Use of sulphopropyl-sephadex chromatography and disc gel electrophoresis for the fractionation and identification of elapid venoms

The cation exchange resin Sulphopropyl (SP)-Sephadex has been used for the preparative isolation of both neurotoxins and cytotoxins from various Elapid venoms. The chromatographic system described affords high resolution of venom components upon elution with simple linear salt gradients. Cytotoxins from the venom of Naja naja naja (India) and neurotoxins from Naja naja atra (Formosa), Naja naja siamensis and Bungarus multicinctus have been isolated in high yield as pure, physiologically-active materials.The resin has also been applied, together with two different discontinuous gel electrophoresis systems, to the characterization of venoms from ten Asian Naja naja subspecies. All of the venoms studied could be identified by their disc gel acidic and basic protein patterns alone, or by a combination of their SP-Sephadex protein elution profiles and the disc gel patterns of their acidic proteins. These studies indicate that the systems reported could be used not only for the identification of specific venoms but could also serve as a basis for a classification system for snake venoms.     

Contamination of commercially obtained Agkistrodon contortrix mokasen snake venom with Bungarus multicinctus and Naja naja atra snake venoms

Carboxymethyl cellulose fractionation of non-neurotoxic Agkistrodon c. mokasen venom contaminated with either Bungarus multicinctus or Naja n. atra venom produced a lethality profile similar to that reported earlier for neurotoxic corpperhead venom. The copperhead neurotoxin co-migrated electrophoretically with α-bungarotoxin, indicating the possibility that the neurotoxic copperhead venom was contaminated with an elapid venom.     

Toxoids and Antivenoms of Venomous Snakes in Taiwan

Abstract

There are in Taiwan six major venomous snakes which can inflict severe bite on human victims. They are three hemorrhagic species i.e. the Taiwan habu (Trimeresurus mucrosquamatus), the green tree viper (Trimeresurus stejnegeri), and the hundred-pace snake (Deinagkistrodon acutus); two neurotoxic species, i.e. the cobra (Naja naja atra) and the krait (Bungarus multicinctus); and the Russell's viper (Daboia r. formosensis) whose venom is both coagulopathy and neurotoxic. Our aim has been the production of highly potent antivenoms for snake-bite treatment in this country. Among individual antivenoms for Taiwan venomous snakes, only those from the pitvipers show partial cross-neutralizing capacity with venoms of other pit vipers.

As all snake venoms are quite lethal to animals, it is important to tame or detoxify the crude venom before using it on the animal to obtain antivenoms. We have demonstrated that glutaraldehyde can be used successfully not only in the detoxification of snake venoms but also improving their immunogenecity. Protocols for toxoid preparation from the crude venoms in the process of manufacturing highly potent antisera have been improved in our institute. Two bivalent equine antivenoms specific for either the combined glutaraldehyde-treated venoms of N. n. atra and B. multicinctus or those of T. mucrosquamatus and T. stejnegeri were successfully produced and proven to be effective and useful. A tetravalent equine antivenom has been prepared likewise against the four major viperid venoms in Taiwan. Recently, we also developed a process to prepare an efficient hexavalent antivenom against all the six major venomous snakes.     

DNA Aptamers against Taiwan Banded Krait α-Bungarotoxin Recognize Taiwan Cobra Cardiotoxins

Bungarus multicinctus α-bungarotoxin (α-Bgt) and Naja atra cardiotoxins (CTXs) share a common structural scaffold, and their tertiary structures adopt three-fingered loop motifs. Four DNA aptamers against α-Bgt have been reported previously. Given that the binding of aptamers with targeted proteins depends on structural complementarity, in this study, we investigated whether DNA aptamers against α-Bgt could also recognize CTXs. It was found that N. atra cardiotoxin 3 (CTX3) reduced the electrophoretic mobility of aptamers against α-Bgt. Analysis of the changes in the fluorescence intensity of carboxyfluorescein-labeled aptamers upon binding toxin molecules revealed that CTX3 and α-Bgt could bind the tested aptamers. Moreover, the aptamers inhibited the membrane-damaging activity and cytotoxicity of CTX3. In addition to CTX3, other N. atra CTX isotoxins also bound to the aptamer against α-Bgt. Taken together, our data indicate that aptamers against α-Bgt show cross-reactivity with CTXs. The findings that aptamers against α-Bgt also suppress the biological activities of CTX3 highlight the potential utility of aptamers in regard to the broad inhibition of snake venom three-fingered proteins.     

