Pharmacological Characterisation of Pseudocerastes and Eristicophis Viper Venoms Reveal Anticancer (Melanoma) Properties and a Potentially Novel Mode of Fibrinogenolysis

Venoms are a rich source of potential lead compounds for drug discovery, and descriptive studies of venom form the first phase of the biodiscovery process. In this study, we investigated the pharmacological potential of crude Pseudocerastes and Eristicophis snake venoms in haematological isorders and cancer treatment. We assessed their antithrombotic potential using fibrinogen thromboelastography, fibrinogen gels with and without protease inhibitors, and colourimetric fibrinolysis assays. These assays indicated that the anticoagulant properties of the venoms are likely induced by the hydrolysis of phospholipids and by selective fibrinogenolysis. Furthermore, while most fibrinogenolysis occurred by the direct activity of snake venom metalloproteases and serine proteases, modest evidence indicated that fibrinogenolytic activity may also be mediated by selective venom phospholipases and an inhibitory venom-derived serine protease. We also found that the Pseudocerastes venoms significantly reduced the viability of human melanoma (MM96L) cells by more than 80%, while it had almost no effect on the healthy neonatal foreskin fibroblasts (NFF) as determined by viability assays. The bioactive properties of these venoms suggest that they contain a number of toxins suitable for downstream pharmacological development as candidates for antithrombotic or anticancer agents

Coagulotoxic effects by brown snake (Pseudonaja) and taipan (Oxyuranus) venoms, and the efficacy of a new antivenom

Snakebite is a neglected tropical disease that disproportionately affects the poor. Antivenom is the only specific and effective treatment for snakebite, but its distribution is severely limited by several factors, including the prohibitive cost of some products. Papua New Guinea (PNG) is a snakebite hotspot but the high costs of Australian antivenoms (thousands of dollars per treatment) makes it unaffordable in PNG. A more economical taipan antivenom has recently been developed at the Instituto Clodomiro Picado (ICP) in Costa Rica for PNG and is currently undergoing clinical trials for the treatment of envenomations by coastal taipans (Oxyuranus scutellatus). In addition to potentially having the capacity to neutralise the effects of envenomations of non-PNG taipans, this antivenom may have the capacity to neutralise coagulotoxins in venom from closely related brown snakes (Pseudonaja spp.) also found in PNG. Consequently, we investigated the cross-reactivity of taipan antivenom across the venoms of all Oxyuranus and Pseudonaja species. In addition, to ascertain differences in venom biochemistry that influence variation in antivenom efficacy, we tested for relative cofactor dependence. We found that the new ICP taipan antivenom exhibited high selectivity for Oxyuranus venoms and only low to moderate cross-reactivity with any Pseudonaja venoms. Consistent with this genus level distinction in antivenom efficacy were fundamental differences in the venom biochemistry. Not only were the Pseudonaja venoms significantly more procoagulant, but they were also much less dependent upon the cofactors calcium and phospholipid. There was a strong correlation between antivenom efficacy, clotting time and cofactor dependence. This study sheds light on the structure-function relationships of the procoagulant toxins within these venoms and may have important clinical implications including for the design of next-generation antivenoms

Factor X activating Atractaspis snake venoms and the relative coagulotoxicity neutralising efficacy of African antivenoms

Atractaspis snake species are enigmatic in their natural history, and venom effects are correspondingly poorly described. Bite reports are scarce but bites have been described as causing severe hypertension, profound local tissue damage leading to amputation, and deaths are on record. Clinical descriptions have largely concentrated upon tissue effects, and research efforts have focused upon the blood-pressure affecting sarafotoxins. However, coagulation disturbances suggestive of procoagulant functions have been reported in some clinical cases, yet this aspect has been uninvestigated. We used a suite of assays to investigate the coagulotoxic effects of venoms from six different Atractaspis specimens from central Africa. The procoagulant function of factor X activation was revealed, as was the pseudo-procoagulant function of direct cleavage of fibrinogen into weak clots. The relative neutralization efficacy of South African Antivenom Producer's antivenoms on Atractaspis venoms were boomslang>polyvalent>saw-scaled viper. While the boomslang antivenom was the most effective on Atractaspis venoms, the ability to neutralize the most potent Atractaspis species in this study was up to 4-6 times less effective than boomslang antivenom neutralizes boomslang venom. Therefore, while these results suggest cross-reactivity of boomslang antivenom with the unexpectedly potent coagulotoxic effects of Atractaspis venoms, a considerable amount of this rare antivenom may be needed. This report thus reveals potent venom actions upon blood coagulation that may lead to severe clinical effects with limited management strategies

