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Plasma in several organisms has components that promote resistance to envenomation by inhibiting specific proteins from snake venoms, such as phospholipases A2 (PLA2s). The major hypothesis for inhibitor's presence would be the protection against self-envenomation in venomous snakes, but the occurrence of inhibitors in non-venomous snakes and other animals has opened new perspectives for this molecule. Thus, this study showed for the first time the structural and functional characterization of the PLA2 inhibitor from the Boa constrictor serum (BoaγPLI), a non-venomous snake that dwells extensively the Brazilian territory. Therefore, the inhibitor was isolated from B. constrictor serum, with 0.63% of recovery. SDS-PAGE showed a band at ~25 kDa under reducing conditions and ~20 kDa under non-reducing conditions. Chromatographic analyses showed the presence of oligomers formed by BoaγPLI. Primary structure of BoaγPLI suggested an estimated molecular mass of 22 kDa. When BoaγPLI was incubated with Asp-49 and Lys-49 PLA2 there was no severe change in its dichroism spectrum, suggesting a non-covalent interaction. The enzymatic assay showed a dose-dependent inhibition, up to 48.2%, when BoaγPLI was incubated with Asp-49 PLA2, since Lys-49 PLA2 has a lack of enzymatic activity. The edematogenic and myotoxic effects of PLA2s were also inhibited by BoaγPLI. In summary, the present work provides new insights into inhibitors from non-venomous snakes, which possess PLIs in their plasma, although the contact with venom is unlikely.

Phospholipase A2 (PLA2) promotes inflammation via lipid mediators and releases arachidonic acid (AA), and these enzymes have been found to be elevated in a variety of diseases, including rheumatoid arthritis, sepsis, and atherosclerosis. The mobilization of AA by PLA2 and subsequent synthesis of prostaglandins are regarded as critical events in inflammation. Inflammatory processes may be treated with drugs that inhibit PLA2, thereby blocking the COX and LOX pathways in the AA cascade. To address this issue, we report herein an efficient method for the synthesis of a series of octahydroquinazolinone compounds (4a–h) in the presence of the catalyst Pd-HPW/SiO2 and their phospholipase A2, as well as protease inhibitory activities. Among eight compounds, two of them exhibited overwhelming results against PLA2 and protease. By using FT-IR, Raman, NMR, and mass spectroscopy, two novel compounds were thoroughly studied. After carefully examining the SAR of the investigated compounds against these enzymes, it was found that compounds (4a, 4b) containing both electron-donating and electron-withdrawing groups on the phenyl ring exhibited higher activity than compounds with only one of these groups. DFT studies were employed to study the electronic nature and reactivity properties of the molecules by optimizing at the BLYP/cc-pVDZ. Natural bond orbitals helped to study the various electron delocalizations in the molecules, and the frontier molecular orbitals helped with the reactivity and stability parameters. The nature and extent of the expressed biological activity of the molecule were studied using molecular docking with human non-pancreatic secretory phospholipase A2 (hnps-PLA2) (PDB ID: 1DB4) and protease K (PDB ID: 2PWB). The drug-ability of the molecule has been tested using ADMET, and pharmacodynamics data have been extracted. Both the compounds qualify for ADME properties and follow Lipinski’s rule of five.

Drugs such as necopidem, saripidem, alpidem, zolpidem, and olprinone contain nitrogen-containing bicyclic, condensed-imidazo[1,2-α]pyridines as bioactive scaffolds. In this work, we report a high-yield one pot synthesis of 1-(2-methyl-8-aryl-substitued-imidazo[1,2-α]pyridin-3-yl)ethan-1-onefor the first-time. Subsequently, we performed in silico mode-of-action analysis and predicted that the synthesized imidazopyridines targets Phospholipase A2 (PLA2). In vitro analysis confirmed the predicted target PLA2 for the novel imidazopyridine derivative1-(2-Methyl-8-naphthalen-1-yl-imidazo [1,2-α]pyridine-3-yl)-ethanone (compound 3f) showing significant inhibitory activity towards snake venom PLA2 with an IC50 value of 14.3 μM. Evidently, the molecular docking analysis suggested that imidazopyridine compound was able to bind to the active site of the PLA2 with strong affinity, whose affinity values are comparable to nimesulide. Furthermore, we estimated the potential for oral bioavailability by Lipinski's Rule of Five. Hence, it is concluded that the compound 3f could be a lead molecule against snake venom PLA2.

