Snake Venom Phospholipase Research Articles

Proteomic characterization of six Taiwanese snake venoms: Identification of species-specific proteins and development of a SISCAPA-MRM assay for cobra venom factors

Deinagkistrodon acutus, Trimeresurus stejnegeri, Protobothrops mucrosquamatus, Daboia russelii siamensis, Bungarus multicinctus and Naja atra are the six medically important venomous snake species in Taiwan. In this study, we characterized and compared their venom protein profiles using proteomic approaches. The major snake venom proteins were identified by GeLC-MS/MS and the total venom proteome was characterized by in-solution digestion coupled with LC-MS/MS. A total of 27–52 proteins, categorized into 23 protein families, were identified in each snake's venom. The major venom components found in Viperidae species (D. acutus, T. stejnegeri, P. mucrosquamatus and D. russelii) were C-type lectin, snake venom serine proteinase, venom metalloproteinase and phospholipase A2, whereas three-finger toxin and phospholipase A2 were the major components detected in the venom of Elapidae snakes (B. multicinctus and N. atra). This study also provided the first demonstration of some low-abundance proteins in these six snake venoms, including 5′-nucleotidase, glutaminyl-peptide cyclotransferase and phosphodiesterase, among others. Furthermore, we found that cobra venom factor (CVF) is a cobra-specific protein. We produced anti-peptide antibodies against CVF and used it to develop a highly sensitive SISCAPA-MRM assay for quantifying CVF. The limit of detection and lower limit of quantification were 3.2 and 9.6 attomoles, respectively. This assay was used to precisely quantify CVF in 1 μg crude venom proteins from three Naja species and king cobra. The amount of CVF varied from 0.9 to 54.36 femtomoles (equivalent to 0.16–10.03 mg/g of venom protein). Biological significanceThere are six medically significant venomous snakes in Taiwan. The venoms of the four Viperidae species (Deinagkistrodon acutus, Trimeresurus stejnegeri, Protobothrops mucrosquamatus and Daboia russelii siamensis) cause local tissue swelling; this symptom is also seen in N. atra envenomation in humans, potentially complicating the differential diagnosis of envenomation by N. atra and Viperidae species. Thus, characterization of the venom proteomes of the six Taiwanese snakes, including the relative abundance of the major components and species-specific protein(s) in each venom type, could be useful for future venom research, including the development of new assay(s) for detecting snake species-specific venom protein(s) and new type(s) of antivenom.

Phospholipases A2 purified from cottonmouth snake venoms display no antibacterial effect against four representative bacterial species

Snake venom phospholipase A2 (PLA2) has widely been reported to possess antibacterial effects, and PLA2 is the major component of cottonmouth snake venoms. We assessed the antibacterial activities of crude venoms from Western cottonmouth (Agkistrodon piscivorus leucostoma), Eastern cottonmouth (Agkistrodon piscivorus piscivorus), and Florida cottonmouth (Agkistrodon piscivorus conanti) snakes against two gram-positive (Bacillus subtilis, Staphylococcus aureus), and two gram-negative (Escherichia coli, Vibrio cholerae) bacteria. Antibacterial activity of PLA2 proteins, AplAsp49 and AplLys49 purified from A. p. leucostoma venom, was also examined. Disk-diffusion assays revealed that A. p. leucostoma crude venom is most effective in inhibiting the growth of the bacteria tested, compared to the other two. Surprisingly, AplAsp49 and AplLys49 PLA2s purified from A. p. leucostoma venom did not display detectable antibacterial activity against any bacteria tested neither by disk-diffusion nor by minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC). The lack of antibacterial activity of cottonmouth venom PLA2s is discussed.

