The World Health Organization (WHO) recognized snakebites as one of the most important Neglected Tropical Diseases in 2017. Nearly 5.4 million snakebites occur each year, and more than 95% of cases occur in tropical or developing countries, mostly in rural areas [1
]. There are between 81,410 and 137,880 deaths per year and around three times as many cases of permanent disabilities including amputations [2
]. A total of 4978 ophidian accidents were reported in Colombia in 2017 [3
Pathophysiological effects observed in snakebite envenomations combine the action of several enzymes, proteins and peptides, such as phospholipases A2
), hemorrhagic metalloproteinases, and other proteolytic enzymes, coagulant components, neurotoxins, cytotoxins and cardiotoxins, among others [4
]. Snake venom metalloproteinases (SVMPs) have been shown to participate in the hemorrhagic process by proteolytic degradation of endothelial cell surface proteins and extracellular matrix components, involved in the maintenance of capillary structure and integrity, leading to capillary network disruption, edema and hemorrhage [5
]. Phospholipases A2
) are calcium-depending enzymes that hydrolyze the sn2 ester bond of glycerophospholipids, inducing systemic and local myotoxicity, myonecrosis, neurotoxicity hemolytic activity, among others [6
Nowadays, the only available and accepted therapy for the treatment of snakebites is the intravenous administration of equine or ovine antivenoms [8
]. Nevertheless, several reports demonstrated the limited efficacy of antivenom therapy against the local tissue damage caused by venoms [9
]. Therefore, there is a need for specific inhibitors of enzymes responsible for local damage to complement conventional parenteral antivenom therapy.
As usual in drug discovery, inhibitors could be found in natural sources or obtained by chemical synthesis, and in some cases in combination with in silico methodologies. This strategy was used for synthesizing naringenin derivatives which have structural similarity with quercetin, a flavonoid that binds to the active site of a PLA2
There are reports of natural and synthetic inhibitors of PLA2
from snake venoms including substituted thiobenzoic acid S-benzyl esters [11
], Aiplai from leaves of Azadirachta indica
] and cholic and ursodeoxycholic acids [13
]. Some of the inhibitors have reported IC50
with the same methodology that we use; Pinostrobin, a flavonone isolated from Renealmia alpinia
, with an IC50
of 1.85 mM [14
]; and Morelloflavone, a biflavonoid from Garcinia madruno
, with IC50
of 0.38 mM [15
In a previous study we found that substituted thiobenzoic acid S-benzyl esters are inhibitors of an Asp49-PLA2
enzyme isolated from the venom of the Colombian Crotalus durissus cumanensis
rattlesnake, but they have solubility drawbacks [11
]. Looking for increasing the polarity and maintaining the biological activity, thioesters derived from 2-sulfenyl ethyl acetate were synthesized changing a phenyl group for an ethyl ester (Figure 1
, Table 1
). The present study aims to evaluate the inhibitory capacity of these compounds on the PLA2
from C. d. cumanensis
rattlesnake and a PI type SVMP from Bothrops atrox
and SVMPs inhibitors with IC50
values in the nano and micromolar range for the neutralization of different activities of these toxins have been reported [11
]. However, some inhibitors have poor water solubility and low specificity that make their clinical applications difficult. Thus, the aim of this work was to synthesize thioester compounds with improved water solubility with respect to substituted thiobenzoic acid S-benzyl esters reported in a previous work [11
], while maintaining the expected biological activity as PLA2
inhibitors. The condensation reaction is a high-yielding synthesis (97%) and the strategy was to utilize the same benzoyl chlorides used in the previous study, with a different thiol. The new selected reactant gives us the possibility to obtain the thioester moiety, and also an additional ester moiety in its structure. This change allows the increase in the number of hydrogen acceptors, improving the solubility (lower values of calculated partition coefficient Log P
). The calculated Log P
for the previous reported thiobenzoic acid S-benzyl esters were between 3.84 and 4.58, and for compounds I–II were between 2.48 and 3.23.
