Anti-Snake Venom Properties of Medicinal Plants: A Comprehensive Systematic Review of Literature

Abstract Snakebite is one of the major health issues posing considerable morbidity and mortality. According to an estimate of World Health Organization (WHO) (World health organization, 2021) approximately 5 million people are bitten by several species of snakes resulting in up to 2.5 million envenomation cases annually. The mainstay of treatment for envenomation is intravenous administration of anti-snake venom. Although antivenom neutralizes the systemic effects but it does not relieve the symptoms such as venom-induced hemorrhage, necrosis and nephrotoxicity. Moreover, the use of antivenoms is associated with hypersensitivity reactions including urticaria, anaphylaxis, or serum sickness due to their heterologous property. Furthermore, stringent storage conditions and narrow specificity of antivenoms limit their use in both developed as well as developing countries. In this context, researchers have been searching for natural products and plant extracts to explore their antivenom activity along with anti-myotoxic, anti-hemorrhagic and anti-inflammatory properties. Plant remedies may prove to be an effective alternate for antivenom sera with less adverse events and better tolerability. To the best of our knowledge, this is the first comprehensive review of medicinal plants possessing anti-snake venom activities against certain species of snakes. The current review highlights the investigated plants with their phytochemical analysis to integrate the available information for future research and development of antivenom sera.

In vitro anti-venom potential of various solvent based leaf extracts of Andrographis serpyllifolia (Rottler ex Vahl) Wight against Naja naja and Daboia russelli.

ETHNOPHARMACOLOGICAL RELEVANCE: Snake bite is a major occupational hazard in tropical and subtropical countries including India as per the World Health Organization. Naja naja (Indian cobra) and Daboia russelli (Russell's viper) are the two poisonous snakes commonly associated with human mortality in India. Andrographis serpyllifolia (Rottler ex Vahl) Wight has been documented in ethnobotanical records as a plant possessing potent anti-snake venom activity. AIM OF THE STUDY: The present study is aimed for systematic evaluation of in vitro anti-venom potential of various solvent based leaf extracts of A. serpyllifolia against toxic venom enzymes of Naja naja and Daboia russelli. MATERIALS AND METHODS: Different solvent based leaf extracts of A. serpyllifolia were tested against the snake venoms of Naja naja and Daboia russelli obtained from Irula Snake Catchers Industrial Co-operative Society Limited, Kancheepuram, Tamil nadu, India. Three different in vitro neutralization assays such as indirect hemolysis, procoagulent and lytic activities and seven in vitro enzyme inhibition assays such as protease, acetylcholinesterase, phosphomonoesterase, phosphodiesterase, 5'nucleotidase, phospholipase A2, hyaluronidase and post synaptic acetylcholine receptor binding activity were carried out according to standard protocols. The results were analyzed using the standard ANOVA procedures. RESULTS: Among various solvent based leaf extracts of A. serpyllifolia tested, aqueous extract showed maximum neutralizing and inhibitory activities against Naja naja and Daboia russelli venoms. CONCLUSIONS: The various in vitro enzymatic studies reveal that the aqueous leaf extract of A. serpyllifolia plant could inhibit most of the toxic enzymes of the Naja naja and Daboia russelli venoms which could be further confirmed by in vivo studies.

The Fraction of the Snake Venom, Its Leishmanicidal Effect, and the Stimulation of an Anti-Leishmania Response in Infected Macrophages.

BACKGROUND AND AIMS: Due to the lack of an effective vaccine and complexity of the control measures against vectors and reservoir hosts, the control of leishmaniasis depends primarily on chemotherapy. This study was aimed to assess the snake venom, Naja naja oxiana fraction 11(NNOVF11) on Leishmania infantum and its broad mode of action. METHODS: A wide range of in vitro advanced assays including high-performance liquid chromatography (HPLC), MTT (3-[4, 5-Dimethylthiazol-2-yl]-2, 5diphenyltetrazolium bromide; Thiazolyl blue), macrophage assays, quantitative real-time polymerase chain reaction (qPCR), flow cytometry and enzyme- linked immunosorbent assay (ELISA) on L. infantum promastigote and amastigote stages were used. IC50 values of L. infantum stages, CC50 value, and apoptosis were also analyzed. RESULTS: The NNOV-F11 demonstrated strong antileishmanial activity against L. infantum stages in a dose-dependent manner compared to the untreated control group. Interleukin (IL)-12, TNF-α, and iNOS genes expression as the indicators of T helper(h)1 response significantly increased; in contrast, the expression level of IL-10, as the representative of Th2 response significantly decreased (p < 0.001). Reactive oxygen species (ROS) detection showed a significant increase (p < 0.001) after treatment with different concentrations of NNOV-F11, unlike arginase (ARG) activity, which displayed a significant reduction (p < 0.001). CONCLUSION: NNOV-F11 possessed a potent inhibitory effect on L. infantum stages with the multifunctional and broad mode of actions, which promoted the immunomodulatory role, induced ROS production, stimulated apoptotic-like mechanisms, and inhibited L-ARG activity, which collectively led to the parasite death. Further studies are crucial to assess the effect of the NNOV-F11 on animal models or clinical settings.

