Biology subjects

Casewell, N. R.

Publications and source records attributed to Casewell, N. R..

At least 19 recordsLinked to original sources

bioRxiv2026

Recombinant Echis disintegrins reveal distinct inhibitory profiles on platelet aggregation and endothelial cell migration

Disintegrins are snake venom toxins that bind integrins and modulate platelet aggregation and cell migration. Isolation from venom often yields insufficient quantities for comprehensive study, making recombinant expression essential for detailed toxin characterisation. Here, we produced and characterised four disintegrins from Echis coloratus and Echis ocellatus in Escherichia coli. These homologous proteins contain distinct integrin-binding motifs: EcDis_RGD, EoDis_RGD, EcDis_KGD, and EcDis_VGD. Functional activities were evaluated using human platelet aggregation and endothelial cell scratch wound-healing assays. RGD-containing disintegrins exhibited the strongest inhibition of platelet aggregation, with EcDis_RGD showing the highest potency. The EcDis_KGD disintegrin inhibited platelet aggregation less potently, whereas EcDis_VGD showed no detectable activity in our assays. In endothelial cells, EcDis_RGD disintegrin markedly impaired cell migration, while EoDis_RGD displayed weaker anti-migratory activity. Neither KGD- nor VGD-containing disintegrins significantly affected wound closure. EoDis_RGD exhibited similar antiplatelet activity to the native, venom-purified disintegrin ocellatusin, validating our recombinant expression strategy. However, the corresponding PII-SVMP preparation displayed reduced inhibitory activity, consistent with incomplete generation of fully mature disintegrins species. Our findings demonstrate the importance of both integrin-binding motif identity and surrounding sequence context in determining disintegrin function, and support further use of recombinant toxins in toxinology and as therapeutic lead molecules.

biochemistry↗

bioRxiv2026

Disintegrin-like and Cysteine-rich Domains Govern Enzymatic Activity and Substrate Recognition in Echis Snake Venom Metalloproteinases

Snake venom metalloproteinases (SVMPs) are major drivers of pathology following viper envenomation and represent important targets for the development of next-generation recombinant antivenoms. PIII SVMPs are among the most potent haemorrhagic toxins and contain disintegrin-like (Dis) and cysteine-rich (C-rich) accessory domains. Despite their biomedical importance, the mechanistic roles of these accessory domains in substrate recognition and catalysis remain poorly understood. We produced recombinant full-length and domain-deletion variants of two functionally distinct PIII SVMPs: the broadly proteolytic, cytotoxic cPIII and the highly specific prothrombin activator Ecarin. Proteins were expressed as latent zymogens in insect cells, auto-activated by Zn2+, and analysed using enzymatic, blood clotting, and cell-based assays. Progressive removal of the C-rich and Dis domains reduced zymogen auto-activation and markedly diminished catalytic activity in both toxins. In cPIII, domain deletion caused a stepwise loss of proteolytic and cytotoxic activity without altering substrate preference. In Ecarin, removal of the accessory domains strongly impaired prothrombin activation, and thus plasma clotting, demonstrating a critical role in substrate recognition. Conversely, deletion of the C-rich domain increased fibrinogenolytic activity, revealing a substrate-dependent gatekeeping function. Deglycosylation showed that N-linked glycans modulate SVMP activity in a construct-dependent manner. Recombinant Ecarin closely recapitulated the biochemical properties of the native venom-derived toxin. Our data support a model in which PIII SVMP accessory domains enhance substrate positioning and catalytic efficiency while selectively restricting access to non-cognate substrates. These findings establish accessory-domain-mediated substrate recognition as a key determinant of SVMP function, informing rational antivenom design.

biochemistry↗

bioRxiv2026

Repurposed small molecule toxin inhibitors neutralise a diversity of venoms from the Neotropical viperid snake genus Bothrops

Snakebite globally claims more than 100,000 lives per year and results in morbidity for 400,000 survivors. Current treatment uses antibody-based antivenoms which are constrained by their efficacy, safety and cost. In this study we evaluated the efficacy of previously described repurposed drugs against viperid snakes of the medically important Bothrops genus. Despite variable toxin representation and bioactivity across this central and south American genus, we found that the lead inhibitors targeting metalloproteinases (marimastat and DMPS) and phospholipases (varespladib), demonstrated pan-species neutralisation in enzymatic assays, whilst nafamostat (serine protease inhibitor) had variable activity. The metalloproteinase inhibitors protected against the procoagulant and haemorrhagic effects of several venoms in phenotypic assays. Collectively these findings demonstrate that repurposed drugs may be of great value as early interventions for the treatment of bothropic envenoming in the Neotropics and thus provides a strong rationale for their progression into future preclinical and clinical evaluation for snakebite indication.