Compounds I–III have both thioester and ester moiety in their structure. Thioester (X=S) and ester (X=O) moieties usually present a synperiplanar configuration around the δO=C-X-C dihedral angle as the more stable conformer. The results obtained for compounds I, II and III are in agreement with previous reports [11
Envenomations induced by viperid snakebites are characterized by local and systemic bleeding. Local effects are associated with a pronounced local tissue damage, while hemodynamic alterations predominate in the systemic effects [18
]. Both enzymes studied in this work, PLA2
s and SVMPs contribute to this pathogenesis inducing hemorrhage, myonecrosis, dermonecrosis, blister formation and edema [5
]. The described effects are difficult to neutralize by antibodies due to their rapid symptoms after envenomation [21
]. Therefore, it is important to find SVMPs and PLA2
s inhibitors, like synthetic compounds I–III, that can be administered at the bite site.
The enzymatic activity of a PLA2
is determined by three principal factors: the integrity of the active site (residues His48, Asp49, Tyr52, Asp99), coordination of Ca2+
cofactor (residues Tyr28, Gly30, Gly32 and Asp49) and the adsorption of the enzyme onto the lipid–water interface of the phospholipids membrane bilayer (interfacial binding surface) [22
], hence they are crucial to study the inhibition mechanism. The molecular docking study suggests that both stable conformations of the studied compounds may interact by either van der Waals or H-bond with amino acids His48 and Asp49 blocking catalytic cycle of PLA2
. The catalytic mechanism implies water activation by His48 for the subsequent nucleophilic attack of sn2 ligation of the glycerophospholipids that will be hydrolyzed, and carboxylate of the Asp49 side chain stabilizes the oxyanion formed after the nucleophilic attack [22
]. Catalytic activity and substrate binding of snake venom phospholipases need submicromolar concentrations of calcium ions. Recently, molecular dynamic studies have demonstrated that calcium induces atomistic movements and conformational changes in snake venom PLA2
which led to the formation of a widened cleft at the active site of calcium bound PLA2
when compared with free PLA2
]. This ion is also available for binding the substrate phosphate group [22
]. Molecular docking also suggested van der Waals interactions between compounds I, II and III with Gly30 and coordination with Ca2+
that could destabilize metal coordination and block enzymatic catalysis.
The interfacial binding surface of the PLA2
s mediates the adsorption of the enzyme onto the lipid–water interface of the phospholipid membrane bilayer. Residues Trp31 and Lys69 are part of this structure. Our molecular docking results suggested that all compounds may form van der Waals or π-alkyl interactions with the mentioned residues, thus, the binding of the substrate to the PLA2
active site may be blocked. Similar findings were reported in substituted thiobenzoic acid S-benzyl esters at 50 μM and obtained inhibition percentages higher than 50% for three of the four assayed compounds [11
There is a type of snake venom PLA2
s catalytically inactive (PLA2
-like myotoxins) due to lack of Ca2+
coordination by a natural mutation in which the residue aspartate at position 49 is substituted with a lysine generating the loss of the catalytic activity. However, these toxins are able to induce local mionecrosis by a mechanism dependent of two different sites forming a putative membrane-docking site (MDoS) and a putative membrane disruption site (MDiS) [24
]. Different small molecules inhibitors have been described by their inhibition against PLA2-like myotoxins through the binding at just one critical region related to the myotoxic mechanism: the MDoS, MDiS or hydrophobic channel. Recently was demonstrated that chicoric acid binds at the entrance of the hydrophobic channel and clusters of a PLA2
-like myotoxin that participate in membrane disruption [25
]. We hypothesized that thioester compounds could inhibit PLA2
-like myotoxic activity interacting with amino acids located at the hydrophobic channel, since some amino acids of this structure (Leu2 and Phe5) are involved in the interaction with the Asp49-PLA2
tested in this study (Figure 5
). Nevertheless, this must be confirmed in future studies.
