Snake Venom Phospholipase Research Articles

21st century Latin American synthetic peptides for their application in antivenom production

Envenomation caused by snakes, scorpions, and spiders represents a serious public health problem in Latin America. The antivenoms used for its treatment are produced by immunizing horses repeatedly with sublethal doses of animal venoms along with the adjuvant. However, venom availability is a bottleneck. Furthermore, toxin-neutralizing antibodies are only a few of the total produced with this classical method. Therefore, high doses of antivenom are required to achieve the neutralization power, which usually causes adverse reactions in the patient. With the aim of obtaining a higher proportion of toxin-neutralizing antibodies while reducing the dependency on venom availability, alternative immunization protocols have been explored using synthetic peptides with epitopes from clinically relevant toxins. The process to design an immunogenic peptide entitles: (a) choice of the medical relevant toxins in the venom; (b) identification of the epitopes in the selected toxins; (c) improvement of peptide immunogenicity; (d) immunogen synthesis; and e) in vitro and in vivo evaluation. The present article aims to review the advances in the design of immunogenic synthetic peptides for their application in antivenom production in Latin America during the 21st century. Epitopes have been identified from many clinically important toxins in Latin American snakes (snake venom metalloproteinases, snake venom serine proteases, crotamine, phospholipases A2, and three-finger toxins), scorpions (beta-mammal/insect toxin Ts1, alpha-mammal toxin Ts2, alpha-mammal toxin Ts3, toxin Ts4, and beta-mammal Tt1g neurotoxin), and spiders (dermonecrotic toxin and delta-ctenitoxin-Pn2a). Nevertheless, their application is still experimental, even though they are ideal for large-scale and low-cost antivenom production, factors that are necessary to meet national and regional demands.

Doxycycline-Mediated Inhibition of Snake Venom Phospholipase and Metalloproteinase.

Warfighters are exposed to life-threatening injuries daily and according to the Joint Trauma System Military Clinical Practice Guideline-Global Snake Envenomation Management snakebites are a concerning threat in all theaters of operation. Snake venom is a complex mixture of toxins including phospholipases A2 (PLA2) and snake venom metalloproteinases (SVMP) that produce myotoxic, hemotoxic, and cytotoxic injuries. Antibody-based antivenom is the standard of care but new approaches including small-molecule inhibitors have gained attention in recent years. Doxycycline is an effective inhibitor of human metalloproteinases and PLA2. The enzymatic activities of 3 phylogenetically distinct snakes: Agkistrodon piscivorus, Naja kaouthia, and Daboia russelii were tested under inhibitory conditions using doxycycline. Enzymatic activity of PLA2 and SVMP was measured in N. kaouthia, D. russelii, and A. piscivorus venom alone and with doxycycline using EnzChek Phospholipase A2 and Gelatinase Assay Kits. A 1-way ANOVA with Tukey's post-hoc test was used to conduct comparative analysis. The median lethal dose of the venoms, the effective dose of doxycycline, and creatine kinase (CK) inhibition levels were measured in a murine model with adult Bagg Albino (BALB/c) mice using intramuscular injections. Median lethal and effective doses were determined using Spearman-Karber's method and a 1-way ANOVA with Tukey's post-hoc test was used to compare CK inhibition levels. Phospholipases A2 activity was reduced to 1.5% to 44.0% in all 3 venoms in a dose-dependent manner using 0.32, 0.16, and 0.08 mg/mL doxycycline when compared to venom-only controls (P < .0001) (Fig.1A). Snake venom metalloproteinases activity was reduced to 4% to 62% in all 3 venoms in a dose-dependent manner using 0.32, 0.16, and 0.08 mg/mL doxycycline (P < .0001) (Fig.1B). The lethal dose (LD50) values of the venoms in the murine model were calculated as follows: A. piscivorus = 20.29 mg/kg (Fig.2A), N. kaouthia = 0.38 mg/kg (Fig.2B), and D. russelii = 7.92 mg/kg (Fig.2C). The effective dose (ED50) of doxycycline in A. piscivorus was calculated to be 20.82 mg/kg and 72.07 mg/kg when treating D. russelii venom. No ED50 could be calculated when treating N. kaouthia venom (Fig.3). Creatine kinase activity was significantly decreased in all 3 venoms treated with doxycycline (P < .0001) (Fig.4). Doxycycline reduced PLA2- and SVMP-related lethality, particularly in A. piscivorus envenomings and in a limited capacity with D. russelii revealing its promise as a treatment for snakebites. In addition, CK activity, a common indicator of muscle damage was inhibited in mice that received doxycycline-treated venom. The doxycycline concentrations identified in the ED50 studies correspond to 1,456 to 5,061 mg dosages for a 70 kg human. Factors including venom yield and snake species would affect the actual dosage needed. Studies into high-dose doxycycline safety and its effectiveness against several snake species is needed to fully translate its use into humans. Based on this work, doxycycline could be used as a treatment en route to higher echelons of care, providing protection from muscle damage and reducing lethality in different snake species.