molecular biology↗

bioRxiv2025

Efficient Recombinant Production and Functional Characterisation of Cytotoxic and Haemotoxic Snake Venom Metalloproteinases

Snake venoms contain diverse mixtures of toxins that evolved to incapacitate prey, but in humans they cause extensive pathology following snakebite envenomation. In viper venom, some of the most potent toxins are the haemorrhagic and coagulopathic snake venom metalloproteinases (SVMPs). Because venoms contain a SVMP cocktail, and due to their cytotoxicity, SVMP characterizations have been hampered by the lack of purified enzymes. By incorporating their prodomain, which blocks the active SVMP site, we overcame their cytotoxicity and enabled recombinant production of zymogens from all three structurally variable SVMP classes (PI, PII and PIII) using our baculovirus/insect cell expression system. Zymogens were auto-activated by incubation with Zn2+ ions, resulting in prodomain cleavage, PII disintegrin cleavage and PIII prodomain proteolysis. Auto-activated SVMPs were characterized using protein substrate degradation, platelet aggregation and blood coagulation assays, benchmarked to native venom-purified SVMP. Our recombinant zymogen production protocol is generically applicable for the expression of SVMPs, unlocking biomedical use in haematology, and discovery of novel snakebite therapeutics.

pharmacology and toxicology↗

bioRxiv2025

Structural and functional characterisation of isolated puff adder (B. arietans) serine proteases

Serine proteases are known to play a major role in the haemotoxic actions of viper venom, but compared with those from other vipers, the serine proteases of puff adder venoms have not been extensively characterised. To address this, we isolated, identified and characterised the bioactivity of the serine proteases within the venom of the Nigerian puff adder which we had previously shown to be especially rich in this class of toxin. Two distinct groups were identified, each with different protein substrate specificities. Both had similar molecular weights of 52-62 kDa, with 4-6 N-glycans, but one group consisted of trypsin-like acidic SVSPs and the other were chymotrypsin-like basic SVSPs. Each acted differently on fibrinogen: the acidic SVSPs showed thrombin-like alpha/beta-fibrinogenase activity, whereas the basic forms were shown to be alpha-fibrinogenases. The acidic SVSPs possess gelatinase activity - a novel activity for SVSPs and the first example of an SVSP acting on proteins other than those of the haemostatic system. Analysis of the transcripts of both sets of SVSPs revealed structural details of the substrate-binding sites that supported the experimental findings. The activity and sequences of the basic SVSPs show that they are very like the alpha-fibrinogenase ML-AF of M. lebetina, which until now was considered to be a unique SVSP. Thus, this basic SVSP and the acidic SVSP with its gelatinase activity can be considered to be atypical viper serine proteases. The gelatinase activity of the acidic SVSPs was found to vary geographically and this, alongside the regional variation in the SVMP activities that we observed previous study, is discussed with reference to the potential implications on pathology of envenoming and the development of therapeutic interventions.

biochemistry↗

bioRxiv2025

Discovery and Development of DC-174 as a Novel Oral Snakebite Treatment

Snakebite envenoming is a neglected tropical disease that causes high mortality and morbidity. The current treatment, intravenous antivenom, comes with numerous disadvantages making new therapeutics important. Optimised small molecules offer the possibility for oral use at the onset of envenoming, and the highly pathogenic, zinc-dependent, snake venom metalloproteinase toxin family represents an attractive target for drug discovery. Through systematic chemical modification guided by molecular modelling, we describe the development of hydroxamic acid DC-174, a molecule that displays potent broad spectrum metalloproteinase inhibition and neutralises the procoagulant activities of multiple snake venoms. In oral-dosing studies, DC-174 showed preclinical efficacy in a mouse model of severe envenoming, with efficacy boosted by a pharmacokinetically-informed multiple dosing regimen. This rationally designed metalloproteinase inhibitor offers a potential paradigm shift from delayed treatment with antivenom in tertiary hospitals to a contemporary approach using oral drugs amenable for rapid use in snakebite-affected communities.

pharmacology and toxicology↗

bioRxiv2025

A drug combinational therapy consisting of toxin phospholipase A2 and metalloproteinase inhibitors provides preclinical protection against North American Crotalid snakebite envenoming