Comments on Proteomic Investigations of Two Pakistani Naja Snake Venoms Species Unravel the Venom Complexity, Posttranslational Modifications, and Presence of Extracellular Vesicles. Toxins 2020, 12, 669.

We read with interest the article by Manuwar et al [...].

Unusual quaternary structure of a homodimeric synergistic-type toxin from mamba snake venom defines its molecular evolution.

Snake venoms are complex mixtures of enzymes and nonenzymatic proteins that have evolved to immobilize and kill prey animals or deter predators. Among them, three-finger toxins (3FTxs) belong to the largest superfamily of nonenzymatic proteins. They share a common structure of three ß-stranded loops extending like fingers from a central core containing all four conserved disulfide bonds. Most 3FTxs are monomers and through subtle changes in their amino acid sequences, they interact with different receptors, ion channels and enzymes to exhibit a wide variety of biological effects. The 3FTxs have further expanded their pharmacological space through covalent or noncovalent dimerization. Synergistic-type toxins (SynTxs) isolated from the deadly mamba venoms, although nontoxic, have been known to enhance the toxicity of other venom proteins. However, the details of three-dimensional structure and molecular mechanism of activity of this unusual class of 3FTxs are unclear. We determined the first three-dimensional structure of a SynTx isolated from Dendroaspis jamesoni jamesoni (Jameson's mamba) venom. The SynTx forms a unique homodimer that is held together by an interchain disulfide bond. The dimeric interface is elaborate and encompasses loops II and III. In addition to the inter-subunit disulfide bond, the hydrogen bonds and hydrophobic interactions between the monomers contribute to the dimer formation. Besides, two sulfate ions that mediate interactions between the monomers. This unique quaternary structure is evolved through noncovalent homodimers such as κ-bungarotoxins. This novel dimerization further enhances the diversity in structure and function of 3FTxs.

Isolation and pharmacological characterization of α-Elapitoxin-Na1a, a novel short-chain postsynaptic neurotoxin from the venom of the Chinese Cobra (Naja atra).

The Chinese Cobra (Naja atra) is an elapid snake of major medical importance in southern China. Although previous studies have shown that postsynaptic neurotoxins account for 11-23% of N. atra venom, envenomed patients do not display marked signs of neurotoxicity. We have previously shown that the lack of clinical neurotoxicity following snake envenoming by some species with 'neurotoxic' venoms may be related to the high prevalence of short-chain postsynaptic neurotoxins in these venoms. In this study, we describe the isolation and characterization of α-Elapitoxin-Na1a (α-EPTX-Na1a; 6949 Da), a short-chain postsynaptic neurotoxin, which accounts for approximately 9% of N. atra crude venom. α-EPTX-Na1a (30-300 nM) produced concentration-dependent inhibition of indirect-twitches, with a t90 value of 17 ± 2 min at 300 nM, and abolished contractile responses to exogenous acetylcholine and carbachol, in the chick biventer cervicis nerve-muscle preparation. The prior addition of either Chinese N. atra monovalent antivenom (0.3 U/ml) or Australian polyvalent snake antivenom (2.4 U/ml), prevented the in vitro neurotoxic effects of α-EPTX-Na1a (30 nM). Addition of each of these antivenoms at the t90 time point partially reversed the in vitro neurotoxicity caused by α-EPTX-Na1a (30 nM). The inhibition of indirect twitches by α-EPTX-Na1a (30 nM) was not reversed by repeatedly washing the tissue. α-EPTX-Na1a displayed pseudo-irreversible antagonism of concentration-response curves to carbachol with a pA2 value of 8.21. De novo protein sequencing of α-EPTX-Na1a revealed a typical short-chain postsynaptic neurotoxin profile of 62 amino acids which shared >98% amino acid sequence similarity with short-chain postsynaptic neurotoxins from other Naja species. When compared to short-chain neurotoxins isolated from cobras in China, α-EPTX-Na1a contained novel residues K47Q (i.e. lysine to glutamine), N48T (i.e. asparagine to threonine) and G49A (i.e. glycine to alanine). In conclusion, α-EPTX-Na1a is a potent, pseudo-irreversible, short-chain neurotoxin. The high prevalence of α-EPTX-Na1a in Chinese N. atra venom is likely to explain the mild neurotoxicity experienced by envenomed patients.