Exploring nature's antidote: unveiling the inhibitory potential of selected medicinal plants from Kisumu, Kenya against venom from some snakes of medical significance in sub-Saharan Africa.

Background: The present study investigated the efficacy of Conyza bonariensis, Commiphora africana, Senna obtusifolia, Warburgia ugandensis, Vernonia glabra, and Zanthoxylum usambarense against Bitis arietans venom (BAV), Naja ashei venom (NAV), and Naja subfulva venom (NSV). Methods: 40 extracts and fractions were prepared using n-hexane, dichloromethane, ethyl acetate, and methanol. In vitro efficacy against snake venom phospholipase A2 (svPLA2) was determined in 96-well microtiter and agarose-egg yolk coagulation assays. in vivo efficacy against venom-induced cytotoxicity was determined using Artemia salina. Two commercial antivenoms were used for comparison. Results: The 96-well microtiter assay revealed poor svPLA2 inhibition of BAV by antivenom (range: 20.76% ± 13.29% to 51.29% ± 3.26%) but strong inhibition (>90%) by dichloromethane and hexane fractions of C. africana, hexane and ethyl acetate extracts and fraction of W. ugandensis, dichloromethane fraction of V. glabra, and the methanol extract of S. obtusifolia. The methanol extract and fraction of C. africana, and the hexane extract of Z. usambarense strongly inhibited (>90%) svPLA2 activity in NAV. The hexane and ethyl acetate fractions of V. glabra and the dichloromethane, ethyl acetate, and methanol extracts of C. africana strongly inhibited (>90%) svPLA2 in NSV. The agarose egg yolk coagulation assay showed significant inhibition of BAV by the dichloromethane fraction of C. africana (EC50 = 3.51 ± 2.58μg/mL), significant inhibition of NAV by the methanol fraction of C. africana (EC50 = 7.35 ± 1.800μg/mL), and significant inhibition of NSV by the hexane extract of V. glabra (EC50 = 7.94 ± 1.50μg/mL). All antivenoms were non-cytotoxic in A. salina but the methanol extract of C. africana and the hexane extracts of V. glabra and Z. usambarense were cytotoxic. The dichloromethane fraction of C. africana significantly neutralized BAV-induced cytotoxicity, the methanol fraction and extract of C. africana neutralized NAV-induced cytotoxicity, while the ethyl acetate extract of V. glabra significantly neutralized NSV-induced cytotoxicity. Glycosides, flavonoids, phenolics, and tannins were identified in the non-cytotoxic extracts/fractions. Conclusion: These findings validate the local use of C. africana and V. glabra in snakebite but not C. bonariensis, S. obtusifolia, W. ugandensis, and Z. usambarense. Further work is needed to isolate pure compounds from the effective plants and identify their mechanisms of action.

Toxicological analyses of the venoms of Nigerian vipers Echis ocellatus and Bitis arietans

