Efficacy and Limitations of Chemically Diverse Small-Molecule Enzyme-Inhibitors against the Synergistic Coagulotoxic Activities of Bitis Viper Venoms.

Snakebite remains a significant public health burden globally, disproportionately affecting low-income and impoverished regions of the world. Recently, researchers have begun to focus on the use of small-molecule inhibitors as potential candidates for the neutralisation of key snake venom toxins and as potential field therapies. Bitis vipers represent some of the most medically important as well as frequently encountered snake species in Africa, with a number of species possessing anticoagulant phospholipase A2 (PLA2) toxins that prevent the prothrombinase complex from inducing clot formation. Additionally, species within the genus are known to exert pseudo-procoagulant activity, whereby kallikrein enzymatic toxins cleave fibrinogen to form a weak fibrin clot that rapidly degrades, thereby depleting fibrinogen levels and contributing to the net anticoagulant state. Utilising well-validated coagulation assays measuring time until clot formation, this study addresses the in vitro efficacy of three small molecule enzyme inhibitors (marimastat, prinomastat and varespladib) in neutralising these aforementioned activities. The PLA2 inhibitor varespladib showed the greatest efficacy for the neutralisation of PLA2-driven anticoagulant venom activity, with the metalloproteinase inhibitors prinomastat and marimastat both showing low and highly variable degrees of cross-neutralisation with PLA2 anticoagulant toxicity. However, none of the inhibitors showed efficacy in neutralising the pseudo-procoagulant venom activity exerted by the venom of B. caudalis. Our results highlight the complex nature of snake venoms, for which single-compound treatments will not be universally effective, but combinations might prove highly effective. Despite the limitations of these inhibitors with regards to in vitro kallikrein enzyme pseudo-procoagulant venom activity, our results further support the growing body of literature indicating the potential use of small molecule inhibitors to enhance first-aid treatment of snakebite envenoming, particularly in cases where hospital and thus antivenom treatment is either unavailable or far away.

Widespread and Differential Neurotoxicity in Venoms from the Bitis Genus of Viperid Snakes.

Research into the neurotoxic activity of venoms from species within the snake family Viperidae is relatively neglected compared with snakes in the Elapidae family. Previous studies into venoms from the Bitis genus of vipers have identified the presence of presynaptic phospholipase A2 neurotoxins in B. atropos and B. caudalis, as well as a postsynaptic phospholipase A2 in B. arietans. Yet, no studies have investigated how widespread neurotoxicity is across the Bitis genus or if they exhibit prey selectivity of their neurotoxins. Utilising a biolayer interferometry assay, we were able to assess the binding of crude venom from 14 species of Bitis to the neuromuscular α-1 nAChR orthosteric site across a wide range of vertebrate taxa mimotopes. Postsynaptic binding was seen for venoms from B. arietans, B. armata, B. atropos, B. caudalis, B. cornuta, B. peringueyi and B. rubida. To further explore the types of neurotoxins present, venoms from the representatives B. armata, B. caudalis, B. cornuta and B. rubida were additionally tested in the chick biventer cervicis nerve muscle preparation, which showed presynaptic and postsynaptic activity for B. caudalis and only presynaptic neurotoxicity for B. cornuta and B. rubida, with myotoxicity also evident for some species. These results, combined with the biolayer interferometry results, indicate complex neurotoxicity exerted by Bitis species, which varies dramatically by lineage tested upon. Our data also further support the importance of sampling across geographical localities, as significant intraspecific variation of postsynaptic neurotoxicity was reported across the different localities.

Varespladib (LY315920) neutralises phospholipase A2 mediated prothrombinase-inhibition induced by Bitis snake venoms.