BackgroundAcross North America an estimated 3,800-6,500 snakebite envenomings occur annually, resulting in 7-15 deaths and an unknown number of disfigurements and disabilities. Most bites are caused by Crotalid snake species. The variable diversity and toxin complexity of crotalid venoms presents a considerable challenge to developing broadly effective small molecule therapeutics to better treat snakebite in this region. MethodsWe evaluated the ability of three small molecule, toxin inhibiting, repurposed drugs to inhibit the venom activities of six medically important crotalid snake species (Agkistrodon contortrix, Crotalus atrox, C. adamanteus, C. horridus, C. scutulatus and Sistrurus miliarius). These drugs target two pathologically relevant venom toxin families, the snake venom metalloproteinases (SVMPs; marimastat and DMPS) and phospholipases A2 (PLA2s; varespladib), and venom inhibition was measured using in vitro enzymatic and phenotypic plasma coagulation assays. Thereafter we evaluated the efficacy of individual drugs and dual drug combinations in in vivo preclinical models of snakebite envenoming, using both preincubation and rescue model formats. ResultsIn vitro bioassays demonstrated that the selected small molecules showed potent inhibition of the enzymatic activity of different toxin families to the nanomolar (varespladib vs PLA2 and marimastat vs SVMP) or micromolar (DMPS vs SVMP) level. Three of the venoms had anticoagulant activity, which varespladib restored to normal coagulation profiles, suggesting this activity is mostly driven by PLA2 toxins. Preclinical experiments revealed that pre-incubation of representative venoms with single drugs was insufficient to completely protect against lethality, except for varespladib against C. scutulatus. Superior efficacy was observed when drugs were used in a combination approach, with the combination of marimastat and varespladib providing greatest protection against lethality in both pre-incubation and rescue models. ConclusionsVenom variation among snake species makes the development of generic snakebite therapeutics challenging. In this study we showed that while SVMP and PLA2 inhibiting drugs show inhibitory potency against diverse North American snake venoms, drug combinations consisting of an SVMP inhibitor together with a PLA2 inhibitor are required to confer broad in vivo protection against lethality caused by envenoming. This study highlights the potential long-term value of drug combinations as next-generation therapeutics for snakebite envenoming.

molecular biology↗

bioRxiv2025

Establishing a preclinical chronic wound model to characterise the pathophysiology of snakebite envenoming

Snakebite claims 138,000 lives a year with an additional 400,000 patients left permanently disabled or disfigured1. Morbidity following envenoming includes the development of chronic wounds around the bite site. The understanding of the underlying pathophysiology of chronic snakebite wounds has been severely limited by the historical reliance on a preclinical model that only captures acute local envenoming pathology. Through the application of three medically important snake venoms (Echis ocellatus, Bothrops atrox and Naja nigricollis) to a recently developed preclinical model of chronic wounds, we have been able to characterise key features of venom wounds. We have been able to show that venom wounds share consistencies with non-venom induced preclinical wounds, and also display unique characteristics such as extracellular matrix degradation and eosinophilic infiltrate. This model will not only serve to increase our understanding the underlying pathophysiology of venom wounds, but will also provide a platform for exploring therapeutic interventions to reduce or resolve snakebite wounds.

pathology↗

bioRxiv2024

Snake venom metalloproteinases are predominantly responsible for the cytotoxic effects of certain African viper venoms

AbstractSnakebite envenoming is a neglected tropical disease that causes substantial mortality and morbidity globally. The puff adder (Bitis arietans) and saw-scaled viper (Echis romani) have cytotoxic venoms that cause permanent injury via tissue-destructive dermonecrosis around the bite site. Identification of cytotoxic toxins within these venoms will allow development of targeted treatments, such as small molecule inhibitors or monoclonal antibodies to prevent snakebite morbidity. Venoms from both species were fractionated using gel filtration chromatography, and a combination of cell-based cytotoxicity approaches, SDS-PAGE gel electrophoresis, and enzymatic assays were applied to identify venom cytotoxins in the resulting fractions. Our results indicated that snake venom metalloproteinase (SVMP) toxins are predominately responsible for causing cytotoxic effects across both venoms, but that the PII subclass of SVMPs are likely the main driver of cytotoxicity following envenoming by B. arietans, whilst the structurally distinct PIII subclass of SVMPs are responsible for conveying this effect in E. romani venom. Identification of distinct SVMPs as the primary cytotoxicity-causing toxins in these two African viper venoms will facilitate the future design and development of novel therapeutics targeting these medically important venoms, which in turn could help to mitigate the severe life and limb threatening consequences of tropical snakebite. Key ContributionSVMP toxins were identified as the primary cytotoxicity-causing toxins in the venoms of the puff adder (Bitis arietans) and saw-scaled viper (Echis romani); PII and PIII SVMPs, respectively. This cytotoxicity can be prevented using the metalloproteinase-inhibiting chelator EDTA, suggesting targeted drugs/antibodies may be a viable option for future treatment.

pharmacology and toxicology↗

bioRxiv2024

Isolation and characterisation of serine proteases and metalloproteases from the venom of African puff adders