Snake venom peptides: Potential therapeutic agents in thrombotic diseases

The snake venoms are constituted of a true biochemical arsenal, consisting of several proteins and peptides with activities that have aroused the curiosity of researchers for centuries, in an attempt to understand its systemic action in order to get pharmacological applications. A number of snake venom proteins that interfere on platelet aggregation have been isolated from these venoms. However, there are no reports in the literature of small peptides interfering in aggregation. In the present work, we identify and characterize, for the first time, a heptapeptide (BaltPAi: platelet aggregation inhibitor from B. alternatus snake venom) and a decapeptide (BmooPAF: platelet-activating factor from B. moojeni snake venom), which potentially inhibits and induces the platelet aggregation, respectively. BmooPAi shows a rather specific inhibitory effect on collagen-induced platelet aggregation in human platelet-rich plasma, whereas it has little or no effect on platelet aggregation induced by adenosine diphosphate. The results presented here suggest that the BaltPAi consists of an amino acid sequence present in the C-terminal region of snake venom phospholipase A2 enzymes. This sequence would be responsible for the inhibition of platelet aggregation as well as for the cytotoxicity effects of tumor cells caused by these enzymes. Assays with monoclonal antibodies (anti-integrin α2b and anti-GP1BA) show a significant inhibitory effect on BmooPAF-induced platelet aggregation. On the other hand, anti-GPVI antibody shows no effect on platelet function. These findings, associated with molecular docking, indicate that BmooPAF induces platelet aggregation via binding to the GPIbα platelet receptor leading to αIIbβ3 integrin activation. These toxins could be of medical interest as tools for the development of novel therapeutic agents for the prevention and treatment of thrombotic disorders. The snake venoms are constituted of a true biochemical arsenal, consisting of several proteins and peptides with activities that have aroused the curiosity of researchers for centuries, in an attempt to understand its systemic action in order to get pharmacological applications. A number of snake venom proteins that interfere on platelet aggregation have been isolated from these venoms. However, there are no reports in the literature of small peptides interfering in aggregation. In the present work, we identify and characterize, for the first time, a heptapeptide (BaltPAi: platelet aggregation inhibitor from B. alternatus snake venom) and a decapeptide (BmooPAF: platelet-activating factor from B. moojeni snake venom), which potentially inhibits and induces the platelet aggregation, respectively. BmooPAi shows a rather specific inhibitory effect on collagen-induced platelet aggregation in human platelet-rich plasma, whereas it has little or no effect on platelet aggregation induced by adenosine diphosphate. The results presented here suggest that the BaltPAi consists of an amino acid sequence present in the C-terminal region of snake venom phospholipase A2 enzymes. This sequence would be responsible for the inhibition of platelet aggregation as well as for the cytotoxicity effects of tumour cells caused by these enzymes. Assays with monoclonal antibodies (anti-integrin α2b and anti-GP1BA) show a significant inhibitory effect on BmooPAF-induced platelet aggregation. On the other hand, anti-GPVI antibody shows no effect on platelet function. These findings, associated with molecular docking, indicate that BmooPAF induces platelet aggregation via binding to the GPIbα platelet receptor leading to αIIbβ3 integrin activation. These toxins could be of medical interest as tools for the development of novel therapeutic agents for the prevention and treatment of thrombotic disorders.

Identification of the Molecular Determinants of the Antibacterial Activity of LmutTX, a Lys49 Phospholipase A2 Homologue Isolated from Lachesis muta muta Snake Venom (Linnaeus, 1766).

Snake venom phospholipases A2 (PLA2 s) are responsible for numerous pathophysiological effects in snakebites; however, their biochemical properties favour antimicrobial actions against different pathogens, thus constituting a true source of potential microbicidal agents. This study describes the isolation of a Lys49 PLA2 homologue from Lachesis muta muta venom using two chromatographic steps: size exclusion and reverse phase. The protein showed a molecular mass of 13,889 Da and was devoid of phospholipase activity on an artificial substrate. The primary structure made it possible to identify an unpublished protein from L. m. muta venom, named LmutTX, that presented high identity with other Lys49 PLA2 s from bothropic venoms. Synthetic peptides designed from LmutTX were evaluated for their cytotoxic and antimicrobial activities. LmutTX was cytotoxic against C2C12 myotubes at concentrations of at least 200 μg/mL, whereas the peptides showed a low cytolytic effect. LmutTX showed antibacterial activity against Gram-positive and Gram-negative bacteria; however, S. aureusATCC 29213 and MRSA strains were more sensitive to the toxin's action. Synthetic peptides were tested on S. aureus, MRSA and P. aeruginosaATCC 27853 strains, showing promising results. This study describes for the first time the isolation of a Lys49 PLA2 from Lachesis snake venom and shows that peptides from specific regions of the sequence may constitute new sources of molecules with biotechnological potential.