BackgroundAmong the medically important snakes in Nigeria, Echis ocellatus and Bitis arietans have the most lethal venom. These venoms were classified according to the presence of snake venom metalloproteinases (SVMPs), snake venom phospholipase A2 (PLA2s), and snake venom serine proteases (SVSPs). Toxicological analyzes were performed to understand the significance of different protein families in venoms.MethodsProteins were separated from venom using column chromatography. The skin and footpad of mice were used to determine hemorrhagic and edematogenic activities. Caprine blood plasma was used to test fibrinolytic activity in vitro.ResultsThe results showed that, compared to the crude venom, the SVMP fraction induced hemorrhagic effects with a diameter of 26.00 ± 1.00 mm in E. ocellatus and 21.33 ± 1.52 mm in B. arietans. Both SVSP and SVMP had anticoagulant effects; however, the SVSP fraction had a stronger effect, with a longer anticoagulation time of 30.00 ± 3.00 min in E. ocellatus and 26.00 ± 2.00 min in B. arietans. These main venom toxins, SVMPs, SVSPs, and PLA2, were found to have edema-forming effects that were optimal at 2 h after envenomation. PLA2s had the highest edema-inducing activity, with onset 30 min after envenomation.ConclusionsGiven the importance of SVMPs in altering the integrity of the membrane structure and impairing the blood coagulation system, an antivenom that can specifically neutralize its activity could inhibit the hemorrhage effects of the venoms.

Alterations of the skeletal muscle contractile apparatus in necrosis induced by myotoxic snake venom phospholipases A2: a mini-review

Skeletal muscle necrosis is a common clinical manifestation of snakebite envenoming. The predominant myotoxic components in snake venoms are catalytically-active phospholipases A2 (PLA2) and PLA2 homologs devoid of enzymatic activity, which have been used as models to investigate various aspects of muscle degeneration. This review addresses the changes in the contractile apparatus of skeletal muscle induced by these toxins. Myotoxic components initially disrupt the integrity of sarcolemma, generating a calcium influx that causes various degenerative events, including hypercontraction of myofilaments. There is removal of specific sarcomeric proteins, owing to the hydrolytic action of muscle calpains and proteinases from invading inflammatory cells, causing an initial redistribution followed by widespread degradation of myofibrillar material. Experiments using skinned cardiomyocytes and skeletal muscle fibers show that these myotoxins do not directly affect the contractile apparatus, implying that hypercontraction is due to cytosolic calcium increase secondary to sarcolemmal damage. Such drastic hypercontraction may contribute to muscle damage by generating mechanical stress and further sarcolemmal damage.

Unraveling snake venom phospholipase A2: an overview of its structure, pharmacology, and inhibitors.

Snake bite is a neglected disease that affects millions of people worldwide. WHO reported approximately 5million people are bitten by various species of snakes each year, resulting in nearly 1million deaths and an additional three times cases of permanent disability. Snakes utilize the venom mainly for immobilization and digestion of their prey. Snake venom is a composition of proteins and enzymes which is responsible for its diverse pharmacological action. Snake venom phospholipase A2 (SvPLA2) is an enzyme that is present in every snake species in different quantities and is known to produce remarkable functional diversity and pharmacological action like inflammation, necrosis, myonecrosis, hemorrhage, etc. Arachidonic acid, a precursor to eicosanoids, such as prostaglandins and leukotrienes, is released when SvPLA2 catalyzes the hydrolysis of the sn-2 positions of membrane glycerophospholipids, which is responsible for its actions. Polyvalent antivenom produced from horses or lambs is the standard treatment for snake envenomation, although it has many drawbacks. Traditional medical practitioners treat snake bites using plants and other remedies as a sustainable alternative. More than 500 plant species from more than 100 families reported having venom-neutralizing abilities. Plant-derived secondary metabolites have the ability to reduce the venom's adverse consequences. Numerous studies have documented the ability of plant chemicals to inhibit the enzymes found in snake venom. Research in recent years has shown that various small molecules, such as varespladib and methyl varespladib, effectively inhibit the PLA2 toxin. In the present article, we have overviewed the knowledge of snake venom phospholipase A2, its classification, and the mechanism involved in the pathophysiology of cytotoxicity, myonecrosis, anticoagulation, and inflammation clinical application and inhibitors of SvPLA2, along with the list of studies carried out to evaluate the potency of small molecules like varespladib and secondary metabolites from the traditional medicine for their anti-PLA2 effect.