Anticoagulant toxicity is a common function of venoms produced by species within the Bitis genus. Potent inhibition of the prothrombinase complex is an identified mechanism of action for the dwarf species B. cornuta and B. xeropaga, along with some localities of B. atropos and B. caudalis. Snake venom phospholipase A2 toxins that inhibit the prothrombinase complex have been identified in snake venom, including an isolated phospholipase A2 toxin from B. caudalis. Current research is investigating the ability of the drug varespladib to inhibit snake venom phospholipase A2 toxins and reduce their toxicity. In particular, varespladib is being investigated as a treatment that could be administered prior to hospital referral which is a major necessity for species such as those from the genus Bitis, due to envenomations often occurring in remote regions of Africa where antivenom is unavailable. Using previously validated coagulation assays, this study aimed to determine if the toxins responsible for inhibition of the prothrombinase complex in the venom of four Bitis species are phospholipase A2 toxins, and if varespladib is able to neutralise this anticoagulant activity. Our results demonstrate that varespladib strongly neutralises the prothrombinase-inhibiting effects of all venoms tested in this study, and that this prothrombinase-inhibiting mechanism of anticoagulant activity is driven by phospholipase A2 class toxins in these four species. This study extends previous reports demonstrating varespladib has broad efficacy for treatment of phospholipase A2 rich snake venoms, indicating it also inhibits their anticoagulant effects mediated by prothrombinase-inhibition.

Fangs for the Memories? A Survey of Pain in Snakebite Patients Does Not Support a Strong Role for Defense in the Evolution of Snake Venom Composition.

Animals use venoms for multiple purposes, most prominently for prey acquisition and self-defense. In snakes, venom composition often evolves as a result of selection for optimization for local diet. However, whether selection for a defensive function has also played a role in driving the evolution of venom composition has remained largely unstudied. Here, we use an online survey of snakebite victims to test a key prediction of a defensive function, that envenoming should result in the rapid onset of severe pain. From the analysis of 584 snakebite reports, involving 192 species of venomous snake, we find that the vast majority of bites do not result in severe early pain. Phylogenetic comparative analysis shows that where early pain after a bite evolves, it is often lost rapidly. Our results, therefore, do not support the hypothesis that natural selection for antipredator defense played an important role in the origin of venom or front-fanged delivery systems in general, although there may be intriguing exceptions to this rule.

Venomous Landmines: Clinical Implications of Extreme Coagulotoxic Diversification and Differential Neutralization by Antivenom of Venoms within the Viperid Snake Genus Bitis.

The genus Bitis comprises 17 snake species that inhabit Africa and the Arabian Peninsula. They are responsible for a significant proportion of snakebites in the region. The venoms of the two independent lineages of giant Bitis (B. arietans and again in the common ancestor of the clade consisting of B. gabonica, B. nasicornis, B. parviocula and B. rhinoceros) induce an array of debilitating effects including anticoagulation, hemorrhagic shock and cytotoxicity, whilst the dwarf species B. atropos is known to have strong neurotoxic effects. However, the venom effects of the other species within the genus have not been explored in detail. A series of coagulation assays were implemented to assess the coagulotoxic venom effects of fourteen species within the genus. This study identified procoagulant venom as the ancestral condition, retained only by the basal dwarf species B. worthingtoni, suggesting anticoagulant venom is a derived trait within the Bitis genus and has been secondarily amplified on at least four occasions. A wide range of anticoagulant mechanisms were identified, such as coagulant and destructive activities upon fibrinogen in both giant and dwarf Bitis and the action of inhibiting the prothrombinase complex, which is present in a clade of dwarf Bitis. Antivenom studies revealed that while the procoagulant effects of B. worthingtoni were poorly neutralized, and thus a cause for concern, the differential mechanisms of anticoagulation in other species were all well neutralized. Thus, this study concludes there is a wide range of coagulotoxic mechanisms which have evolved within the Bitis genus and that clinical management strategies are limited for the procoagulant effects of B. worthingtoni, but that anticoagulant effects of other species are readily treated by the South African polyvalent antivenom. These results therefore have direct, real-work implications for the treatment of envenomed patients.

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 Bß-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.

Coagulotoxic Cobras: Clinical Implications of Strong Anticoagulant Actions of African Spitting Naja Venoms That Are Not Neutralised by Antivenom but Are by LY315920 (Varespladib).