Computational and in vitro insights on snake venom phospholipase A2 inhibitor of phytocompound ikshusterol3-O-glucoside of Clematis gouriana Roxb. ex DC.

Ikshusterol3-O-glucoside was isolated from Clematis gouriana Roxb. ex DC. root. A structure of the isolated compound was determined on the basis of various spectroscopic interpretations (UV, NMR, FTIR, and GC-MS-EI). This structure was submitted in the PubChem compound database (SID 249494133). SID 249494133 was carried out by density functional theory calculation to observe the chemical stability and electrostatic potential of this compound. The absorption, distribution, metabolism, and excretion property of this compound was predicted to evaluate the drug likeness and toxicity. In addition, molecular docking, quantum polarized ligand docking, prime MMGBSA calculation, and induced fit docking were performed to predict the binding status of SID 249494133 with the active site of phospholipase A2 (PLA2) (PDB ID: 1A3D). The stability of the compound in the active site of PLA2 was carried out using molecular dynamics simulation. Further, the anti-venom activity of the compound was assessed using the PLA2 assay against Naja naja (Indian cobra) crude venom. The results strongly show that Ikshusterol3-O-glucoside has a potent snake-venom neutralizing capacity and it might be a potential molecule for the therapeutic treatment for snakebites.

Can Inhibitors of Snake Venom Phospholipases A₂ Lead to New Insights into Anti-Inflammatory Therapy in Humans? A Theoretical Study.

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.

Preparation of monoclonal antibodies against gamma-type phospholipase A2 inhibitors and immunodetection of these proteins in snake blood

BackgroundThe gamma-type phospholipase A2 inhibitor (PLIγ) is a natural protein commonly found in snake serum, which can neutralize pathophysiological effects of snake venom phospholipases A2. Therefore, this protein is a potential candidate to the development of a novel antivenom. To the best of our knowledge, there is no antibody currently available for PLIγ identification and characterization.MethodsBioinformatics prediction of epitope using DNAStar software was performed based on the sequence of Sinonatrix annularis PLIγ (SaPLIγ). The best epitope 151CPVLRLSNRTHEANRNDLIKVA172 was chosen and synthesized, and then conjugated to keyhole limpet hemocyanin and bovine serum albumin for use as an immunogen and plate-coating antigen, respectively.ResultsEighteen IgG anti-PLIγ mAb hybridoma cell strains were obtained, and all the mAbs had positive interaction with recombinant His6-PLIγ and natural SaPLIγ. Moreover, the mAb from 10E9 strain was also successfully used for the immunodetection of other snake serum PLIγs. cDNA sequence alignment of those PLIγs from different snake species showed that their epitope segments were highly homologous.ConclusionsThe successful preparation of anti-PLIγmAb is significant for further investigation on the relationship between the structure and function of PLIγs, as well as the interaction between PLIγs and PLA2s.

Cross-recognition of a pit viper (Crotalinae) polyspecific antivenom explored through high-density peptide microarray epitope mapping.

Snakebite antivenom is a 120 years old invention based on polyclonal mixtures of antibodies purified from the blood of hyper-immunized animals. Knowledge on antibody recognition sites (epitopes) on snake venom proteins is limited, but may be used to provide molecular level explanations for antivenom cross-reactivity. In turn, this may help guide antivenom development by elucidating immunological biases in existing antivenoms. In this study, we have identified and characterized linear elements of B-cell epitopes from 870 pit viper venom protein sequences by employing a high-throughput methodology based on custom designed high-density peptide microarrays. By combining data on antibody-peptide interactions with multiple sequence alignments of homologous toxin sequences and protein modelling, we have determined linear elements of antibody binding sites for snake venom metalloproteases (SVMPs), phospholipases A2s (PLA2s), and snake venom serine proteases (SVSPs). The studied antivenom antibodies were found to recognize linear elements in each of the three enzymatic toxin families. In contrast to a similar study of elapid (non-enzymatic) neurotoxins, these enzymatic toxins were generally not recognized at the catalytic active site responsible for toxicity, but instead at other sites, of which some are known for allosteric inhibition or for interaction with the tissue target. Antibody recognition was found to be preserved for several minor variations in the protein sequences, although the antibody-toxin interactions could often be eliminated completely by substitution of a single residue. This finding is likely to have large implications for the cross-reactivity of the antivenom and indicate that multiple different antibodies are likely to be needed for targeting an entire group of toxins in these recognized sites.