In Vitro Immunological Cross-Reactivity of Thai Polyvalent and Monovalent Antivenoms with Asian Viper Venoms

The intravenous administration of polyclonal antibodies known as antivenom is the only effective treatment for snakebite envenomed victims, but because of inter-specific variation in the toxic components of snake venoms, these therapies have variable efficacies against different snake species and/or different populations of the same species. In this study, we sought to characterize the in vitro venom binding capability and in vitro cross-neutralizing activity of antivenom, specifically the Hemato Polyvalent antivenom (HPAV; The Queen Saovabha Memorial Institute (QSMI) of the Thai Red Cross Society, Thailand) and three monovalent antivenoms (QSMI) specific to Daboia siamensis, Calloselasma rhodostoma, and Trimeresurus albolabris venoms, against a variety of South Asian and Southeast Asian viper venoms (Calloselasma rhodostoma, Daboia russelii, Hypnale hypnale, Trimeresurus albolabris, Trimeresurus purpureomaculatus, Trimeresurus hageni, and Trimeresurus fucatus). Using ELISA and immunoblotting approaches, we find that the majority of protein components in the viper venoms were recognized and bound by the HPAV polyvalent antivenom, while the monospecific antivenom made against T. albolabris extensively recognized toxins present in the venom of related species, T. purpureomaculatus, T. hageni, and T. fucatus. In vitro coagulation assays using bovine plasma revealed similar findings, with HPAV antivenom significantly inhibiting the coagulopathic activities of all tested viper venoms and T. albolabris antivenom inhibiting the venoms from Malaysian arboreal pit vipers. We also show that the monovalent C. rhodostoma antivenom exhibits highly comparable levels of immunological binding and in vitro venom neutralization to venom from both Thailand and Malaysia, despite previous reports of considerable intraspecific venom variation. Our findings suggest that Thai antivenoms from QSMI may by useful therapeutics for managing snake envenomings caused by a number of Southeast Asian viper species and populations for which no specific antivenom currently exists and thus should be explored further to assess their clinical utility in treating snakebite victims.     

In Vitro Neurotoxicity of Chinese Krait (Bungarus multicinctus) Venom and Neutralization by Antivenoms

Bungarus multicinctus, the Chinese krait, is a highly venomous elapid snake which causes considerable morbidity and mortality in southern China. B. multicinctus venom contains pre-synaptic PLA₂ neurotoxins (i.e., β-bungarotoxins) and post-synaptic neurotoxins (i.e., α-bungarotoxins). We examined the in vitro neurotoxicity of B. multicinctus venom, and the efficacy of specific monovalent Chinese B. multicinctus antivenom, and Australian polyvalent elapid snake antivenom, against venom-induced neurotoxicity. B. multicinctus venom (1–10 μg/mL) abolished indirect twitches in the chick biventer cervicis nerve-muscle preparation as well as attenuating contractile responses to exogenous ACh and CCh, but not KCl. This indicates a post-synaptic neurotoxic action but myotoxicity was not evident. Given that post-synaptic α-neurotoxins have a more rapid onset than pre-synaptic neurotoxins, the activity of the latter in the whole venom will be masked. The prior addition of Chinese B. multicinctus antivenom (12 U/mL) or Australian polyvalent snake antivenom (15 U/mL), markedly attenuated the neurotoxic actions of B. multicinctus venom (3 μg/mL) and prevented the inhibition of contractile responses to ACh and CCh. The addition of B. multicinctus antivenom (60 U/mL), or Australian polyvalent snake antivenom (50 U/mL), at the t₉₀ time point after the addition of B. multicinctus venom (3 μg/mL), did not restore the twitch height over 180 min. The earlier addition of B. multicinctus antivenom (60 U/mL), at the t₂₀ or t₅₀ time points, also failed to prevent the neurotoxic effects of the venom but did delay the time to abolish twitches based on a comparison of t₉₀ values. Repeated washing of the preparation with physiological salt solution, commencing at the t₂₀ time point, failed to reverse the neurotoxic effects of venom or delay the time to abolish twitches. This study showed that B. multicinctus venom displays marked in vitro neurotoxicity in a skeletal muscle preparation which is not reversed by antivenom. This does not appear to be related to antivenom efficacy, but due to the irreversible/pseudo-irreversible nature of the neurotoxins.     