Increased delivery and cytotoxicity of doxorubicin in HeLa cells using the synthetic cationic peptide pEM-2 functionalized liposomes

HypothesisDue to the inability of nano-carriers to passively cross the cell membrane, cell penetration enhancers are used to accelerate cytoplasmic delivery of antineoplastic drugs. In this regard, snake venom phospholipase A2 peptides are known for their ability to destabilize natural and artificial membranes. In this context, functionalized liposomes with peptide pEM-2 should favor the incorporation of doxorubicin and increase its cytotoxicity in HeLa cells compared to free doxorubicin, and doxorubicin encapsulated in non-functionalized liposomes. ExperimentsSeveral characteristics were monitored, including doxorubicin loading capacity of the liposomes, as well as the release and uptake before and after functionalization. Cell viability and half-maximal inhibition concentrations were determined in HeLa cells. FindingsIn vitro studies showed that functionalization of doxorubicin-loaded PC-NG liposomes with pEM-2 not only improved the amount of doxorubicin delivered compared to free doxorubicin or other doxorubicin-containing formulations, but also showed enhanced cytotoxicity against HeLa cells. The PC-NG liposomes loaded with doxorubicin improved treatment efficacy by reducing the IC50 value and incubation time. This increase in cell toxicity was directly related to the concentration of pEM-2 peptide bound to the liposomes. We conclude that the cytotoxicity observed in HeLa cells due to the action of doxorubicin was strongly favored when encapsulated in synthetic liposomes and functionalized with the pEM-2 peptide.

INHIBITORY ACTIVITY OF LUPEOL FROM Cryptolepis oblongifolia (MEINS) SCHLTR ON PARTIALLY PURIFIED PHOSPHOLIAPASE A2 VENOM OF Naja nigricollis (ELAPIDAE)

The research is design to provide scientific evidence for the use of Cryptolepis oblongifolia in traditional treatment of inflammation and also to examine whether it have some snake venom phospholipase A2 inhibition potential. A triterpene was isolated from C. oblongifolia using column chromatography and identified via spectroscopic means. The inhibitory activity of the isolate on phospholipase A2 (PLA2) of Naja nigricollis venom was evaluated. The proton nuclear magnetic resonance (1H NMR) of the isolated compound showed the presence of olefinic protons while the carbon nuclear magnetic resonance (13C NMR) showed the presence of exomethylene carbon atoms. By comparison with the reported literature the compound was found to be lupeol, the inhibitory activity of the isolate on PLA2 of Naja nigricollis venom showed that, the isolate inhibits the PLA2 at dose dependent manner with inhibition binding constant (ki) of 0.015 mg/ml. The activity of the compound isolated supports the local usage of C. oblongifolia in traditional treatment of inflammation.

Bactericidal Effects of Snake Venom Phospholipases A2: A Systematic Review and Analysis of Minimum Inhibitory Concentration

Background: Infections caused by multi-drug resistance (MDR) strains are potentially fatal public health issues worldwide that need pressing attention. Previous reports suggested using snake venom fractions as an effective alternative mechanism to the already available antibacterial drugs. In this study, we conducted a systematic review to analyze the bactericidal effects of snake venom phospholipases (PLA2s). Methods: From the beginning through 30 March 2022, we searched the PubMed and Embase databases in accordance with the most recent PRISMA recommendations. We also conducted a manual search to identify relevant reports to improve literature coverage. Results: A total of 24 studies were included based on the selection criteria to compile this review. Of them, 16 studies were obtained from the abovementioned databases and eight through manual searches. The other 8 studies were obtained through the references of the included studies. According to the review, we reported that some PLA2s showed more vigorous bactericidal activity on some Gram-negative and a moderate effect on Gram-negative and Gram-positive. Furthermore, we reported that the presence of p-bromophenacyl bromide (p-BPP) showed a significant decrease in enzymatic and associated antibacterial activities. Moreover, we observed that about 80% of the PLA2s reported in our systematic review study were those from the Viperidae family, whereas 20% came from the Elapidae family. Moreover, some variations were revealed in the current study regarding the mechanism of actions of the snake venom PLA2s (svPLA2s). Conclusion: This systematic review provides a comprehensive overview of the bactericidal effect of snake venom PLA2s and the analysis of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of PLA2s for bacterial strains. Varying bactericidal effects from various snake species and South American rattlesnakes were reported, presenting compelling concepts to the alternative search for therapies against bacterial resistance. Thus, further analysis of the bactericidal effects of other snake venoms PLA2s considering different strains is needed. Moreover, more data are needed to investigate other bacteria of public health priority using peptides and other purified snake toxins.