Molecular dynamics simulations reveal structural insights into inhibitor binding modes and functionality in human Group IIA phospholipase A2.

Human Group IIA phospholipase A2 (hGIIA) promotes inflammation in immune-mediated pathologies by regulating the arachidonic acid pathway through both catalysis-dependent and -independent mechanisms. The hGIIA crystal structure, both alone and inhibitor-bound, together with structures of closely related snake-venom-derived secreted phospholipase enzymes has been well described. However, differentiation of biological and nonbiological contacts and the relevance of structures determined from snake venom enzymes to human enzymes are not clear. We employed molecular dynamics (MD) and docking approaches to understand the binding of inhibitors that selectively or nonselectively block the catalysis-independent mechanism of hGIIA. Our results indicate that hGIIA behaves as a monomer in the solution environment rather than a dimer arrangement that is in the asymmetric unit of some crystal structures. The binding mode of a nonselective inhibitor, KH064, was validated by a combination of the experimental electron density and MD simulations. The binding mode of the selective pentapeptide inhibitor FLSYK to hGIIA was stipulated to be different to that of the snake venom phospholipases A2 of Daboia russelli pulchella (svPLA2 ). Our data suggest that the application of MD approaches to crystal structure data is beneficial in evaluating the robustness of conclusions drawn based on crystal structure data alone. Proteins 2017; 85:827-842. © 2016 Wiley Periodicals, Inc.

Primary structures and partial toxicological characterization of two phospholipases A2 from Micrurus mipartitus and Micrurus dumerilii coral snake venoms

Snake venom phospholipases A2 (PLA2) share high sequence identities and a conserved structural scaffold, but show important functional differences. Only a few PLA2s have been purified and characterized from coral snake (Micrurus spp.) venoms, and their role in envenomation remains largely unknown. In this report, we describe the isolation, sequencing and partial functional characterization of two Micrurus PLA2s: MmipPLA2 from Micrurus mipartitus and MdumPLA2 from Micrurus dumerilii, two species of clinical importance in Colombia. MmipPLA2 consisted of 119 amino acid residues with a predicted pI of 8.4, whereas MdumPLA2 consisted of 117 residues with a pI of 5.6. Both PLA2s showed the conserved 'group I' cysteine pattern and were enzymatically active, although MdumPLA2 had higher activity. The two enzymes differed notably in their toxicity, with MmipPLA2 being highly lethal to mice and mildly myotoxic, whereas MdumPLA2 was not lethal (up to 3 μg/g body weight) but strongly myotoxic. MdumPLA2 displayed higher anticoagulant activity than MmipPLA2in vitro and caused more sustained edema in the mouse footpad assay. Neither of these enzymes was cytolytic to cultured skeletal muscle C2C12 myotubes. Based on their structural differences, the two enzymes were placed in separate lineages in a partial phylogeny of Micrurus venom PLA2s and this classification agreed with their divergent biological activities. Overall, these findings highlight the structural and functional diversity of Micrurus venom PLA2s.

Influence of phospholipasic inhibition on neuromuscular activity of Bothrops fonsecai snake venom