Differential procoagulant effects of saw-scaled viper (Serpentes: Viperidae: Echis ) snake venoms on human plasma and the narrow taxonomic ranges of antivenom efficacies

Saw-scaled vipers (genus Echis) are one of the leading causes of snakebite morbidity and mortality in parts of Sub-Saharan Africa, the Middle East, and vast regions of Asia, constituting a public health burden exceeding that of almost any other snake genus globally. Venom-induced consumption coagulopathy, owing to the action of potent procoagulant toxins, is one of the most relevant clinical manifestations of envenomings by Echis spp. Clinical experience and prior studies examining a limited range of venoms and restricted antivenoms have demonstrated for some antivenoms an extreme lack of antivenom cross-reactivity between different species of this genus, sometimes resulting in catastrophic treatment failure. This study undertook the most comprehensive testing of Echis venom effects upon the coagulation of human plasma, and also the broadest examination of antivenom potency and cross-reactivity, to-date. 10 Echis species/populations and four antivenoms (two African, two Asian) were studied. The results indicate that the venoms are, in general, potently procoagulant but that the relative dependence on calcium or phospholipid cofactors is highly variable. Additionally, three out of the four antivenoms tested demonstrated only a very narrow taxonomic range of effectiveness in preventing coagulopathy, with only the SAIMR antivenom displaying significant levels of cross-reactivity. These results were in conflict with previous studies using prolonged preincubation of antivenom with venom to suggest effective cross-reactivity levels for the ICP Echi-Tab antivenom. These findings both inform upon potential clinical effects of envenomation in humans and highlight the extreme limitations of available treatment. It is hoped that this will spur efforts into the development of antivenoms with more comprehensive coverage for bites not only from wild snakes but also from specimens widely kept in zoological collections

Extensive Variation in the Activities of and Viper Venoms Suggests Divergent Envenoming Strategies Are Used for Prey Capture.

Snakes of the genera and (Viperidae: Viperinae) are known as the desert vipers due to their association with the arid environments of the Middle East. These species have received limited research attention and little is known about their venom or ecology. In this study, a comprehensive analysis of desert viper venoms was conducted by visualising the venom proteomes via gel electrophoresis and assessing the crude venoms for their cytotoxic, haemotoxic, and neurotoxic properties. Plasmas sourced from human, toad, and chicken were used as models to assess possible prey-linked venom activity. The venoms demonstrated substantial divergence in composition and bioactivity across all experiments. venom activated human coagulation factors X and prothrombin and demonstrated potent procoagulant activity in human, toad, and chicken plasmas, in stark contrast to the potent neurotoxic venom of . The venom of also induced coagulation, though this did not appear to be via the activation of factor X or prothrombin. The coagulant properties of and venoms varied among plasmas, demonstrating strong anticoagulant activity in the amphibian and human plasmas but no significant effect in that of bird. This is conjectured to reflect prey-specific toxin activity, though further ecological studies are required to confirm any dietary associations. This study reinforces the notion that phylogenetic relatedness of snakes cannot readily predict venom protein composition or function. The significant venom variation between these species raises serious concerns regarding antivenom paraspecificity. Future assessment of antivenom is crucial

Characterising venom variation and antivenom effectiveness within the “True” vipers (Viperinae: Viperidae)