Structural and functional studies of a snake venom phospholipase A2-like protein complexed to an inhibitor from Tabernaemontana catharinensis

Snake envenomation is an ongoing global health problem and tropical neglected disease that afflicts millions of people each year. The only specific treatment, antivenom, has several limitations that affects its proper distribution to the victims and its efficacy against local effects, such as myonecrosis. The main responsible for this consequence are the phospholipases A2 (PLA2) and PLA2-like proteins, such as BthTX-I from Bothrops jararacussu. Folk medicine resorts to plants such as Tabernaemontana catharinensis to palliate these and other snakebite effects. Here, we evaluated the effect of its root bark extract and one of its isolated compounds, 12-methoxy-4-methyl-voachalotine (MMV), against the in vitro paralysis and muscle damage induced by BthTX-I. Secondary and quaternary structures of BthTX-I were not modified by the interaction with MMV. Instead, this compound interacted in an unprecedented way with the region inside the toxin hydrophobic channel and promoted a structural change in Val31, loop 58–71 and Membrane Disruption Site. Thus, we hypothesize that MMV inhibits PLA2-like proteins by preventing entrance of fatty acid into the hydrophobic channel. These data may explain the traditional use of T. catharinensis extract and confirm MMV as a promising candidate to complement antivenom or a structural guide to develop more effective inhibitors.

In silico screening of the phytochemicals present in Clitoria ternatea L. as the inhibitors of snake venom phospholipase A2 (PLA2)

Millions of people suffer from snake bite envenomation, and its management is a challenge, even today. Medicinal plants have attracted the researcher’s attention for their outstanding advantages in treating many diseases, including snake venom poisoning. Clitoria ternatea L, is a plant popularly known for its various pharmacological effects especially, anti-snake venom property. However, the molecular mechanism behind this is poorly understood. It is reported that snake venom PLA2 is an extensively studied toxic factor. This study is meant to screen the compound’s capability to act as inhibitors of the Daboia russelli snake venom PLA2 through molecular docking and dynamics studies. Our results show that among the 27 compounds taken for the study, only Kaempferol showed good interaction profile with the conserved catalytic active site residues, His48 and Asp49. The pharmacophore features of the compound also demonstrate its exact fitting at the binding pocket. Further RMSD, RMSF, Rg, and hydrogen bond analysis confirmed the stable binding of Kaempferol with PLA2 through molecular dynamic simulations for 100 ns. In addition, the MM/PBSA binding free energy calculation of the complex was also affirming the docking results. The binding free energy (BFE) of Kaempferolis better than the reference compound. ADME and Lipinski’s rule of five reveals its drug like properties. Communicated by Ramaswamy H. Sarma

The relative efficacy of chemically diverse small-molecule enzyme-inhibitors against anticoagulant activities of Black Snake (Pseudechis spp.) venoms

Snakebite remains a worldwide public health burden and a severely neglected tropical disease. Recent research has begun to focus on the potential use of repurposed small-molecule enzyme-inhibitors as early treatments to neutralise the effects of snake venoms. Black snakes (Pseudechis spp.) are a widespread and dangerously venomous group found throughout Australia and New Guinea. Utilising validated coagulation assays, our study assessed the efficacy of two chemically different small molecule inhibitors, a phospholipase A2 inhibitor (varespladib) and a metalloproteinase inhibitor (prinomastat), in vitro neutralisation of the anticoagulant prothrombinase-inhibiting activity of venom from seven species within the Pseudechis genus (P. australis, P. butleri, P. coletti, P. guttatus, P. papuanus, P.rossignolii, P. sp (NT).). Varespladib was shown to be highly effective at neutralising this anticoagulant activity for all seven species, but with P. coletti notably less so than the others. In contrast, prinomastat showed strong neutralisation for five out of the seven species, but was ineffective at neutralising the activity of P. coletti or P. rossignolii venoms. This suggests that varespladib binds to a highly conserved site but that prinomastat binds to a more variable site. These results build upon recent literature indicating that metalloproteinase inhibitors have cross-neutralising potential towards snake venom phospholipase A2 toxins, but with higher degrees of variability that PLA2-specific inhibitors. An important caveat is that these are in vitro tests and while suggestive of potential clinical utility, in vivo animal testing and clinical trials are required as future work.