Compounds I–III have IC50
s between 132.7 and 305.4 μM against PLA2
enzymatic activity (p
> 0.05) and are much more active than inhibitors like Pinostrobin and Moreloflavone with IC50
s determined by the same method of 1.85 and 0.38 mM, respectively [14
]. Compounds I and III have an electronegative substitution in the para position of the aromatic ring and comparable IC50
s, whereas compound II is substituted in the meta position. Besides these subtle differences, our docking results suggested that compounds I and III may interact in a similar position with the benzoyl ring at the beginning of the hydrophobic channel and thioester moiety at the middle of the hydrophobic pocket, near to the active site of the enzyme. Instead, compound II was located in an opposite way with phenyl ring near to the active site of the PLA2
, which may explain the difference in its inhibition capacity. In order to improve the inhibitory potency of compounds, some structural modifications, such as the substitution with a metal binding group can be done with the aim to improve their interaction with catalytically active PLA2
Synthesized compounds were also tested to inhibit the enzymatic activity of a SVMP, however, only II and III showed some ability to inhibit the enzyme with IC50 values about 10 times higher than those found for PLA2. This talk about their specificity for PLA2s, however these compounds may give the possibility to partially also inhibit SVMPs present in snake venoms. Nevertheless, this hypothesis should be addressed in future studies with whole venom and in vivo assays.
4. Materials and Methods
Reagents were purchased from Merck, Sigma-Aldrich and Acros organics with commercial, analytic or HPLC grade.
Solvents were evaporated from solutions in a rotary evaporator Heidolph Laborota 4010 equipped with a ROTAVAP valve control.
Ethyl 2-((4-chlorobenzoyl)thio)acetate (I), Ethyl 2-((3-nitrobenzoyl)thio)acetate (II) and Ethyl 2-((4-nitrobenzoyl)thio)acetate (III) (Figure 1
) were prepared with Ethyl 2-mercaptoacetate (1 mmol) dissolved in pyridine and the corresponding substituted-benzoyl chloride (1 mmol) was added to this dissolution. (4-chlorobenzoyl chloride, 3-nitrobenzoyl chloride and of 4-nitrobenzoyl chloride for I, II and III respectively). The reaction mixture was stirred for 1 h at room temperature. The reaction mixture was treated with 1 M hydrochloric acid (5 mL) and methylene chloride (5 mL) and washed thoroughly with distilled water. The organic layer was dried over sodium sulfate anhydride, the solvent was removed in a rotatory evaporator and the residue was recrystallized from methanol and dried under vacuum.
4.3. Spectroscopical Characterization
Melting points (m.p.) were recorded in an Electrothermal 9100 apparatus. Infrared spectra in KBr pellets were measured between 4000 and 400 cm−1 (4 cm−1 resolution) with and FT-IR spectrometer Thermo-Nicolet IR200. The mass spectra were measured with a CG-MS Shimadzu QP-2010 spectrometer with a HP-5 column. NMR spectra were measured at 298 K on a Bruker DPX 200 spectrometer. The compounds were dissolved in CDCl3. Chemical shifts, δ, are given in ppm relative to TMS (δ = 0 ppm) and are referenced by using the residual undeuterated solvent signal. Coupling constants, J, are reported in Hz, multiplicities being marked as: singlet (s), doublet (d), triplet (t), double triplet (dt) of multiplet (m).
4.4. Toxins Isolation
The PLA2 from C. d. cumanensis was obtained from a pool venom of four specimens maintained in captivity at the serpentarium of the University of Antioquia (Medellín, Colombia).
The enzyme was purified through reverse-phase HPLC on C-18 column eluted at 1.0 mL/min with a gradient from 0% to 100% of acetonitrile in 0.1% trifluoroacetic acid (v
). The absorbance in the effluent solution was recorded at wavelength of 280 nm [26
Baxt-I was isolated from a venom pool collected from adult specimens of B. atrox
from Meta, southeastern Colombia, via ion-exchange chromatography (CM-Sephadex C25) following the protocol described by Patiño et al. (2010) [27
]. For both proteins, the purity was judged by RP-HPLC and SDS-PAGE.