King cobra peptide OH-CATH30 as a potential candidate drug through clinic drug-resistant isolates.

Cationic antimicrobial peptides (AMPs) are considered as important candidate therapeutic agents, which exert potent microbicidal properties against bacteria, fungi and some viruses. Based on our previous findings king cobra cathelicidin (OH-CATH) is a 34-amino acid peptide that exerts strong antibacterial and weak hemolytic activity. The aim of this research is to evaluate the efficacy of both OH-CATH30 and its analog D-OH-CATH30 against clinical isolates comparing with routinely utilized antibiotics in vitro. In this study, 584 clinical isolates were tested (spanning 2013-2016) and the efficacy of the candidate peptides and antibiotics were determined by a broth microdilution method according to the CLSI guidelines. Among the 584 clinical isolates, 85% were susceptible to OH-CATH30 and its analogs. Both L- and D-OH-CATH30 showed higher efficacy against (toward) Gram-positive bacteria and stronger antibacterial activity against nearly all Gram-negative bacteria tested compare with antibiotics. The highest bactericidal activity was detected against Acinetobacter spp., including multi-drug-resistant Acinetobacter baumannii (MRAB) and methicillin-resistant Staphylococcus aureus (MRSA). The overall efficacy of OH-CATH30 and its analogs was higher than that of the 9 routinely used antibiotics. OH-CATH30 is a promising candidate drug for the treatment of a wide variety of bacterial infections which are resistant to many routinely used antimicrobial agents.

Actiflagelin, a new sperm activator isolated from Walterinnesia aegyptia venom using phenotypic screening

Background Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia. Methods Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. Results Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4- C6, C7-C8 and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom. Conclusions This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.

Non-neurotoxic activity of Malayan krait (Bungarus candidus) venom from Thailand

Envenoming by kraits (genus Bungarus) is a medically significant issue in South Asia and Southeast Asia. Malayan krait (Bungarus candidus) venom is known to contain highly potent neurotoxins. In recent years, there have been reports on the non-neurotoxic activities of krait venom that include myotoxicity and nephrotoxicity. However, research on such non-neurotoxicity activities of Malayan krait venom is extremely limited. Thus, the aim of the present study was to determine the myotoxic, cytotoxic and nephrotoxic activities of B. candidus venoms from northeastern (BC-NE) and southern (BC-S) Thailand in experimentally envenomed rats. Methods: Rats were administered Malayan krait (BC-NE or BC-S) venom (50 µg/kg, i.m.) or 0.9% NaCl solution (50 µL, i.m.) into the right hind limb. The animals were sacrificed 3, 6 and 24 h after venom administration. The right gastrocnemius muscle and both kidneys were collected for histopathological analysis. Blood samples were also taken for determination of creatine kinase (CK) and lactate dehydrogenase (LDH) levels. The human embryonic kidney cell line (HEK-293) was used in a cell proliferation assay to determine cytotoxic activity. Results: Administration of BC-NE or BC-S venom (50 µg/kg, i.m.) caused time-dependent myotoxicity, characterized by an elevation of CK and LDH levels. Histopathological examination of skeletal muscle displayed marked muscle necrosis and myofiber disintegration 24 h following venom administration. Both Malayan krait venoms also induced extensive renal tubular injury with glomerular and interstitial congestion in rats. BC-NE and BC-S venoms (100­0.2 µg/ mL) caused concentration-dependent cytotoxicity on the HEK-293 cell line. However, BC-NE venom (IC50 =8 ± 1 µg/mL; at 24 h incubation; n = 4) was found to be significantly more cytotoxic than BC-S venom (IC50 =15 ± 2 µg/mL; at 24 h incubation; n = 4). In addition, the PLA2 activity of BC-NE venom was significantly higher than that of BC-S venom. Conclusions: This study found that Malayan krait venoms from both populations possess myotoxic, cytotoxic and nephrotoxic activities. These findings may aid in clinical diagnosis and treatment of envenomed patients in the future.(AU)

Actiflagelin, a new sperm activator isolated from Walterinnesia aegyptia venom using phenotypic screening

Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia. Methods Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. Results Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4- C6, C7-C8 and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom. Conclusions This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.(AU)

Delineating residues for haemolytic activities of snake venom cardiotoxin 1 from Naja naja as probed by molecular dynamics simulations and in vitro validations.