Distribution of proteinase inhibitors in snake venoms

Potent proteinase inhibitors, which inactivate bovine pancreatic trypsin, α-chymotrypsin, bovine plasma kallikrein and plasmin, were found in several snake venoms. The content of the inhibitor was highest in Vipera russelli venom. Proteinase inhibitors of the same type were demonstrated also in the venoms of five snakes of the Elapidae family; Hemachatus haemachatus (Ringhals cobra), Dendroaspis angusticeps (Green mamba), Dendroaspis polylepis (Black mamba), Naja nivea (Cape cobra) and Naja haje (Egyptian cobra). No proteinase inhibitors were demonstrated in several Crotalidae and Hydrophiidae venoms. Toxic polypeptides such as α-bungarotoxin, cytotoxin I and II (Naja naja venom) and cardiotoxin (Naja naja atra venom) did not show any inhibitory action on typical mammalian proteinases including trypsin, α-chymotrypsin, plasmin and kallikrein.     

Isolation of “ceruleotoxin” from Bungarus fasciatus venoms

Ceruleotoxin is a potent neurotoxin which irreversibly blocks the neuromuscular transmission at a postsynaptic level, without preventing the binding of acetylcholine to its receptor. We have originally purified this toxin from a batch of venom obtained from the Pasteur Institute, which was thought to be Bungarus caeruleus venom. Recently Noble and co-workers have observed that the protein composition of Bungarus caeruleus provided by Miami Serpentarium significantly differed from that of the Pasteur Institute batch, which they concluded therefore to be of a different origin.In order to clarify this point, the venom composition of various Bungarus species from several origins have been analysed by electrophoresis and by electrofocusing on polyacrylamide gels. Although individual variations exist between samples of the same snake species, the venom from Bungarus caeruleus, Bungarus fasciatus and Bungarus multicinctus possess distinct and characteristic protein compositions. The results of this study allowed us to identify unambiguously the batch used to purify the ceruleotoxin, as a Bungarus fasciatus venom.We identified a neurotoxin similar to ceruleotoxin in each of the five samples of Bungarus fasciatus venoms that we tested. On the contrary we did not detect such a toxin either in Bungarus caeruleus or in Bungarus multicinctus venoms. All purified ceruleotoxins are acidic proteins with a high toxicity (their LD₅₀ by intravenous injection in mice are from 0.04 to 0.06 mg per kg), which irreversibly block the postsynaptic response of Electrophorus electricus electroplaque to cholinergic agonists. They also possess a phospholipase A₂ activity (200 nmoles of egg lecithins hydrolysed per min per mg of protein). In this respect, ceruleotoxin is analogous to crotoxin and β-bungarotoxin.     

Venom of Bothrops asper from Mexico and Costa Rica: Intraspecific variation and cross-neutralization by antivenoms

Bothrops asper is the species that induces the highest incidence of snakebite envenomation in southern Mexico, Central America and parts of northern South America. The intraspecies variability in HPLC profile and toxicological activities between the venoms from specimens collected in Mexico (Veracruz) and Costa Rica (Caribbean and Pacific populations) was investigated, as well as the cross-neutralization by antivenoms manufactured in these countries. Venoms differ in their HPLC profiles and in their toxicity, since venom from Mexican population showed higher lethal and defibrinogenating activities, whereas those from Costa Rica showed higher hemorrhagic and in vitro coagulant activities. In general, antivenoms were more effective in the neutralization of homologous venoms. Overall, both antivenoms effectively neutralized the various toxic effects of venoms from the two populations of B. asper. However, antivenom raised against venom from Costa Rican specimens showed a higher efficacy in the neutralization of defibrinogenating and coagulant activities, thus highlighting immunochemical differences in the toxins responsible for these effects associated with hemostatic disturbances in snakebite envenoming. These observations illustrate how intraspecies venom variation may influence antivenom neutralizing profile.     