4.5. Inhibition of PLA2 Activity Using 4-nitro-3-octanoyloxybenzoic acid (4N3OBA) as Monodispersed Substrate
The measurements of enzymatic activity using the monodispersed substrate 4N3OBA were performed according to the method described by Holzer and Mackessy (1996), and adapted for a 96-well ELISA plate. The standard assay contained 200 μL of buffer (10 mM Tris–HCl, 10 mM CaCl2, 100 mM NaCl, pH 8.0), 20 μL of 10 mM of substrate (4NO3BA), 20 μL of sample (20 μg PLA2 or 20 μg PLA2 + several concentrations of compounds) and 20 μL of water. The negative control was only buffer. The inhibitory effect of the studied thioesters on PLA2 activity was determined through co-incubation of the enzyme with each concentration of the 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 IC50 value was determined from a logistic-dose response curve.
4.6. Inhibition of Proteolytic Activity
The inhibition of proteolytic activity was measured using the EnzCheck®
Gelatinase/Collagenase assay kit (Molecular Probes Inc., Eugene, Oregon, USA) following the protocol described by Preciado et al. (2017) with modification [28
]. Briefly, aliquots of 80 μL of each triterpenic acid in concentrations from 15 to 500 μM, or buffer (0.05 M Tris-HCl, 0.15 M NaCl, 5 mM CaCl2
, 0.2 mM sodium azide) as positive control, were added to each well of a 96-well plate. Then, 20 μL of DQ-gelatin followed by 100 μL of active Batx-I (1 μg/μL) were added, and the fluorescence intensity was measured by a Synergy HT Multi-Mode Microplate Reader (BioTek Instruments, Inc.; Winooski, USA) for excitation at 485 nm and emission detection at 515 nm at each minute for 60 min. Each reaction was performed in triplicate.
4.7. Computational Studies
Compounds (I–III) were built using Gauss View 5 [29
]. The geometric parameters for the more stable conformers were calculated at the B3LYP/6-31++G (d,p) level of approximation using GAUSSIAN 09 [30
]. Molecular docking was carried out on a personal computer using Autodock Vina [31
]. The structure of the PLA2
(PDB code 2QOG) from Crotalus durissus terrificus
that showed 57% of homology with the PLA2
from C. d. cumanensis
] was used in this study.
Protein structure was prepared using the Protein Preparation module implemented in the Maestro program and uploaded without water molecules. Hydrogen atoms were automatically added to the protein according to the chemical nature of each amino acid, on the basis of the ionized form expected in physiological condition. This module also controls the atomic charges assignment. The 3D structure of the protein was relaxed through constrained local minimization, using the OPLS force fields in order to remove possible structural mismatches due to the automatic procedure employed to add the hydrogen atoms. When necessary, bonds, bond orders, hybridizations and hydrogen atoms were added, charges were assigned (a formal charge of +2 for Ca ion) and flexible torsions of ligands were detected.
To perform molecular docking experiments of compounds I–III with PLA2
, the α-carbon of His48 was used as the center of the grid (X = 44.981, Y = 27.889 and Z = 46.392), whose size was 24 Å3
. Exhaustiveness = 20. Finally, the ligand poses with best affinity were chosen, and a visual inspection of the interactions at the active site was performed and recorded using the open functionalities of Discovery Studio Visualizer and UCSF Chimera (www.cgl.ucsf.edu/chimera/
). Physicochemical properties with importance in oral bioavailability for compounds I–III were calculated using Molinspiration [32
4.8. Statistical Analysis
In order to determine significant differences between the concentrations of the compounds I–III used in the inhibition of the enzymes metalloproteinase and PLA2, two-way ANOVA followed by Bonferroni’s test was applied. In all cases, a difference with a p < 0.05 was considered significant.