Cardiotoxins (CTXs) are single polypeptide chain consisting of 59-62 amino acids with four disulfide bridges and globular proteins of simple ß-sheet folds. The CTXs are one of principal toxic components causing haemolysis and damaging various cells and belong to three-finger toxin (TFT) superfamily of snake venoms. However, there is no natural or synthetic small molecular inhibitor to the protein toxins to date. In the present study, modes of interaction of cardiotoxin 1 (CTX1) from Indian cobra (Naja naja) with heterogeneous erythrocyte membrane (EM) model system have been extensively examined by using all-atom molecular dynamics (MD) simulations in near physiological conditions and comprehensive analyses of the MD data revealed two distinct principal regions ('head groove' and 'loop groove') of the protein toxin for establishing structural interactions with the EM system. Moreover, combined analyses of data from high-throughput virtual screening of NCI small molecular database, in vitro haemolytic assays for top-hits of the chemical compounds against crude venom of Naja naja and as well CTXs purified from the venom and pharmacokinetic examinations on the chemical compounds retarding haemolytic activities of CTXs suggested that Etidronic acid and Zoledronic acid are promising prototypic chemical inhibitors to CTXs of snake venoms.

Hydrostatin-TL1, an Anti-Inflammatory Active Peptide from the Venom Gland of Hydrophis cyanocinctus in the South China Sea.

Tumor necrosis factor (TNF)-α is a pleiotropic cytokine with intense pro-inflammatory and immunomodulatory properties, and anti-TNF-α biologics are effective therapies for various inflammatory diseases such as inflammatory bowel disease (IBD) and sepsis. Snake venom, as a traditional Chinese medicine, has been used in the treatment of inflammatory diseases in China for centuries. In this research, we constructed a venom gland T7 phage display library of the sea snake Hydrophis cyanocinctus to screen bioactive compounds that antagonize TNF-α and identified a novel nine-amino-acid peptide, termed hydrostatin-TL1 (H-TL1). In enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) analyses, H-TL1 inhibited the interaction between TNF-α and TNF receptor 1 (TNFR1). Further, H-TL1 attenuated the cytotoxicity of TNF-α in L929 cells as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. H-TL1 also decreased the mRNA expression of TNF-α/TNFR1 downstream targets and suppressed the phosphorylation of well-characterized proteins of downstream signal transduction pathways in HEK-293 cells. In vivo data demonstrated that H-TL1 protects animals against dextran sodium sulfate (DSS)-induced acute colitis and lipopolysaccharide (LPS)-induced acute shock. Given its significant anti-inflammatory activity in vitro and in vivo, H-TL1 is a potential peptide for the development of new agents to treat TNF-α-associated inflammatory diseases.

Lemnitoxin, the major component of Micrurus lemniscatus coral snake venom, is a myotoxic and pro-inflammatory phospholipase A2.

The venom of Micrurus lemniscatus, a coral snake of wide geographical distribution in South America, was fractionated by reverse-phase HPLC and the fractions screened for phospholipase A2 (PLA2) activity. The major component of the venom, a PLA2, here referred to as 'Lemnitoxin', was isolated and characterized biochemically and toxicologically. It induces myotoxicity upon intramuscular or intravenous injection into mice. The amino acid residues Arg15, Ala100, Asn108, and a hydrophobic residue at position 109, which are characteristic of myotoxic class I phospholipases A2, are present in Lemnitoxin. This PLA2 is antigenically related to M. nigrocinctus nigroxin, Notechis scutatus notexin, Pseudechis australis mulgotoxin, and Pseudonaja textilis textilotoxin, as demonstrated with monoclonal and polyclonal antibodies. Lemnitoxin is highly selective in its targeting of cells, being cytotoxic for differentiated myotubes in vitro and muscle fibers in vivo, but not for undifferentiated myoblasts or endothelial cells. Lemnitoxin is not lethal after intravenous injection at doses up to 2µg/g in mice, evidencing its lack of significant neurotoxicity. Lemnitoxin displays anticoagulant effect on human plasma and proinflammatory activity also, as it induces paw edema and mast cell degranulation. Thus, the results of this work demonstrate that Lemnitoxin is a potent myotoxic and proinflammatory class I PLA2.