Venom is a functional adaptation that has evolved independently in almost every major animal lineage for use in defence, predation, and/or competition. The complexity and diversity of components formulating the venom mixture has resulted in a phenomenon whereby the composition of a species’ venom can vary down to the level of the individual. Venom variation produces an undesirable outcome from an anthropogenic perspective as the efficacy of an antivenom may be compromised due to variation in the antigenicity of venoms. This complicates the research efforts that seek to ameliorate the global impact of snakebite. The Viperinae subfamily of vipers contains some of the most medically significant snakes world-wide, often responsible for majority proportions of snakebite in the regions they inhabit.\ua0Despite their medical importance, Viperinae is an understudied clade. There is a\ua0need to characterise the bioactivity, composition, and variation of venom in these snakes in order to develop a deeper understanding of the complex interactions between snake ecology, evolution, venom, and snakebite envenoming.\ua0Accordingly, the aim of this work is threefold: 1. To characterise the venom activity of select viperine genera that are underrepresented in the existing literature, focussing on venom variation; 2. To explore links in the evolutionary relationships between snake ecology and venom biochemistry; and 3. To investigate the effectiveness of antivenoms relative to venom activity and variability. The thesis begins with a broad overview of the evolutionary history, biology, and ecology of the viperines and their venom in Chapter 1. Chapter 2 contains study protocols. Chapter 3, which comprises three sub-chapters, focusses on the genus\ua0Daboia. It begins with a characterisation of the proteolytic and haemotoxic properties of venoms from twelve geographical variants of five\ua0Daboia\ua0species, followed by an assessment of antivenom cross-reactivity, and finishes with a critical comparative analysis of the limitations and advantages offered by a suite of commonly used antivenom assessment protocols. Chapter 4 consists of two sub-chapters focussing on the neglected genera\ua0Pseudocerastes\ua0and\ua0Eristicophis. The first section of the chapter entails a comprehensive analysis of the neurotoxic, haemotoxic, and cytotoxic bioactivities of the venoms, and the second section evaluates antivenom cross-reactivity for the clade. The thesis is concluded in chapter 5.These research aims were achieved by adopting an integrated proteomic, bioactivity, and immunogenic assay approach, and the venoms of\ua0Daboia, Pseudocerastes, and\ua0Eristicophis\ua0were evaluated using all or a combination of the following methods: Venom protein composition was assessed via SDS-PAGE gel analysis. Generalised proteolytic activity, factor X and prothrombin activation, and total thrombin generation by the venoms were assessed via kinetic fluorescence assays. Coagulant activity of venoms on human plasma was quantified using an automated coagulation analyser. Venom-induced kinetics and structure of human, amphibian, and avian plasma clots were assessed via thromboelastography. Activity of venoms on fibrinogen was evaluated by thromboelastography and imaged via fibrinogen gels. Interactions of the venoms with phospholipid, tissue factor, and plasminogen activator were testing using chromogenic assays. Neurotoxicity testing was conducted via chick biventer assays. Cytopathic activity testing was conducted on PaTu-T, NFF, and MM96L cells, quantified via MTT assays, and visualised via light microscopy. Antivenom effectiveness was assessed functionally by using an automated coagulation analyser to measure shifts in coagulation time, as well as immunogenically via western blotting and ELISAs (endpoint titration and chaotropic).\ua0\ua0The advantages and limitations of a range of antivenom assessment assays were also evaluated.The previously unknown venom activities of\ua0Pseudocerastes urarachnoides\ua0were revealed to exert strong procoagulant activity, activating both prothrombin and factor X. Most other species tested also possessed strong, procoagulant venoms, with exception of the Afro-Arabian species of\ua0Daboia\ua0(D. palaestinae\ua0and\ua0D. deserti) and two species of\ua0Pseudocerastes (P. persicus\ua0and\ua0P. fieldi). Fibrinogenolytic activity across the clade was extremely diverse. Intragenus and intraspecies variation in venom activity was extreme throughout all assays. The variability of these venoms typically had a detrimental impact on the effectiveness of antivenoms, whereby many antivenoms proved inadequate. Their neutralisation of venom activities was inconsistent and ranged from good coverage for some species’ venoms to no coverage for others.\ua0These studies present the first comprehensive functional analyses of\ua0Pseudocerastes\ua0and\ua0Eristicophis\ua0venoms, representing a key contribution to our knowledge of viper venom composition and activity. Crucially, antivenom effectiveness and cross-reactivity for this clade has been described for the first time. In addition, this work has given greater depth and breadth to the scope of research that exists surrounding the geographic variation in venom composition of\ua0Daboia\ua0and the impact this has on antivenom effectiveness. This will aid in the treatment of envenomings by thesespecies, and thus positively contributes to the global efforts seeking to address snakebite mortality and morbidity. This series of studies provides a critical data set which informs antivenom manufacturers, clinicians, and evolutionary toxinologists in the field of venom research

Malaysian and Chinese King Cobra Venom Cytotoxicity in Melanoma and Neonatal Foreskin Fibroblasts Is Mediated by Age and Geography