Cloning of cDNAs encoding C-type lectins from Elapidae snakes Bungarus fasciatus and Bungarus multicinctus

A number of C-type lectins with various biological activities have been purified and characterized from Viperidae snake venoms. In contrast, only a few reports could be found in literature concerning the C-type lectins in Elapidae snake venoms. Based on the published cDNA sequences of C-type lectins from Viperidae snake venoms, oligonucleotide primers were designed and used to screen the cDNA libraries made from the venom glands of Bungarus fasciatus and Bungarus multicinctus. This allowed the cloning of three full length cDNAs encoding C-type lectins. The encoded proteins, named BFL-1, BFL-2 and BML, exhibit high degrees of sequence identities with Viperidae snake venom saccharide-binding lectins (around 60% with Trimeresurus stejnegeri venom lectin, Crotalus atrox venom lectin and Agkistrodon piscivorus venom lectin). They show much less identities with other venom C-type lectin-like proteins (around 30% with the platelet glycoprotein Ib-binding protein from Agkistrodon blomhoffi venom and the factor IX/X-binding protein from Trimeresurus flavoviridis venom). The cDNAs revealed that the precursors contain potential signal peptides characterized by a hydrophobic core. To our knowledge, these are the first cDNA cloning of group VII C-type lectins (Drickamer K. 1993. Prog. Nucleic Acid Res. Mol. Biol. 45, 207–232) from Elapidae snake venom glands.     

Preclinical assessment of the efficacy of a new antivenom (EchiTAb-Plus-ICP®) for the treatment of viper envenoming in sub-Saharan Africa

A preclinical assessment was performed on the neutralizing efficacy of a whole IgG polyspecific antivenom (EchiTAb-Plus-ICP^®), designed for the treatment of snakebite envenomings in Nigeria. It was generated by immunizing horses with the venoms of Echis ocellatus, Bitis arietans and Naja nigricollis, the most medically important species in Nigeria. Antivenom was tested against the venoms of E. ocellatus, Echis leucogaster, Echis pyramidum leakeyi, B. arietans, Bitis gabonica, Bitis rhinoceros and Bitis nasicornis. The neutralization of the venom toxins responsible for the lethal, hemorrhagic, coagulant and local necrotizing activities was assessed, since these are the most significant effects that characterize envenoming by these species. Echis sp venoms exerted lethal, hemorrhagic, coagulant and necrotizing effects, whereas the Bitis sp venoms tested induced lethality, hemorrhage and necrosis, but were devoid of coagulant activity. The antivenom was effective in the neutralization of all effects tested in all venoms. Highest neutralization was achieved against the venoms of E. ocellatus and B. arietans, and the lowest neutralizing potency was against the venom of B. nasicornis, a species that has a low clinical relevance. It is concluded that EchiTAb-Plus-ICP^®, whilst specifically designed for Nigeria, has a good preclinical neutralizing profile against homologous and heterologous viperid venoms from other sub-Saharan African locations. It therefore constitutes a promising therapeutic option for the treatment of snakebite envenoming in this region.     

Snake Venom Proteomics,Immunoreactivity and Toxicity Neutralization Studies for the Asiatic Mountain Pit Vipers,Ovophis convictus,Ovophis tonkinensis,and Hime Habu,Ovophis okinavensis

