I.C. Henao Castañeda et al. / Journal of Molecular Structure 1028 (2012) 7–12
9
and kept in captivity at the serpentarium of the Universidad de
Antioquia (Medellín, Colombia). PLA2 was purified by molecular
exclusion chromatography on Sephadex G-75 and reverse-phase
HPLC on C-18 column eluted at 1.0 mL/min with a gradient from
0% to 100% acetonitrile in 0.1% trifluoroacetic acid (v/v). Absor-
bance in effluent solution was recorded at wavelength of 280 nm
[13].
nature of each amino acid, on the basis of the ionized form, ex-
pected in physiological condition. This module also controls the
atomic charges assignment. Second, each 3D structure of the pro-
tein was relaxed through constrained local minimization, using
the OPLS force fields in order to remove possible structural mis-
matches due to the automatic procedure employed to add the
hydrogen atoms. When necessary, bonds, bond orders, hybridiza-
tions, and hydrogen atoms were added, charges were assigned (a
formal charge of +2 for Ca ion) and flexible torsions of ligands were
2.5. Inhibition of phospholipase A2 activity using phosphatidilcholine
as substrate
detected. The
a-carbon of His48 was used as center of the grid
(X = 44.981, Y = 27.889 and Z = 46.392), whose size was 24 Å3.
Exhaustiveness = 20. Then, the ligand poses with best affinity were
chosen, and a visual inspection of the interactions at the active site
was performed and recorded. MMV [20] was used to generate
docking images.
This activity was assayed according to the method reported by
Dole [14], with titration of free fatty acids (FA) released from phos-
phatidylcholine (from dried egg yolk, Sigma) suspended in 1% Tri-
ton X-100, 0.1 M Tris–HCl, 0.01 M CaCl2, pH 8.5 buffer, using
20
The amount of protein was selected from the linear region of activ-
ity curves. For inhibition experiments, 2 M of each compound
lg/10 lL of PLA2. The time of reaction was 15 min at 37 °C.
3. Results and discussion
l
were pre-incubated for 30 min at 37 °C before PLA2 activity deter-
mination. The results are indicated as inhibition percentage, where
100% is the activity induced by PLA2 alone.
3.1. Vibrational analysis
Compounds I, II, III and IV present 78, 84, 84 and 87 normal
modes of vibration, respectively. Normal modes from the benzyl
ring were quite similar in the four compounds, and the normal
modes originated in the benzoate ring showed some differences
due to the different substitution pattern. Vibrational frequencies
calculated at B3LYP/6–31+G(d,p) were scaled by 0.964 [21]. The
spectral position of the C@O carbonyl experimental bands ap-
2.6. Inhibition of phospholipase A2 activity using 4-nitro-3-
octanoyloxybenzoic acid (4N3OBA) as substrate
The measurements of enzymatic activity using the linear sub-
strate 4N3OBA were performed according to the method described
by Holzer and Mackessy [15] and adapted for a 96-well ELISA plate.
peared at 1664, 1653, 1643 and 1653 cmꢀ1; while calculated
mC@O
The standard assay contained 200
10 mM CaCl2, 100 mM NaCl, pH 8.0), 20
(4NO3BA), 20 L of sample (20 g PLA2 or 20
each compound) and 20 L of water. The negative control was only
l
L of buffer (10 mM Tris–HCl,
L of 10 mM of substrate
g PLA2 + 50 M of
bands appeared at 1661, 1663, 1662 and 1660 cmꢀ1 for com-
pounds I–IV, respectively. The C8–S stretching vibration was
assigned to the bands at 920, 916, 927 and 908 cmꢀ1; and the
calculated bands appeared at 892, 925, 897 and 870 cmꢀ1, for
compounds I–IV respectively. These data are in agreement with
previous studies of organic thioester compounds [22].
l
l
l
l
l
l
buffer. The inhibitory effect of the compounds I–IV on PLA2 activity
was determined through co-incubation of the enzyme with each
compound for 30 min at 37 °C. After the incubation period, the
sample was added to the assay and the reaction was monitored
at 425 nm for 40 min (at 10 min intervals) at 37 °C. The quantity
of chromophore released (4-nitro-3-hydroxy benzoic acid) was
proportional to the enzymatic activity, and the initial velocity
(Vo) was calculated considering the absorbance measured at
20 min.
3.2. Biological, conformational and docking analysis
Phospholipases A2-induced myotoxicity occurs in two clinical
patterns: local and systemic myotoxicity [21]. The action of these
enzymes may result in irreversible lesions, which in addition to
edema, hemorrhage and blistering may lead to amputation of the
affected limb [23–25]. Moreover, it has been demonstrated that
antivenoms, the current therapy for snakebite, generally have a
limited efficacy against the local tissue damaging activities of ven-
oms [26]. In addition, enzymatic activity of the PLA2s is a key step
on the induction of myonecrosis, inflammation and neurotoxicity
induced by PLA2 [24,27,28]. Thus, it is necessary to search for addi-
tional inhibitors and approaches that may be useful counterparts
to conventional antivenom therapy. In this direction, we assayed
the ability of the compounds I–IV to inhibit the enzymatic activity
of a venom PLA2. As described in Table 2, we used two substrates,
in both cases compound II showed the best inhibitory ability;
whereas, compounds I and III displayed comparable inhibition
2.7. Computational studies
Quantum chemical calculations were carried out with the
GAUSSIAN 09 [16] program package, implemented on a personal
computer. The geometric structures for the more stable conformers
were calculated at the B3LYP/6-31+G(d,p) level of approximation
and the same method was used to determine the vibration mode
frequencies of the free molecules. Assignment of vibrational modes
was obtained by visual inspection of displacement vectors using
Gauss View 5 [17]. The potential energy curve around the dihedral
angle dC60–C10–C7–S was calculated for compounds I–IV employ-
ing the B3LYP/6-31+G(d,p) approximation with structure optimi-
zation for the torsion angle in steps of 30°. The calculations were
performed for molecules in vacuum (gas phase) and therefore
environmental effects were not considered. Some physicochemical
properties of compounds (I–IV) were obtained from Molinspiration
[18] and are showed in Table 1. Molecular docking was carried out
on a personal computer using Autodock Vina [19]. The PLA2 (PDB
code 2QOG) from Crotalus durissus terrificus showed 57% homology
in the N-terminal with the PLA2 used on in vitro studies [13]. Pro-
tein was used without water molecules. The structure of the pro-
tein was prepared using the Protein Preparation module
implemented in the Maestro program. First, hydrogen atoms were
automatically added to each protein according to the chemical
Table 1
Physicochemical properties and ligand–PLA2 affinity.
Compound Affinity
Molecular
Number of HB Number of HB LogPb
(kJ/mol)a weight (Da)b acceptorsb
donorsb
I
II
III
IV
ꢀ30.5
ꢀ32.6
ꢀ30.5
ꢀ31.8
262.8
273.3
273.3
242.3
1
4
4
1
0
0
0
0
4.58
3.84
3.86
4.35
a
Calculated with Autodock Vina.
Calculated using Molinspiration.
b
Henao Casta?eda