Interpopulational variation and ontogenetic shift in the venom composition of Lataste's viper (Vipera latastei, Boscá 1878) from northern Portugal

Lataste's viper (Vipera latastei) is a venomous European viper endemic to the Iberian Peninsula, recognised as medically important by the World Health Organization. To date, no comprehensive characterisation of this species' venom has been reported. Here, we analysed the venoms of juvenile and adult specimens of V. latastei from two environmentally different populations from northern Portugal. Using bottom-up venomics, we produced six venom proteomes (three per population) from vipers belonging to both age classes (i.e., two juveniles and four adults), and RP-HPLC profiles of 54 venoms collected from wild specimens. Venoms from juveniles and adults differed in their chromatographic profiles and relative abundances of their toxins, suggesting the occurrence of ontogenetic changes in venom composition. Specifically, snake venom metalloproteinase (SVMP) was the most abundant toxin family in juvenile venoms, while snake venom serine proteinases (SVSPs), phospholipases A2 (PLA2s), and C-type lectin-like (CTLs) proteins were the main toxins comprising adult venoms. The RP-HPLC venom profiles were found to vary significantly between the two sampled localities, indicating geographic variability. Furthermore, the presence/absence of certain peaks in the venom chromatographic profiles appeared to be significantly correlated also to factors like body size and sex of the vipers. Our findings show that V. latastei venom is a variable phenotype. The intraspecific differences we detected in its composition likely mirror changes in the feeding ecology of this species, taking place during different life stages and under different environmental pressures. SignificanceLataste's viper (Vipera latastei) is a medically important viper endemic to the Iberian Peninsula, inhabiting different habitats and undergoing a marked ontogenetic dietary shift. In the current study, we report the first proteomic analysis of V. latastei venom from two environmentally different localities in northern Portugal. Our bottom-up venomic analyses show that snake venom serine proteinases (SVSPs), phospholipases A2 (PLA2s), and C-type lectin-like (CTLs) proteins are the major components of adult V. latastei venom. The comparative analysis of young and adult venoms suggests the occurrence of ontogenetic shift in toxin abundances, with snake venom metalloproteinases (SVMPs) being the predominant toxins in juvenile venoms. Moreover, geographic venom variation between the two studied populations is also detected, with our statistical analyses suggesting that factors like body size and sex of the vipers are possibly at play in its determination.Our work represents the first assessment of the composition of V. latastei venom, and the first step towards a better understanding of the drivers behind its variability.

The Unusual Metalloprotease-Rich Venom Proteome of the Australian Elapid Snake Hoplocephalus stephensii.

The Australasian region is home to the most diverse elapid snake radiation on the planet (Hydrophiinae). Many of these snakes have evolved into unique ecomorphs compared to elapids on other continents; however, their venom compositions are poorly known. The Australian elapid Hoplocephalus stephensii (Stephen’s banded snake) is an arboreal snake with a unique morphology. Human envenoming results in venom-induced consumption coagulopathy, without neurotoxicity. Using transcriptomics and a multi-step fractionation method involving reverse-phase high-performance liquid chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis and bottom-up proteomics, we characterized the venom proteome of H. stephensii. 92% of the total protein component of the venom by weight was characterized, and included all dominant protein families and 4 secondary protein families. Eighteen toxins made up 76% of the venom, four previously characterized and 14 new toxins. The four dominant protein families made up 77% of the venom, including snake venom metalloprotease (SVMP; 36.7%; three identified toxins), phospholipase A2 (PLA2; 24.0%; five identified toxins), three-finger toxin (3FTx; 10.2%; two toxins) and snake venom serine protease (SVSP; 5.9%; one toxin; Hopsarin). Secondary protein families included L-amino acid oxidase (LAAO; 10.8%; one toxin), natriuretic peptide (NP; 0.8%; two toxins), cysteine-rich secretory protein (CRiSP; 1.7%; two toxins), c-type lectin (CTL; 1.1%; one toxin), and one minor protein family, nerve growth factor (NGF; 0.8%; one toxin). The venom composition of H. stephensii differs to other elapids, with a large proportion of SVMP and LAAO, and a relatively small amount of 3FTx. H. stephensii venom appeared to have less toxin diversity than other elapids, with only 18 toxins making up three-quarters of the venom.