Snakebite envenomation is a serious neglected tropical disease, and its management is often complicated by the diversity of snake venoms. In Asia, pit vipers of the Ovophis species complex are medically important venomous snakes whose venom properties have not been investigated in depth. This study characterized the venom proteomes of Ovophis convictus (West Malaysia), Ovophis tonkinensis (northern Vietnam, southern China), and Ovophis okinavensis (Okinawa, Japan) by applying liquid chromatography-tandem mass spectrometry, which detected a high abundance of snake venom serine proteases (SVSP, constituting 40–60% of total venom proteins), followed by phospholipases A₂, snake venom metalloproteinases of mainly P-III class, L-amino acid oxidases, and toxins from other protein families which were less abundant. The venoms exhibited different procoagulant activities in human plasma, with potency decreasing from O. tonkinensis > O. okinavensis > O. convictus. The procoagulant nature of venom confirms that consumptive coagulopathy underlies the pathophysiology of Ovophis pit viper envenomation. The hetero-specific antivenoms Gloydius brevicaudus monovalent antivenom (GbMAV) and Trimeresurus albolabris monovalent antivenom (TaMAV) were immunoreactive toward the venoms, and cross-neutralized their procoagulant activities, albeit at variably limited efficacy. In the absence of species-specific antivenom, these hetero-specific antivenoms may be useful in treating coagulotoxic envenomation caused by the different snakes in their respective regions.     

Anticoagulant Activity of Naja nigricollis Venom Is Mediated by Phospholipase A2 Toxins and Inhibited by Varespladib

Bites from elapid snakes typically result in neurotoxic symptoms in snakebite victims. Neurotoxins are, therefore, often the focus of research relating to understanding the pathogenesis of elapid bites. However, recent evidence suggests that some elapid snake venoms contain anticoagulant toxins which may help neurotoxic components spread more rapidly. This study examines the effects of venom from the West African black-necked spitting cobra (Naja nigricollis) on blood coagulation and identifies potential coagulopathic toxins. An integrated RPLC-MS methodology, coupled with nanofractionation, was first used to separate venom components, followed by MS, proteomics and coagulopathic bioassays. Coagulation assays were performed on both crude and nanofractionated N. nigricollis venom toxins as well as PLA₂s and 3FTx purified from the venom. Assays were then repeated with the addition of either the phospholipase A₂ inhibitor varespladib or the snake venom metalloproteinase inhibitor marimastat to assess whether either toxin inhibitor is capable of neutralizing coagulopathic venom activity. Subsequent proteomic analysis was performed on nanofractionated bioactive venom toxins using tryptic digestion followed by nanoLC-MS/MS measurements, which were then identified using Swiss-Prot and species-specific database searches. Varespladib, but not marimastat, was found to significantly reduce the anticoagulant activity of N. nigricollis venom and MS and proteomics analyses confirmed that the anticoagulant venom components mostly consisted of PLA₂ proteins. We, therefore, conclude that PLA₂s are the most likely candidates responsible for anticoagulant effects stimulated by N. nigricollis venom.     

Immunological Cross-Reactivity and Neutralisation of European Viper Venoms with the Monospecific Vipera berus Antivenom ViperaTAb

Medically important cases of snakebite in Europe are predominately caused by European vipers of the genus Vipera. The mainstay of snakebite therapy is polyclonal antibody therapy, referred to as antivenom. Here we investigate the capability of the monospecific V. berus antivenom, ViperaTAb^®, to cross-react with, and neutralise lethality induced by, a variety of European vipers. Using ELISA and immunoblotting, we find that ViperaTAb^® antibodies recognise and bind to the majority of toxic components found in the venoms of the Vipera species tested at comparably high levels to those observed with V. berus. Using in vivo pre-clinical efficacy studies, we demonstrate that ViperaTAb^® effectively neutralises lethality induced by V. berus, V. aspis, V. ammodytes and V. latastei venoms and at much higher levels than those outlined by regulatory pharmacopoeial guidelines. Notably, venom neutralisation was found to be superior to (V. berus, V. aspis and V. latastei), or as equally effective as (V. ammodytes), the monospecific V. ammodytes “Zagreb antivenom”, which has long been successfully used for treating European snake envenomings. This study suggests that ViperaTAb^® may be a valuable therapeutic product for treating snakebite by a variety of European vipers found throughout the continent.     