Snake venoms constitute a complex, rapidly evolving trait, whose composition varies between and within populations depending on geographical location, age and preys (diets). These factors have determined the adaptive evolution for predatory success and link venom heterogeneity with prey specificity. Moreover, understanding the evolutionary drivers of animal venoms has streamlined the biodiscovery of venom-derived compounds as drug candidates in biomedicine and biotechnology. The king cobra (Ophiophagus hannah; Cantor, 1836) is distributed in diverse habitats, forming independent populations, which confer differing scale markings, including between hatchlings and adults. Furthermore, king cobra venoms possess unique cytotoxic properties that are used as a defensive trait, but their toxins may also have utility as promising anticancer-agent candidates. However, the impact of geographical distribution and age on these potential venom applications has been typically neglected. In this study, we hypothesised that ontogenetic venom variation accompanies the morphological distinction between hatchlings and adults. We used non-transformed neonatal foreskin (NFF) fibroblasts to examine and compare the variability of venom cytotoxicity between adult captive breeding pairs from Malaysian and Chinese lineages, along with that of their progeny upon hatching. In parallel, we assessed the anticancer potential of these venoms in human-melanoma-patient-derived cells (MM96L). We found that in a geographical distribution and gender-independent manner, venoms from hatchlings were significantly less cytotoxic than those from adults (NFF; ~Log EC50: 0.5–0.6 vs. 0.2–0.35 mg/mL). This is consistent with neonates occupying a semifossorial habitat, while adults inhabit more above-ground habitats and are therefore more conspicuous to potential predators. We also observed that Malaysian venoms exhibited a slightly higher cytotoxicity than those from the Chinese cobra cohorts (NFF; Log EC50: 0.1–0.3 vs. 0.3–0.4 mg/mL), which is consistent with Malaysian king cobras being more strongly aposematically marked. These variations are therefore suggestive of differential anti-predator strategies associated with the occupation of distinct niches. However, all cobra venoms were similarly cytotoxic in both melanoma cells and fibroblasts, limiting their potential medical applications in their native forms

A genus‐wide bioactivity analysis of daboia (Viperinae: Viperidae) viper venoms reveals widespread variation in haemotoxic properties

The snake genus Daboia (Viperidae: Viperinae; Oppel, 1811) contains five species: D. deserti, D. mauritanica, and D. palaestinae, found in Afro‐Arabia, and the Russell’s vipers D. russelii and D. siamensis, found in Asia. Russell’s vipers are responsible for a major proportion of the medically important snakebites that occur in the regions they inhabit, and their venoms are notorious for their coagulopathic effects. While widely documented, the extent of venom variation within the Russell’s vipers is poorly characterised, as is the venom activity of other species within the genus. In this study we investigated variation in the haemotoxic activity of Daboia using twelve venoms from all five species, including multiple variants of D. russelii, D. siamensis, and D. palaestinae. We tested the venoms on human plasma using thromboelastography, dose‐response coagulometry analyses, and calibrated automated thrombography, and on human fibrinogen by thromboelas-tography and fibrinogen gels. We assessed activation of blood factors X and prothrombin by the venoms using fluorometry. Variation in venom activity was evident in all experiments. The Asian species D. russelii and D. siamensis and the African species D. mauritanica possessed procoagulant venom, while D. deserti and D. palaestinae were net‐anticoagulant. Of the Russell’s vipers, the venom of D. siamensis from Myanmar was most toxic and D. russelli of Sri Lanka the least. Activation of both factor X and prothrombin was evident by all venoms, though at differential levels. Fibrino-genolytic activity varied extensively throughout the genus and followed no phylogenetic trends. This venom variability underpins one of the many challenges facing treatment of Daboia snakebite envenoming. Comprehensive analyses of available antivenoms in neutralising these variable venom activities are therefore of utmost importance

Differential destructive (non-clotting) fibrinogenolytic activity in Afro-Asian elapid snake venoms and the links to defensive hooding behavior

Envenomations by venomous snakes have major public health implications on a global scale. Despite its medical importance, snakebite has long been a neglected tropical disease by both governments and medical science. Many aspects of the resulting pathophysiology have been largely under-investigated. Most research on snake venom has focused on the neurological effects, with coagulotoxicity being relatively neglected, especially for venoms in the Elapidae snake family. In order to fill the knowledge gap regarding the coagulotoxic effects of elapid snake venoms, we performed functional activity tests to determine the fibrinogenolytic activity of 29 African and Asian elapid venoms across eight genera. The results of this study revealed that destructive (non-clotting) fibrinogenolytic activity is widespread across the African and Asian elapids. This trait evolved independently twice: once in the Hemachatus/Naja last common ancestor and again in Ophiophagus. Further, within Naja this trait was amplified on several independent occasions and possibly explains some of the clinical symptoms produced by these species. Species within the Hemachatus/Naja with fibrinogenolytic activity only cleaved the Aα-chain of fibrinogen, whereas Ophiophagus venoms degraded both the Aα- and the Bβ-chain of fibrinogen. All other lineages tested in this study lacked significant fibrinogenolytic effects. Our systematic research across Afro-Asian elapid snake venoms helps shed light on the various molecular mechanisms that are involved in coagulotoxicity within Elapidae