4
,5 disubstituted 1, 3,
-oxadiazole derivatives
BBB HIA
level
Solubility Hepatotoxicity CYP2D6 PPB
probability probability level
AlogP98 PSA-2D
level level
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
1.00
0.00
1.00
1.00
0.00
0.00
1.00
0.00
1.00
1.00
1.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
4.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
1.00
0.00
1.00
1.00
1.00
0.00
1.00
1.00
2.00
2.00
2.00
2.00
2.00
1.00
2.00
1.00
2.00
2.00
2.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
3.00
0.83
0.91
0.74
0.74
0.82
0.93
0.78
0.83
0.81
0.77
0.77
0.74
0.88
0.95
0.89
0.90
0.33
0.49
0.32
0.44
0.93
0.77
0.76
0.80
0.80
0.52
0.39
0.52
0.40
0.60
0.73
0.73
0.39
0.71
0.64
0.72
0.67
0.44
0.42
0.49
0.47
0.55
2.00
2.00
2.00
2.00
2.00
2.00
2.00
2.00
2.00
2.00
2.00
1.00
2.00
2.00
2.00
2.00
2.00
2.00
2.00
2.00
2.00
4.20
4.87
4.03
4.51
4.69
5.36
4.51
5.00
4.03
4.51
4.51
5.00
5.78
6.44
5.60
6.08
5.87
6.53
5.69
6.17
1.63
35.08
35.08
35.08
35.08
35.08
35.08
35.08
35.08
35.08
35.08
35.08
35.08
44.01
44.01
44.01
44.01
35.08
35.08
35.08
35.08
130.31
g
h
i
j
k
l
m
n
o
p
q
r
s
t
Quercetin
(
standard)
5
m denotes the potent ligand henceforth it has been highlighted with bold
1
23

Mol Cell Biochem
Hence, 5m possesses chlorine molecule in its R group
(P \ 0.0001) (Fig. 6). 5m inhibited edema-forming activ-
ity in a dose-dependent manner. When VRV-PL-VIIIa was
injected (i.m) with half of the LD50 dose, tissue showed
bundles of skeletal muscle fibers with areas of myolysis
around the injected spot in contrast to the normal healthy
control. Microscopic results were confirmed by elevated
levels of cytosolic markers such as LDH, AST, and CK
activities in case of positive control which received VRV-
PL-VIIIa alone [37]. Treated group showed decrease in
area of myolysis around the injected spot. VRV-PL-VIIIa
induced lung hemorrhage, when injected intraperitoneally
to the mice [5]. 5m showed decrease in areas of interstitial,
alveolar hemorrhage associated with intense rupture of
blood vessels in VRV-PL-VIIIa-induced group. It was also
evident by microscopic observation that infiltration of
polymorphonuclear leukocytes was reduced, when com-
pared to the positive control.
(
Scheme 1). Hence, we have evaluated whether 5m inter-
acts with choline group in DMPC. Our result showed that
m exerts very weak interaction with DMPC (Fig. 2b, c).
5
Hence 5m does not attribute significant fluorescence
intensity of binary (5m-DMPC) complex, thereby not
triggering drug-induced phospholipidosis, which is a major
concern of sPLA inhibitors [28].
2
Biophysical study indicates that 5m interacts with
sPLA enzyme to bring about inhibition, which is further
2
confirmed by circular dichroism (CD) spectral study. CD
data suggest that major secondary changes are substanti-
ated at random coil and a-helix (Fig. 3a); hence it is evi-
dent that 5m is bringing major changes at active site.