Viperidae snake venom phospholipase A2 . Biochemical targets for the action of protein in the human blood circulatory system. Part 1 (review of literature)

Snake venoms have been fervently studied for decades for two reasons: 1) high death rate due to snake bites; 2) numerous components of snake venoms prove useful in medicine and treatment of diverse pathologies. Snake venom phospholipases A2 are among the most aggressive toxic proteins often playing the main role in immobilization and killing of snakebite victims. These enzymes catalyze the hydrolysis of glycerophospholipids to produce lysoglycerophospholipids and free fatty acids. But the diversity of phospholipase A2 effects is not limited to their catalytic action. In this review (Part 1), we describe the features of the structure and properties of Viperidae snake venom phospholipase A2 .

Exploring the potent inhibitor β-stigmasterol from Pittosporum dasycaulon Miq. leaves against snake venom phospholipase A2 protein through in vitro and molecular dynamics behavior approach

β-Stigmasterol, responsible for antivenom activity against abundant PLA2 protein of Viper (Daboia russelii) and Cobra (Naja naja) venom, was identified, characterized, and isolated from Pittosporum dasycaulon leaves extract. The compound purity was checked by RP-HPLC analysis, and structure was elucidated by FTIR and NMR analysis, followed by in-vitro as well as in-silico studies. The structural stability of the docked complexes was evaluated by molecular dynamic simulation, and the binding free energies were calculated by MM-PBSA analysis. The in-vitro analysis revealed that the isolated compound β-SS is effective against D. russelii PLA2 (IC50 40.903 ± 0.479 μg/mL) than N. naja venom PLA2 (42.340 ± 0.11 μg/mL). The in-silico approaches helps to conclude that the β-SS-PLA2 D. russelii complex showed a stable conformation with a reduced degree of flexibility throughout the dynamics simulation time period with high-affinity docking energy −10.60 kcal/mol than β-SS-PLA2 N. naja complex, which observed docking energy of −10.39 kcal/mol.

Biochemical and proteomic analyses of venom from a new pit viper, Protobothrops kelomohy.

Background: A new pit viper, Protobothrops kelomohy, has been recently discovered in northern and northwestern Thailand. Envenoming by the other Protobothrops species across several Asian countries has been a serious health problem since their venom is highly hematotoxic. However, the management of P. kelomohy bites is required as no specific antivenom is available. This study aimed to investigate the biochemical properties and proteomes of P. kelomohy venom (PKV), including the cross-neutralization to its lethality with antivenoms available in Thailand.Methods: PKV was evaluated for its neutralizing capacity (ER50), lethality (LD50), procoagulant and hemorrhagic effects with three monovalent antivenoms (TAAV, DSAV, and CRAV) and one polyvalent (HPAV) hematotoxic antivenom. The enzymatic activities were examined in comparison with venoms of Trimeresurus albolabris (TAV), Daboia siamensis (DSV), Calloselasma rhodostoma (CRV). Molecular mass was separated on SDS-PAGE, then the specific proteins were determined by western blotting. The venom protein classification was analyzed using mass spectrometry-based proteomics.Results: Intravenous LD50 of PKV was 0.67 µg/g. ER50 of HPAV, DSAV and TAAV neutralize PKV at 1.02, 0.36 and 0.12 mg/mL, respectively. PKV exhibited procoagulant effect with a minimal coagulation dose of 12.5 ± 0.016 µg/mL and hemorrhagic effect with a minimal hemorrhagic dose of 1.20 ± 0.71 µg/mouse. HPAV was significantly effective in neutralizing procoagulant and hemorrhagic effects of PKV than those of TAAV, DSAV and CRAV. All enzymatic activities among four venoms exhibited significant differences. PKV proteome revealed eleven classes of putative snake venom proteins, predominantly metalloproteinase (40.85%), serine protease (29.93%), and phospholipase A2 (15.49%).Conclusions: Enzymatic activities of PKV are similarly related to other viperid venoms in this study by quantitatively hematotoxic properties. Three major venom toxins were responsible for coagulopathy in PKV envenomation. The antivenom HPAV was considered effective in neutralizing the lethality, procoagulant and hemorrhagic effects of PKV.

New multienzymatic complex formed between human cathepsin D and snake venom phospholipase A2.