Lancehead pitvipers, snakes, or, popularly, \"jararacas\", are common and broadly distributed in the Americas, especially in Brazil, where they are responsible for causing a high number of snakebite accidents. Their venoms are able to induce local and systemic effects, such as hemorrhaging, acute kidney failure, and shock, that can be fatal. Among the compounds of the venom are phospholipases A (PLA s), which are abundant in some species. PLA s can perform different activities during envenoming, such as neurotoxicity, myotoxicity, and cytotoxicity, among others, through the hydrolysis of the ester bond at the sn-2 position of phospholipids, producing free fatty acids and lysophospholipids. Although different PLA s can be classified into different PLA groups and subgroups, according to structure, function, size, localization and Ca dependence, they converge to be available in biotechnological and therapeutic applications, such as antiviral and antitumor, among others, being relevant molecules to be deeply studied. Here, we provide the state of the art of PLA s, found in snake venoms, focusing on venoms, as well as their potential applications, beyond their inhibitors, that also receive attention due their importance in PLA studies and diverse applications.

Arachidonic acid metabolism leads to the generation of key lipid mediators which play a fundamental role during inflammation. The inhibition of enzymes involved in arachidonic acid metabolism has been considered as a synergistic anti-inflammatory effect with enhanced spectrum of activity. A series of 1,3-diphenyl-2-propen-1-one derivatives were investigated for anti-inflammatory related activities involving inhibition of secretory phospholipase A2, cyclooxygenases, soybean lipoxygenase, and lipopolysaccharides-induced secretion of interleukin-6 and tumor necrosis factor-alpha in mouse RAW264.7 macrophages. The results from the above mentioned assays exhibited that the synthesized compounds were effective inhibitors of pro-inflammatory enzymes and cytokines. The results also revealed that the chalcone derivatives with 4-methlyamino ethanol substitution seem to be significant for inhibition of enzymes and cytokines. Molecular docking experiments were carried out to elucidate the molecular aspects of the observed inhibitory activities of the investigated compounds. Present findings increase the possibility that these chalcone derivatives might serve as a beneficial starting point for the design and development of improved anti-inflammatory agents.

We aimed to investigate the effects of snake-derived phospholipase A2 inhibitor (PLA2) from and on acute pancreatitis in vivo and in vitro and assess the mechanisms. The levels of platelet-activating factor (PAF) and tumor necrosis factor (TNF)-α were detected by ELISA, and the characteristics of autophagy were detected by transmission electron microscopy and Western blotting (LC3, p62, and ATG5). In vitro experiments showed that PLA2 treatment caused obvious formation of autophagic bodies. By contrast, and peptides reduced the number of autophagic bodies. The concentrations of PAF and TNF-α, and the expressions of p62, autophagy-related 5 (ATG5), and microtubule-associated protein 1A/1B-light chain 3 (LC3)II/LC3I in the PLA2-treated group were significantly higher than in the control group (P<0.05). The concentrations of PAF and TNF-α, and the expressions of p62, ATG5, and LC3II/LC3I in the or peptide treatment groups were significantly lower than in the PLA2-treated cells (P<0.05). In the pancreatic tissue, autophagic bodies were observed in the model group; autophagic bodies were remarkably reduced in or peptide-treated groups compared with the model group. In vivo experiments also showed that the levels of PAF and TNF-α, and the expressions of p62, ATG5, and LC3II/LC3I were significantly higher in the model group than in the control group (P<0.05). The levels of PAF and TNF-α in the model group, and the expressions of p62, ATG5, and LC3II/LC3I in or peptide-treated groups were significantly lower than in the model group (P<0.05). or peptide could ameliorate the features of acute pancreatitis, likely through regulating autophagy.

Ophidian envenomation is an important health problem in Brazil and other South American countries. In folk medicine, especially in developing countries, several vegetal species are employed for the treatment of snakebites in communities that lack prompt access to serum therapy. However, the identification and characterization of the effects of several new plants or their isolated compounds, which are able to inhibit the activities of snake venom, are extremely important and such studies are imperative. Snake venom contains several organic and inorganic compounds; phospholipases A2 (PLA2s) are one of the principal toxic components of venom. PLA2s display a wide variety of pharmacological activities, such as neurotoxicity, myotoxicity, cardiotoxicity, anticoagulant, hemorrhagic, and edema-inducing effects. PLA2 inhibition is of pharmacological and therapeutic interests as these enzymes are involved in several inflammatory diseases. This review describes the results of several studies of plant extracts and their isolated active principles, when used against crude snake venoms or their toxic fractions. Isolated inhibitors, such as steroids, terpenoids, and phenolic compounds, are able to inhibit PLA2s from different snake venoms. The design of specific inhibitors of PLA2s might help in the development of new pharmaceutical drugs, more specific antivenom, or even as alternative approaches for treating snakebites.