Bothrops fonsecai (B. fonsecai), a pitviper endemic to southeastern Brazil, has a venom mainly composed by snake venom phospholipases (PLA2) and metalloproteases, compounds that could interfere with neuromuscular junction in vitro. In this work, we investigated the role of PLA2 in the myotoxicity and neuromuscular blockade caused by B. fonsecai venom using different procedures frequently associated with PLA2 activity inhibition: 24 °C bath temperature, Ca2+ - Sr2+ replacement and chemical modification with p-bromophenacyl bromide (p-BPB). Mice extensor digitorum longus preparations (EDL) were incubated with usual or modified Tyrode solution (prepared with Ca2+ or Sr2+ respectively) at 24 °C or 37 °C (as controls) and in addition of B. fonsecai venom (100 μg/mL) alone or after its incubation with buffer (24 h, 23 °C) on the absence (alkylation control) and presence of p-BPB; all muscle were processed for histological analysis. The PLA2, proteolytic and amidolytic activities under the same conditions (24 °C or 37 °C, Ca2+ - Sr2+ replacement, absence or presence p-BPB) were also assessed. The B. fonsecai venom caused total neuromuscular blockade after 100 min of incubation, in Ca2+ Tyrode solution at 37 °C (usual conditions); on Sr2+ Tyrode solution (37 °C) the twitch height were 31.7 ± 7.4% of basal, and at 24 °C (Ca2+ Tyrode solution) were 53.6 ± 7.0% of basal. The alkylation of PLA2 with p-BPB promoted a great blockade decrease at 100 min of incubation (88.7 ± 5.7% of basal), but it was also observed on alkylation control preparations (66.2 ± 6.6%). The venom produced 50% of blockade at 40.5 ± 5.9 min, in Ca2+ Tyrode solution at 37 °C. The protocols delayed the time for 50% blockade: 105.7 ± 7.1 min (at 24 °C, in Ca2+ Tyrode solution) and 71.1 ± 9.0 min (at 37 °C, in Sr2+ Tyrode solution). Regarding p-BPB incubation and alkylation control preparations, 50% of blockade was not reached during the 120 min of venom incubation. Regarding to enzymatic activities, the 24 °C protocol reduced not only PLA2 (to 62.3%) but also proteolytic (52.3%) and amidolytic (73.4%) activities, as well as observed on p-BPB alkylation protocol which markedly inhibited all enzymes (<10%). The alkylation control promoted the same proteolytic and amidolytic inhibition but no reduction of PLA2 activity; Ca2+ - Sr2+ replacement reduced only the PLA2 activity (to 15.3%). We observed a strict relation between the inhibition of PLA2 activity and the myotoxicity. On the other hand, this relation was not observed with neuromuscular blockade, suggesting that blockade and muscle damage may not be strictly related. It suggests that the neuromuscular blockade may be induced by non-catalytic PLA2 or other venom components, such as metalloproteinases.

Snake Venom PLA2, a Promising Target for Broad-Spectrum Antivenom Drug Development.

Snakebite envenomation is a neglected global health problem, causing substantial mortality, disability, and psychological morbidity, especially in rural tropical and subtropical zones. Antivenin is currently the only specific medicine for envenomation. However, it is restricted by cold storage, snakebite diagnosis, and high price. Snake venom phospholipase A2s (svPLA2s) are found in all kinds of venomous snake families (e.g., Viperidae, Elapidae, and Colubridae). Along with their catalytic activity, svPLA2s elicit a wide variety of pharmacological effects that play a pivotal role in envenomation damage. Hence, neutralization of the svPLA2s could weaken or inhibit toxic damage. Here we overviewed the latest knowledge on the distribution, pathophysiological effects, and inhibitors of svPLA2s to elucidate the potential for a novel, wide spectrum antivenom drug targeting svPLA2s.

Antitumoral Potential of Snake Venom Phospholipases A2 and Synthetic Peptides.

Cancer, a disease that currently affects approximately 14 million people, is characterized by abnormal cell growth with altered replication capacity, which leads to the development of tumor masses without apoptotic control. Resistance to the drugs used in chemotherapy and their side effects stimulate scientific research seeking new therapies to combat this disease. Molecules from flora and fauna with cytotoxic activity against tumor cells have been studied for their potential to become a source of pharmaceutical agents. In this regard, snake venoms have a variety of proteins and peptides that have proven biotechnological potential. In several studies, antibacterial action and antitumor activity have been observed. One of the most widely studied venom components are phospholipases A2. Snake venom phospholipases A2 (svPLA2s) comprise a large class of molecules that catalyze the hydrolysis of the sn-2 position of phospholipids releasing fatty acids and lysophospholipids and are related to a broad spectrum of biotechnological activities. In addition to their specific cytotoxicity against some tumor cell lines, inhibitory activity of angiogenesis, adhesion and cell migration has been described. The antitumor activity of svPLA2s was observed both in vitro and in vivo, but little is known about the mechanism of action of these proteins in promoting this activity. In this review, the main structural and functional characteristics of svPLA2s are discussed, along with the mechanisms proposed, thus far, to explain their antitumor activity, targeting their potential use as a therapeutic alternative against cancer.