Mapping Proteoforms and Protein Complexes From King Cobra Venom Using Both Denaturing and Native Top-down Proteomics.

Characterizing whole proteins by top-down proteomics avoids a step of inference encountered in the dominant bottom-up methodology when peptides are assembled computationally into proteins for identification. The direct interrogation of whole proteins and protein complexes from the venom of Ophiophagus hannah (king cobra) provides a sharply clarified view of toxin sequence variation, transit peptide cleavage sites and post-translational modifications (PTMs) likely critical for venom lethality. A tube-gel format for electrophoresis (called GELFrEE) and solution isoelectric focusing were used for protein fractionation prior to LC-MS/MS analysis resulting in 131 protein identifications (18 more than bottom-up) and a total of 184 proteoforms characterized from 14 protein toxin families. Operating both GELFrEE and mass spectrometry to preserve non-covalent interactions generated detailed information about two of the largest venom glycoprotein complexes: the homodimeric l-amino acid oxidase (∼130 kDa) and the multichain toxin cobra venom factor (∼147 kDa). The l-amino acid oxidase complex exhibited two clusters of multiproteoform complexes corresponding to the presence of 5 or 6 N-glycans moieties, each consistent with a distribution of N-acetyl hexosamines. Employing top-down proteomics in both native and denaturing modes provides unprecedented characterization of venom proteoforms and their complexes. A precise molecular inventory of venom proteins will propel the study of snake toxin variation and the targeted development of new antivenoms or other biotherapeutics.

Anti-osteoarthritic activity of Bungarus fasciatus venom fraction BF-F47 involving molecular markers in the rats.

A heat stable protein BF-F47 was purified from the crude venom of Bungarus fasciatus by CM cellulose ion exchange chromatography and HPLC. Osteoarthritis (OA) was developed in male albino Wistar rats by collagenase injection. BF-F47 treatment significantly restored urinary hydroxyproline and glucosamine in OA rats. Serum acid phosphatase, alkaline phosphatase, creatinine and serum molecular markers TNF-α, IL-1ß, IL-17, cytokine induced neutrophil chemoattractant-1, matrix metalloproteinase-1, cathepsin-K, osteocalcin and PGE2 were also significantly altered. BF-F47 showed partial restoration of osteoarthritis joints. Thus, BF-F47 induced anti-osteoarthritic activity in Wistar rats acted through molecular markers of arthritis and inflammation.

Isolation and Pharmacological Characterization of α-Elapitoxin-Ot1a, a Short-Chain Postsynaptic Neurotoxin from the Venom of the Western Desert Taipan, Oxyuranus temporalis.

Taipans (Oxyuranus spp.) are elapids with highly potent venoms containing presynaptic (ß) and postsynaptic (α) neurotoxins. O. temporalis (Western Desert taipan), a newly discovered member of this genus, has been shown to possess venom which displays marked in vitro neurotoxicity. No components have been isolated from this venom. We describe the characterization of α-elapitoxin-Ot1a (α-EPTX-Ot1a; 6712 Da), a short-chain postsynaptic neurotoxin, which accounts for approximately 30% of O. temporalis venom. α-Elapitoxin-Ot1a (0.1-1 µM) produced concentration-dependent inhibition of indirect-twitches, and abolished contractile responses to exogenous acetylcholine and carbachol, in the chick biventer cervicis nerve-muscle preparation. The inhibition of indirect twitches by α-elapitoxin-Ot1a (1 µM) was not reversed by washing the tissue. Prior addition of taipan antivenom (10 U/mL) delayed the neurotoxic effects of α-elapitoxin-Ot1a (1 µM) and markedly attenuated the neurotoxic effects of α-elapitoxin-Ot1a (0.1 µM). α-Elapitoxin-Ot1a displayed pseudo-irreversible antagonism of concentration-response curves to carbachol with a pA2 value of 8.02 ± 0.05. De novo sequencing revealed the main sequence of the short-chain postsynaptic neurotoxin (i.e., α-elapitoxin-Ot1a) as well as three other isoforms found in O. temporalis venom. α-Elapitoxin-Ot1a shows high sequence similarity (i.e., >87%) with other taipan short-chain postsynaptic neurotoxins.

Neutralization of the Principal Toxins from the Venoms of Thai Naja kaouthia and Malaysian Hydrophis schistosus: Insights into Toxin-Specific Neutralization by Two Different Antivenoms.