Comparison of the effect of Crotalus simus and Crotalus durissus ruruima venoms on the equine antibody response towards Bothrops asper venom: Implications for the production of polyspecific snake antivenoms

Antivenoms are preparations of immunoglobulins purified from the plasma of animals immunized with snake venoms. Depending on the number of venoms used during the immunization, antivenoms can be monospecific (if venom from a single species is used) or polyspecific (if venoms from several species are used). In turn, polyspecific antivenoms can be prepared by purifying antibodies from the plasma of animals immunized with a mixture of venoms, or by mixing antibodies purified from the plasma of animals immunized separately with single venom. The suitability of these strategies to produce polyspecific antibothropic-crotalic antivenoms was assessed using as models the venoms of Bothrops asper, Crotalus simus and Crotalus durissus ruruima. It was demonstrated that, when used as co-immunogen, C. simus and C. durissus ruruima venoms exert a deleterious effect on the antibody response towards different components of B. asper venom and in the neutralization of hemorrhagic and coagulant effect of this venom when compared with a monospecific B. asper antivenom. Polyspecific antivenoms produced by purifying immunoglobulins from the plasma of animals immunized with venom mixtures showed higher antibody titers and neutralizing capacity than those produced by mixing antibodies purified from the plasma of animals immunized separately with single venom. Thus, despite the deleterious effect of Crotalus sp venoms on the immune response against B. asper venom, the use of venom mixtures is more effective than the immunization with separate venoms for the preparation of polyspecific bothropic-crotalic antivenoms.     

Inhibition of the myotoxic activities of three African Bitis venoms (B. rhinoceros,B. arietans and B. nasicornis) by a polyvalent antivenom

A murine model of venom-induced myotoxicity was used to assess the antimyotoxic capacity of a polyvalent antivenom (PAV), rich in F(ab')2 fragments, obtained from horses immunized with Bitis venoms. Intramuscular (i.m.) injection of Bitis rhinoceros, Bitis arietans or Bitis nasicornis into mice induced a time- and dose-dependent increase in plasma CK activity. The area under the plasma CK activity vs. time curve (AUC) between 0 and 48 h was used to quantify the data. Pre-incubation with PAV neutralized the venoms' myotoxicity, in a concentration-dependent manner: 80–100% neutralization occurred when the ratio of the PAV volume to the venom mass was 3-fold that recommended for use in human envenomation. Intravenous administration of PAV 1 h before the i.m. venom injection, afforded significant protection against myotoxicity, especially in the case of B. arietans. An antimyotoxic effect was also observed, albeit reduced, when the PAV treatment was applied 1 h after the venom injection. These data indicate that a PAV developed and manufactured in Brazil protects against the myotoxicity of the venoms of B. rhinoceros, B. arietans or B. nasicornis, which account for a large number of snakebite accidents in the African continent.     

Attempt to Develop Rat Disseminated Intravascular Coagulation Model Using Yamakagashi (Rhabdophis tigrinus) Venom Injection

Disseminated intravascular coagulation, a severe clinical condition caused by an underlying disease, involves a markedly continuous and widespread activation of coagulation in the circulating blood and the formation of numerous microvascular thrombi. A snakebite, including that of the Yamakagashi (Rhabdophis tigrinus), demonstrates this clinical condition. Thus, an animal model using Yamakagashi venom was constructed. Yamakagashi venom was administered to rats, and its lethality and the changes in blood coagulation factors were detected after venom injection. When 300 μg venom was intramuscularly administered to 12-week-old rats, (1) they exhibited hematuria with plasma hemolysis and died within 48 h; (2) Thrombocytopenia in the blood was observed in the rats; (3) irreversible prolongation of prothrombin time in the plasma to the measurement limit occurred; (4) fibrinogen concentration in the plasma irreversibly decreased below the measurement limit; and (5) A transient increase in the plasma concentration of D-dimer was observed. In this model, a fixed amount of Rhabdophis tigrinus venom injection resulted in the clinical symptom similar to the human pathology with snakebite. The use of the rat model is very effective in validating the therapeutic effect of human disseminated intravascular coagulation condition due to snakebite.     