Snakebite is a global tropical disease that has long had huge implications for human health and well-being. Despite its long-standing medical importance, it has been the most neglected of tropical diseases. Reflective of this is that many aspects of the pathology have been underinvestigated. Snakebite by species in the Elapidae family is typically characterised by neurotoxic effects that result in flaccid paralysis. Thus, while clinically significant disturbances to the coagulation cascade have been reported, the bulk of the research to date has focused upon neurotoxins. In order to fill the knowledge gap regarding the coagulotoxic effects of elapid snake venoms, we screened 30 African and Asian venoms across eight genera using in vitro anticoagulant assays to determine the relative inhibition of the coagulation function of thrombin and the inhibition of the formation of the prothrombinase complex through competitive binding to a nonenzymatic site on Factor Xa (FXa), thereby preventing FXa from binding to Factor Va (FVa). It was revealed that African spitting cobras were the only species that were potent inhibitors of either clotting factor, but with Factor Xa inhibited at 12 times the levels of thrombin inhibition. This is consistent with at least one death on record due to hemorrhage following African spitting cobra envenomation. To determine the efficacy of antivenom in neutralising the anticoagulant venom effects, for the African spitting cobras we repeated the same 8-point dilution series with the addition of antivenom and observed the shift in the area under the curve, which revealed that the antivenom performed extremely poorly against the coagulotoxic venom effects of all species. However, additional tests with the phospholipase A2 inhibitor LY315920 (trade name: varespladib) demonstrated a powerful neutralisation action against the coagulotoxic actions of the African spitting cobra venoms. Our research has important implications for the clinical treatment of cobra snakebites and also sheds light on the molecular mechanisms involved in coagulotoxicity within Naja. As the most coagulotoxic species are also those that produce characteristic extreme local tissue damage, future research should investigate potential synergistic actions between anticoagulant toxins and cytotoxins.

How the Cobra Got Its Flesh-Eating Venom: Cytotoxicity as a Defensive Innovation and Its Co-Evolution with Hooding, Aposematic Marking, and Spitting.

The cytotoxicity of the venom of 25 species of Old World elapid snake was tested and compared with the morphological and behavioural adaptations of hooding and spitting. We determined that, contrary to previous assumptions, the venoms of spitting species are not consistently more cytotoxic than those of closely related non-spitting species. While this correlation between spitting and non-spitting was found among African cobras, it was not present among Asian cobras. On the other hand, a consistent positive correlation was observed between cytotoxicity and utilisation of the defensive hooding display that cobras are famous for. Hooding and spitting are widely regarded as defensive adaptations, but it has hitherto been uncertain whether cytotoxicity serves a defensive purpose or is somehow useful in prey subjugation. The results of this study suggest that cytotoxicity evolved primarily as a defensive innovation and that it has co-evolved twice alongside hooding behavior: once in the Hemachatus + Naja and again independently in the king cobras (Ophiophagus). There was a significant increase of cytotoxicity in the Asian Naja linked to the evolution of bold aposematic hood markings, reinforcing the link between hooding and the evolution of defensive cytotoxic venoms. In parallel, lineages with increased cytotoxicity but lacking bold hood patterns evolved aposematic markers in the form of high contrast body banding. The results also indicate that, secondary to the evolution of venom rich in cytotoxins, spitting has evolved three times independently: once within the African Naja, once within the Asian Naja, and once in the Hemachatus genus. The evolution of cytotoxic venom thus appears to facilitate the evolution of defensive spitting behaviour. In contrast, a secondary loss of cytotoxicity and reduction of the hood occurred in the water cobra Naja annulata, which possesses streamlined neurotoxic venom similar to that of other aquatic elapid snakes (e.g., hydrophiine sea snakes). The results of this study make an important contribution to our growing understanding of the selection pressures shaping the evolution of snake venom and its constituent toxins. The data also aid in elucidating the relationship between these selection pressures and the medical impact of human snakebite in the developing world, as cytotoxic cobras cause considerable morbidity including loss-of-function injuries that result in economic and social burdens in the tropics of Asia and sub-Saharan Africa.