Detailed analysis at [h]k (k = 198) of VRV-PL-
VIIIa ? 5m spectral peak showed a maximum saturation,
suggesting a complete abolishment of random coil,
whereas at [h]k (k = 218) of VRV-PL-VIIIa ? 5m peak a
minor change was observed in b-sheet. The change in the
percentage of secondary structure of VRV-PL-VIIIa upon
interaction with IC50 concentration of 5m is summarized
Conclusion
Compounds inhibiting sPLA2 have been implicated as
potential therapeutic agents, in the treatment of inflam-
matory disorders. Recent studies show that over 50% of
patients consuming known NSAIDs like fenoprofen,
diclofenac, ibuprofen, undergo major damage in their small
intestine [14, 38]. Hence, we targeted the primary enzyme
(
Table 3).To analyze the binding of 5m with secondary
structure of enzyme, active site residues were analyzed by
PDB structure and Kabsch–Sander’s secondary structure
prediction models for VRV-PL-VIIIa using Accelrys dis-
covery (Fig. 3b). The results illustrated that these models
are closely related to elucidating a-helical, b-sheet, and
random coils of VRV-PL-VIIIa as reported earlier [26].
These data authenticate that 5m binds chiefly to random
coil and a-helical of active site. Thus, CD study substan-
tiates the fluorescence data that 5m interacts closely with
enzyme for inhibition. Molecular docking studies showed
an interacting map of VRV-PL-VIIIa with compound 5m
(sPLA ), which drives the inflammatory cascade. Results
2
emphasize that 1,3,4-oxadiazole may act as a better pro-
drug to block the release of arachidonic acid, which is a
substrate for COX-2.
Our experimental data revealed that the structure–ac-
tivity relationship studies of 1,3,4-oxadiazoles with sPLA2
correlated with the molecular interaction studies. The
binding mode of 5m involved crucial interactions with
important residues for substrate recognition, such as His48
and consequent perturbation of His48 resulted in down-
regulation of VRV-PL-VIIIa. 5m binds within or close to
the calcium-binding loop of the catalytic triad, wherein
(
Fig. 4a, b). Based on XP glide score, 5m showed a
promising scoring function, when compared to other
structurally related compounds (Table 4). Additionally, the
low E model value indicates the best binding affinity
between protein and ligand [12]. In general, all the ligands
showed interaction with Trp31 which is close to the active
site. Importantly, binding of ligands 5m near the a-helical
random coil clearly suggests that it had interaction with
His48, which resides near the random coil. 5m showed
comparatively significant dose-dependent inhibition,
induced by VRV-PL-VIIIa compared to positive control.
Inhibition of indirect hemolytic activity of VRV-PL-VIIIa
was tested with 5m and found to be more potent when
compared to control (Fig. 5).
2
?
Ca and His48 reside. Hence, this is validated by ANS–
m fluorescent interaction studies, CD and Kabsch–San-
der’s secondary structure prediction models. Thus, the
5
compound 5m showed potential anti-inflammatory activity
in vitro, in situ, and in vivo studies by targeting sPLA . The
2
present study may lead to developing better inhibitors
against venom PLA2.
Acknowledgements SNS thanks UGC, New Delhi [UGC-MRP Vide
No: F. No. 38-220/2009 (SR) dated December 24, 2009]. HKV thanks
CSIR, Govt of India for CSIR-Senior Research Fellowship (08/584
In vivo toxicological study showed that, VRV-PL-VIIIa
affects the vital organs such as the lung, liver, and kidney
by causing hemorrhage in victims. Edema inducing activity
is an unusual activity of VRV-PL-VIIIa [5, 35, 36]. VRV-
PL-VIIIa-induced edema ratio decreased significantly to
(
0001)/2012-EMR-I), and TEQIP-II, SJCE for Ph.D. assistantship.
We thank Dr. Basappa, Dr. Sharath, Mr. Shankar and IOE, University
of Mysore, Mysuru, Schr o¨ dinger team, Mr. Govindaraju -MBU
(IISC,Bengaluru), Dr. Sunila, and Dr. M. Guruswamy, JSS hospital
Mysuru, for their help towards this work.
5
0%, when compared to vehicle edematous leg
123

Mol Cell Biochem
Compliance with ethical standards
12. Husain A, Ajmal M (2009) Synthesis of novel 1,3,4-oxadiazole
derivatives and their biological properties. Acta Pharm
59:223–233. doi:10.2478/v10007-009-0011-1
Conflict of interest The authors have no conflict of interest.
1
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