Studies on the venom proteome of Bothrops asper: Perspectives and applications

Bothrops asper is responsible for the vast majority of snakebite accidents in Central America and several studies have demonstrated that specific toxic and enzymatic activities of its venom vary with the geographic origin and age of the specimens. Variability in venom proteins and enzymes between specimens from the Caribbean and the Pacific versants of Costa Rica has been reported since 1964. Recently, we performed a comparative proteomic characterization of the venoms from one population of each versant. Proteins belonging to several families, including disintegrin, phospholipases A₂, serine proteinases, C-type lectins, CRISP, l-amino acid oxidase, and Zn²⁺-dependent metalloproteinases show a variable degree of relative occurrence in the venoms of both populations. The occurrence of prominent differences in the protein profile between venoms from adults and newborns, and among venom samples from individual specimens of the same region or developmental stage, further demonstrated the existence of geographic, ontogenetic and individual variability in the venom proteome of this species. These findings provide new insights towards understanding the biology of B. asper, contribute to a deeper understanding of the pathology induced by its venom and underscore the importance of the use of venoms pooled from specimens from both regions for producing antivenom exhibiting the broadest cross-reactivity. Furthermore, knowledge of the protein composition of B. asper venom paves the way for detailed future structure–function studies of individual toxins as well as for the development of new protocols to study the reactivity of therapeutic antivenoms.     

Neutralising effects of small molecule toxin inhibitors on nanofractionated coagulopathic Crotalinae snake venoms

Repurposing small molecule drugs and drug candidates is considered as a promising approach to revolutionise the treatment of snakebite envenoming. In this study, we investigated the inhibiting effects of the small molecules varespladib (nonspecific phospholipase A₂ inhibitor), marimastat (broad spectrum matrix metalloprotease inhibitor) and dimercaprol (metal ion chelator) against coagulopathic toxins found in Crotalinae (pit vipers) snake venoms. Venoms from Bothrops asper, Bothrops jararaca, Calloselasma rhodostoma and Deinagkistrodon acutus were separated by liquid chromatography, followed by nanofractionation and mass spectrometry identification undertaken in parallel. Nanofractions of the venom toxins were then subjected to a high-throughput coagulation assay in the presence of different concentrations of the small molecules under study. Anticoagulant venom toxins were mostly identified as phospholipases A₂, while procoagulant venom activities were mainly associated with snake venom metalloproteinases and snake venom serine proteases. Varespladib was found to effectively inhibit most anticoagulant venom effects, and also showed some inhibition against procoagulant toxins. Contrastingly, marimastat and dimercaprol were both effective inhibitors of procoagulant venom activities but showed little inhibitory capability against anticoagulant toxins. The information obtained from this study aids our understanding of the mechanisms of action of toxin inhibitor drug candidates, and highlights their potential as future snakebite treatments.     

Development of a Broad-Spectrum Antiserum against Cobra Venoms Using Recombinant Three-Finger Toxins

Three-finger toxins (3FTXs) are the most clinically relevant components in cobra (genus Naja) venoms. Administration of the antivenom is the recommended treatment for the snakebite envenomings, while the efficacy to cross-neutralize the different cobra species is typically limited, which is presumably due to intra-specific variation of the 3FTXs composition in cobra venoms. Targeting the clinically relevant venom components has been considered as an important factor for novel antivenom design. Here, we used the recombinant type of long-chain α-neurotoxins ({"type":"entrez-protein","attrs":{"text":"P01391","term_id":"128930"}}P01391), short-chain α-neurotoxins ({"type":"entrez-protein","attrs":{"text":"P60770","term_id":"46397632"}}P60770), and cardiotoxin A3 ({"type":"entrez-protein","attrs":{"text":"P60301","term_id":"41688806"}}P60301) to generate a new immunogen formulation and investigated the potency of the resulting antiserum against the venom lethality of three medially important cobras in Asia, including the Thai monocled cobra (Naja kaouthia), the Taiwan cobra (Naja atra), and the Thai spitting cobra (Naja Siamensis) snake species. With the fusion of protein disulfide isomerase and the low-temperature settings, the correct disulfide bonds were built on these recombinant 3FTXs (r3FTXs), which were confirmed by the circular dichroism spectra and tandem mass spectrometry. Immunization with r3FTX was able to induce the specific antibody response to the native 3FTXs in cobra venoms. Furthermore, the horse and rabbit antiserum raised by the r3FTX mixture is able to neutralize the venom lethality of the selected three medically important cobras. Thus, the study demonstrated that the r3FTXs are potential immunogens in the development of novel antivenom with broad neutralization activity for the therapeutic treatment of victims involving cobra snakes in countries.