Antivenom neutralization against cobra venoms is generally low in potency, presumably due to poor toxin-specific immunoreactivity. This study aimed to investigate the effectiveness of two elapid antivenoms to neutralize the principal toxins purified from the venoms of the Thai monocled cobra (Naja kaouthia, Nk-T) and the Malaysian beaked sea snake (Hydrophis schistosus, Hs-M). In mice, N. kaouthia Monovalent Antivenom (NKMAV) neutralization against Nk-T long neurotoxin (LNTX) and cytotoxin was moderate (potency of 2.89-6.44 mg toxin/g antivenom protein) but poor against the short neurotoxin (SNTX) (1.33 mg/g). Its cross-neutralization against Hs-M LNTX of Hs-M is compatible (0.18 mg/g) but much weaker against Hs-M SNTX (0.22 mg/g). Using CSL (Seqirus Limited) Sea Snake Antivenom (SSAV), we observed consistently weak neutralization of antivenom against SNTX of both species, suggesting that this is the limiting factor on the potency of antivenom neutralization against venoms containing SNTX. Nevertheless, SSAV outperformed NKMAV in neutralizing SNTXs of both species (0.61-2.49 mg/g). The superior efficacy of SSAV against SNTX is probably partly attributable to the high abundance of SNTX in sea snake venom used as immunogen in SSAV production. The findings indicate that improving the potency of cobra antivenom may be possible with a proper immunogen formulation that seeks to overcome the limitation on SNTX immunoreactivity.

Insights into the subunit arrangement and diversity of paradoxin and taipoxin.

Paradoxin and taipoxin are neurotoxic phospholipases from the inland and coastal species of Australian taipan. Despite their relatively high sequence homology of 70% and 84% for the acidic and basic chains respectively, they differ substantially in reported assays of neurotoxicity. This study provides the first characterisation of paradoxin, which like taipoxin, is a trimer at physiological pH. More broadly, these toxins were found to be composed of a more diverse range of subunits than previously recognised, including newly discovered γTPx isoforms, which give rise to an additional, major conformation of TPx.

Pr-SNTX, a short-chain three-finger toxin from Papuan pigmy mulga snake, is an antagonist of muscle-type nicotinic acetylcholine receptor (α2ßδε).

Three-finger toxins (3FTxs) are one of the major components in snake venoms. In this study, we isolated a cDNA encoding a short-chain 3FTx, Pr-SNTX, from Pseudechis rossignolii. The amino acid sequence of Pr-SNTX is nearly identical to that of its ortholog in Pseudechis australis. Pr-SNTX protein inhibited muscle-type (α2ßδε), but not neuronal α7 nicotinic acetylcholine receptor (nAChR) activity.

Purification and characterization of tenerplasminin-1, a serine peptidase inhibitor with antiplasmin activity from the coral snake (Micrurus tener tener) venom.

A plasmin inhibitor, named tenerplasminin-1 (TP1), was isolated from Micrurus tener tener (Mtt) venom. It showed a molecular mass of 6542Da, similarly to Kunitz-type serine peptidase inhibitors. The amidolytic activity of plasmin (0.5nM) on synthetic substrate S-2251 was inhibited by 91% following the incubation with TP1 (1nM). Aprotinin (2nM) used as the positive control of inhibition, reduced the plasmin amidolytic activity by 71%. Plasmin fibrinolytic activity (0.05nM) was inhibited by 67% following incubation with TP1 (0.1nM). The degradation of fibrinogen chains induced by plasmin, trypsin or elastase was inhibited by TP1 at a 1:2, 1:4 and 1:20 enzyme:inhibitor ratio, respectively. On the other hand, the proteolytic activity of crude Mtt venom on fibrinogen chains, previously attributed to metallopeptidases, was not abolished by TP1. The tPA-clot lysis assay showed that TP1 (0.2nM) acts like aprotinin (0.4nM) inducing a delay in lysis time and lysis rate which may be associated with the inhibition of plasmin generated from the endogenous plasminogen activation. TP1 is the first serine protease plasmin-like inhibitor isolated from Mtt snake venom which has been characterized in relation to its mechanism of action, formation of a plasmin:TP1 complex and therapeutic potential as anti-fibrinolytic agent, a biological characteristic of great interest in the field of biomedical research. They could be used to regulate the fibrinolytic system in pathologies such as metastatic cancer, parasitic infections, hemophilia and other hemorrhagic syndromes, in which an intense fibrinolytic activity is observed.