Isolation and characterization of bioactive compounds of Clematis gouriana Roxb. ex DC against snake venom phospholipase A2 (PLA2) computational and in vitro insights

Bioactive compounds were isolated from Clematis gouriana Roxb. ex DC. The compounds were separated, characterized, the structures elucidated and submitted to the PubChem Database. The PubChem Ids SID 249494134 and SID 249494135 were tested against phospholipases A2 (PLA2) of Naja naja (Indian cobra) venom for PLA2 activity. Both the compounds showed promising inhibitory activity; computational data also substantiated the results. The two compounds underwent density functional theory calculation to observe the chemical stability and electrostatic potential profile. Molecular interactions between the compounds and PLA2 were observed at the binding pocket of the PLA2 protein. Further, this protein–ligand complexes were simulated for a timescale of 100 ns of molecular dynamics simulation. Experimental and computational results showed significant PLA2 inhibition activity.

Venom Evolution: Gene Loss Shapes Phenotypic Adaptation

Snake venoms are variable protein mixtures with a multitude of bioactivities. New work shows, surprisingly, that it is the loss of toxin-encoding genes that strongly influences venom function in rattlesnakes, highlighting how gene loss can underpin adaptive phenotypic change.

Biochemical and monolayer characterization of Tunisian snake venom phospholipases

The present study investigated the kinetic and interfacial properties of two secreted phospholipases isolated from Tunisian vipers’venoms: Cerastes cerastes (CC-PLA2) and Macrovipera lebetina transmediterranea (MVL-PLA2). Results show that these enzymes have great different abilities to bind and hydrolyse phospholipids. Using egg-yolk emulsions as substrate at pH 8, we found that MVL-PLA2 has a specific activity of 1473U/mg at 37°C in presence of 1mM CaCl2. Furthermore the interfacial kinetic and binding data indicate that MVL-PLA2 has a preference to the zwitterionic phosphatidylcholine monolayers (PC). Conversely, CC-PLA2 was found to be able to hydrolyse preferentially negatively charged head group phospholipids (PG and PS) and exhibits a specific activity 9 times more important (13333U/mg at 60°C in presence of 3mM CaCl2). Molecular models of both CC-PLA2 and MVL-PLA2 3D structures have been built and their electrostatic potentials surfaces have been calculated. A marked anisotropy of the overall electrostatic charge distribution leads to a significantly difference in the dipole moment intensity between the two enzymes explaining the great differences in catalytic and binding properties, which seems to be governed by the electrostatic and hydrophobic forces operative at the surface of the two phospholipases.

The standard aqueous stem bark extract of Mangifera indica L. inhibits toxic PLA2 – NN-XIb-PLA2 of Indian cobra venom

The aqueous extract of Mangifera indica is known to possess diverse medicinal properties, which also includes anti-snake venom activities. However, its inhibitory potency and mechanism of action on multi-toxic snake venom phospholipases A2s are still unknown. Therefore, the objective of this study was to evaluate the modulatory effect of standard aqueous bark extract of M. indica on NN-XIb-PLA2 of Indian cobra venom. The in vitro sPLA2, in situ hemolytic and in vivo edema inhibition effect were carried out as described. Also the effect of substrate and calcium concentration was carried out. M. indica extract dose dependently inhibited the GIA sPLA2 (NN-XIb-PLA2) activity with an IC50 value of 7.6μg/ml. M. indica extract effectively inhibited the indirect hemolytic activity up to 98% at ∼40μg/ml concentration. Further, M. indica extract (0–50μg/ml) inhibited the edema formed in a dose dependent manner. When examined as a function of increased substrate and calcium concentration, there was no relieve of inhibitory effect of M. indica extract on the NN-XIb-PLA2. Further, the inhibition was irreversible as evident from binding studies. The in vitro inhibition is well correlated with in situ and in vivo edema inhibiting activities of M. indica. As the inhibition is independent of substrate and calcium and was irreversible, it can be concluded that M. indica extract mode of inhibition could be due to direct interaction of components present in the extract with the PLA2 enzyme. The aqueous extract of M. indica effectively inhibits svPLA2 enzymatic and its associated toxic activities, which substantiate their anti-snake venom properties. Further in-depth studies on the role and mechanism of the principal constituents present in the extract, responsible for the anti-PLA2 activity will be interesting to develop them into potent antisnake component and also as an anti-inflammatory agent.