Snakebite envenomation is a neglected condition that constitutes a public health problem in tropical and subtropical countries, including Brazil. Interestingly, some animals are resistant to snake envenomation due to the presence of inhibitory glycoproteins in their serum that target toxic venom components. DM64 is an acidic glycoprotein isolated from Didelphis aurita (opossum) serum that has been characterized as an inhibitor of the myotoxicity induced by bothropic toxins bearing phospholipase A2 (PLA2) structures. This antitoxic protein can serve as an excellent starting template for the design of novel therapeutics against snakebite envenomation, particularly venom-induced local tissue damage. Therefore, the aim of this work was to produce a recombinant DM64 (rDM64) in the methylotrophic yeast Pichia pastoris and to compare its biological properties with those of native DM64. Yeast fermentation in the presence of Pefabloc, a serine protease inhibitor, stimulated cell growth (~1.5-fold), increased the rDM64 production yield approximately 10-fold and significantly reduced the susceptibility of rDM64 to proteolytic degradation. P. pastoris fermentation products were identified by mass spectrometry and Western blotting. The heterologous protein was efficiently purified from the culture medium by affinity chromatography (with immobilized PLA2 myotoxin) and/or an ion exchange column. Although both native and recombinant DM64 exhibit different glycosylation patterns, they show very similar electrophoretic mobilities after PNGase F treatment. rDM64 formed a noncovalent complex with myotoxin II (Lys49-PLA2) from Bothrops asper and displayed biological activity that was similar to that of native DM64, inhibiting the cytotoxicity of myotoxin II by 92% at a 1:1 molar ratio.

Human phospholipase A2 (hPLA2) of the IIA group (HGIIA) catalyzes the hydrolysis of membrane phospholipids, producing arachidonic acid and originating potent inflammatory mediators. Therefore, molecules that can inhibit this enzyme are a source of potential anti-inflammatory drugs, with different action mechanisms of known anti-inflammatory agents. For the study and development of new anti-inflammatory drugs with this action mechanism, snake venom PLA2 (svPLA2) can be employed, since the svPLA2 has high similarity with the human PLA2 HGIIA. Despite the high similarity between these secretory PLA2s, it is still not clear if these toxins can really be employed as an experimental model to predict the interactions that occur with the human PLA2 HGIIA and its inhibitors. Thus, the present study aims to compare and evaluate, by means of theoretical calculations, docking and molecular dynamics simulations, as well as experimental studies, the interactions of human PLA2 HGIIA and two svPLA2s, Bothrops toxin II and Crotoxin B (BthTX-II and CB, respectively). Our theoretical findings corroborate experimental data and point out that the human PLA2 HGIIA and svPLA2 BthTX-II lead to similar interactions with the studied compounds. From our results, the svPLA2 BthTX-II can be used as an experimental model for the development of anti-inflammatory drugs for therapy in humans.

The work examines the effect of inhibitors of cytosolic Ca 2+ -dependent and Ca 2+ -independent phospholipases A 2 on bilayer lipid membranes. It was established that trifluoroperazine (TFP) and, to a lesser extent, arachidonyl trifluoromethyl ketone (AACOCF 3 ) and palmitoyl trifluoromethyl ketone (PACOCF 3 ) were able to permeabilize artificial lipid membranes (BLM and liposomes). It was shown that AACOCF 3 lowered the temperature of phase transition of DMPC liposomes, inducing disordering of the hydrophobic region of lipid bilayer. TFP disordered membranes both in the hydrophobic region and in the region of hydrophilic heads, this being accompanied by changes in the membrane permeability: appearance of a channel-like BLM activity and leakage of sulforhodamine B from liposomes. In contrast to AACOCF 3 and TFP, PACOCF 3 increased membrane orderliness in the hydrophobic region (heightened the temperature of phase transition of DMPC liposomes) and in the region of lipid heads. The effectiveness of AACOCF 3 and PACOCF 3 as inductors of BLM and liposome permeabilization was considerably lower comparatively to TFP. As revealed by dynamic light scattering, incorporation of TFP, AACOCF 3 and PACOCF 3 into the membrane of liposomes resulted in the increase of the average size of particles in the suspension, presumably due to their aggregation or fusion. The paper discusses possible mechanisms of the influence of phospholipase A 2 inhibitors on bilayer lipid membranes.