Cathepsin D (CatD) is a lysosomal proteolytic enzyme expressed in almost all tissues and organs. This protease is a multifunctional enzyme responsible for essential biological processes such as cell cycle regulation, differentiation, migration, tissue remodeling, neuronal growth, ovulation, and apoptosis. The overexpression and hypersecretion of CatD have been correlated with cancer aggressiveness and tumor progression, stimulating cancer cell proliferation, fibroblast growth, and angiogenesis. In addition, some studies report its participation in neurodegenerative diseases and inflammatory processes. In this regard, the search for new inhibitors from natural products could be an alternative against the harmful effects of this enzyme. An investigation was carried out to analyze CatD interaction with snake venom toxins in an attempt to find inhibitory molecules. Interestingly, human CatD shows the ability to bind strongly to snake venom phospholipases A2 (svPLA2), forming a stable muti-enzymatic complex that maintains the catalytic activity of both CatD and PLA2. In addition, this complex remains active even under exposure to the specific inhibitor pepstatin A. Furthermore, the complex formation between CatD and svPLA2 was evidenced by surface plasmon resonance (SPR), two-dimensional electrophoresis, enzymatic assays, and extensive molecular docking and dynamics techniques. The present study suggests the versatility of human CatD and svPLA2, showing that these enzymes can form a fully functional new enzymatic complex.

Lessons from a Single Amino Acid Substitution: Anticancer and Antibacterial Properties of Two Phospholipase A2-Derived Peptides.

The membrane-active nature of phospholipase A2-derived peptides makes them potential candidates for antineoplastic and antibacterial therapies. Two short 13-mer C-terminal fragments taken from snake venom Lys49-PLA2 toxins (p-AppK and p-Acl), differing by a leucine/phenylalanine substitution, were synthesized and their bioactivity was evaluated. Their capacity to interfere with the survival of Gram-positive and Gram-negative bacteria as well as with solid and liquid tumors was assessed in vitro. Toxicity to red blood cells was investigated via in silico and in vitro techniques. The mode of action was mainly studied by molecular dynamics simulations and membrane permeabilization assays. Briefly, both peptides have dual activity, i.e., they act against both bacteria, including multidrug-resistant strains and tumor cells. All tested bacteria were susceptible to both peptides, Pseudomonas aeruginosa being the most affected. RAMOS, K562, NB4, and CEM cells were the main leukemic targets of the peptides. In general, p-Acl showed more significant activity, suggesting that phenylalanine confers advantages to the antibacterial and antitumor mechanism, particularly for osteosarcoma lines (HOS and MG63). Peptide-based treatment increased the uptake of a DNA-intercalating dye by bacteria, suggesting membrane damage. Indeed, p-AppK and p-Acl did not disrupt erythrocyte membranes, in agreement with in silico predictions. The latter revealed that the peptides deform the membrane and increase its permeability by facilitating solvent penetration. This phenomenon is expected to catalyze the permeation of solutes that otherwise could not cross the hydrophobic membrane core. In conclusion, the present study highlights the role of a single amino acid substitution present in natural sequences towards the development of dual-action agents. In other words, dissecting and fine-tuning biomembrane remodeling proteins, such as snake venom phospholipase A2 isoforms, is again demonstrated as a valuable source of therapeutic peptides.

Snake venom phospholipase A2s exhibit strong virucidal activity against SARS-CoV-2 and inhibit the viral spike glycoprotein interaction with ACE2.

The COVID-19 pandemic caused by SARS-CoV-2 requires new treatments both to alleviate the symptoms and to prevent the spread of this disease. Previous studies demonstrated good antiviral and virucidal activity of phospholipase A2s (PLA2s) from snake venoms against viruses from different families but there was no data for coronaviruses. Here we show that PLA2s from snake venoms protect Vero E6 cells against SARS-CoV-2 cytopathic effects. PLA2s showed low cytotoxicity to Vero E6 cells with some activity at micromolar concentrations, but strong antiviral activity at nanomolar concentrations. Dimeric PLA2 from the viper Vipera nikolskii and its subunits manifested especially potent virucidal effects, which were related to their phospholipolytic activity, and inhibited cell–cell fusion mediated by the SARS-CoV-2 spike glycoprotein. Moreover, PLA2s interfered with binding both of an antibody against ACE2 and of the receptor-binding domain of the glycoprotein S to 293T/ACE2 cells. This is the first demonstration of a detrimental effect of PLA2s on β-coronaviruses. Thus, snake PLA2s are promising for the development of antiviral drugs that target the viral envelope, and could also prove to be useful tools to study the interaction of viruses with host cells.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-021-03985-6.