Molecular modeling and snake venom phospholipase A2 inhibition by phenolic compounds: Structure–activity relationship

In our earlier study, we have reported that a phenolic compound 2-hydroxy-4-methoxybenzaldehyde from Janakia arayalpatra root extract was active against Viper and Cobra envenomations. Based on the structure of this natural product, libraries of synthetic structurally variant phenolic compounds were studied through molecular docking on the venom protein. To validate the activity of eight selected compounds, we have tested them in in vivo and in vitro models. The compound 21 (2-hydroxy-3-methoxy benzaldehyde), 22 (2-hydroxy-4-methoxybenzaldehyde) and 35 (2-hydroxy-3-methoxybenzylalcohol) were found to be active against venom-induced pathophysiological changes. The compounds 20, 15 and 35 displayed maximum anti-hemorrhagic, anti-lethal and PLA2 inhibitory activity respectively. In terms of SAR, the presence of a formyl group in conjunction with a phenolic group was seen as a significant contributor towards increasing the antivenom activity. The above observations confirmed the anti-venom activity of the phenolic compounds which needs to be further investigated for the development of new anti-snake venom leads.

Inhibitory potential of important phytochemicals from Pergularia daemia (Forsk.) chiov., on snake venom (Naja naja).

Pergularia daemia (Forsk.) chiov., is a milk weed of Asclepiadaceae family. In the present study β-sitosterol, β-amyrin, α-amyrin and lupeol were identified in the leaf by GC–MS. Molecular docking studies were performed to evaluate their activities on phospholipase A2 (PLA2) and l-amino acid oxidase enzymes which constituted a rich source in snake venoms (Naja naja). Snake venom Phospholipase A2 with PDB code 1A3D devoid of co-crystallized ligand was extracted from Protein Data Bank. Using Molegro Virtual Docker two cavities are formed by cocrystallization. l-Amino acid oxidase (PDB code 4E0V) was a receptor model with a co-crystallized ligand FAD. Among the phytochemicals analysed, β-sitosterol displayed high affinity of binding to the active site regions of phospholipase A2 and l-amino acid oxidase, respectively. The affinity of binding was −125.939 and −157.521 kcal/mole identified by gold scores. α-Amyrin and β-amyrin had two hydrogen bond interactions with PLA2. Hence this study suggests that β-sitosterol identified in P. daemia can antagonize PLA2 and LAAO activities and forms a theoretical basis for the folk use of the plant against snake venom.

Orthogonal optimization of prokaryotic expression of a natural snake venom phospholipase A2 inhibitor from Sinonatrix annularis

Phospholipase A2 (PLA2) is a calcium-dependent enzyme that is involved in inflammatory processes such as the liberation of free arachidonic acid from the membrane pool for the biosynthesis of eicosanoids. Snake venom are known containing PLA2s (svPLA2s) which exhibit a wide variety of pharmacological effects including neurotoxicity, cardiotoxicity, myotoxicity and hemorrhage. Therefore, inhibition of svPLA2 would be advantageous to successful envenomation treatment. A gamma type PLI (PLA2 inhibitor) has been extracted from the serum of Sinonatrix annularis, a non-venomous snake indigenous to China. This showed strong inhibition of Deinagkistrrodon acutus PLA2, however, the PLIγ level in the serum and snake resource are not sufficiently sustainable for further research. To overcome these limitations, we constructed a His6-PLIγ pET28 fusion expression vector and transformed Escherichia coli BL21. To improve the expression of PLIγ, an orthogonal experiment [L16(4)5] was performed to optimize induction parameters. The optimized condition was determined to be: induction by 0.4 mM isopropyl-β-d-thiogalactoside (IPTG) for 6 h to the recombinant BL21 after its OD600 was 0.8, with continuous shaking cultivation at 190 rpm and 35 °C. Under these conditions, the amount of expressed protein could reach 57 mg/L. The His6-PLIγ was purified by nickel affinity chromatography and renatured by On-column refolding. The resulting PLIγ showed a good inhibitory effect of enzymatic activities to venom PLA2 isolated from D. acutus. Moreover, the PLIγ had a wide anti-hemorrhage activities to D. acutus, Naja atra and Agkistrodon halys venom.