The puff adder (Bitis arietans) is a highly venomous viper responsible for many fatalities in Africa, yet there have been few comprehensive analyses of its venom proteins, particularly of the proteases that play a key role in pathology of envenoming. To address this, we have isolated, identified and characterised the bioactivity of the venom metalloproteases of puff adders obtained from a wide range of sources. Prominent in all venoms was an SVMP PI, derived from a PII precursor. This protein existed in either of two forms: non-glycosylated (21 kDa) or glycosylated with either one (26 kDa) or two N-glycans (30 kDa). All the venoms we tested here were found to contain either one or the other form: none had both. The 21 kDa form proved to be highly potent, with alpha-, beta- and in some cases gamma-fibrinogenase activities and were very destructive towards laminin. Prothrombin and Factor X were also extensively degraded by the 21 kDa SVMP, but in neither case did this result in generation of the respective active forms of these clotting factors. In contrast, the two-glycan forms were markedly less active against all of these substrates. The one-glycan form isolated from a Kenyan venom possessed activities that was intermediate between the non- and two-glycan forms. Because of the predominance and ubiquity of these SVMPs in puff adders, and their undoubted clinical significance, we propose to name them the arilysins. The SVMP PIII content of the puff adder venoms was, atypically for African vipers, quite low. In some Kenyan venoms, however, there was an abundant SVMP PIII, with strong gelatinase activity. This protein possesses an unusual oligomeric structure, being a 140 kDa homodimer (c.f. SVMPIII-c) but without the disulphide bonds that normally hold the monomers together in this class of SVMP. This diversity in venom metalloprotease activities is discussed with reference to the potential implications on the pathology of envenomation and the development of therapeutic interventions.

biochemistry↗

bioRxiv2024

Intraspecific venom variation in the medically important puff adder (Bitis arietans): comparative venom gland transcriptomics, in vitro venom activity and immunological recognition by antivenom

BackgroundVariation in snake venoms is well documented, both between and within species, with intraspecific venom variation often correlated with geographically distinct populations. The puff adder, Bitis arietans, is found widely distributed across sub-Saharan Africa and into the Arabian Peninsula where it is considered a leading cause of the [~]310,000 annual snakebites across the region, with its venom capable of causing substantial morbidity and mortality. Despite its medical importance and wide geographic distribution, there is little known about venom variation between different B. arietans populations and the potential implications of this variation on antivenom efficacy. MethodologyWe applied a range of analyses, including venom gland transcriptomics, in vitro enzymatic assays and reverse phase chromatography to comparatively analyse B. arietans venoms originating from Nigeria, Tanzania, and South Africa. Immunological assays and in vitro enzymatic neutralisation assays were then applied to investigate the impact of venom variation on the potential efficacy of three antivenom products; SAIMR Polyvalent, EchiTAb-Plus and Fav-Afrique. FindingsThrough the first comparison of venom gland transcriptomes of B. arietans from three geographically distinct regions (Nigeria, Tanzania, and South Africa), we identified substantial variation in toxin expression. Findings of venom variation were further supported by chromatographic venom profiling, and the application of enzymatic assays to quantify the activity of three pathologically relevant toxin families. However, the use of western blotting, ELISA, and in vitro enzymatic inhibition assays revealed that variation within B. arietans venom does not appear to substantially impact upon the efficacy of three African polyvalent antivenoms. ConclusionsThe large distribution and medical importance of B. arietans makes this species ideal for understanding venom variation and the impact this has on therapeutic efficacy. The findings in this study highlight the likelihood for considerable venom toxin variation across the range of B. arietans, but that this may not dramatically impact upon the utility of treatment available in the region. Author SummaryThe puff adder (Bitis arietans) is found across sub-Saharan Africa and the Arabian Peninsula and is capable of causing life threatening pathology due to its potent venom. The extensive range of B. arietans exposes populations to different ecological pressures which may impact upon the composition of venom toxins. In this study, we examined the venom composition of B. arietans from three countries separated by large geographic distance: Nigeria, Tanzanian and South Africa. By integrating venom gland transcriptomes, venom chromatography, and in vitro functional assays to profile B. arietans venom composition, we uncovered extensive variation between the three locales. Given that venom variation can have a significant impact on the efficacy of antivenom treatment, we also investigated the ability of three African antivenoms to recognise and inhibit in vitro venom activity. Through these analyses, we were able to determine that venom variation did not have a substantial impact on the neutralising effect of selected antivenoms. This study has highlighted the potentially extensive venom variation found across the range of B. arietans and initiated valuable investigations into the efficacy of African antivenoms to protect human populations vulnerable to snakebite envenoming.

molecular biology↗

bioRxiv2023

Dermonecrosis caused by spitting cobra snakebite is the result of toxin potentiation and is prevented by the repurposed drug varespladib

Snakebite envenoming is a neglected tropical disease that causes substantial mortality and morbidity globally. The venom of African spitting cobras often causes permanent injury via tissue-destructive dermonecrosis at the bite site, which is ineffectively treated by current antivenoms. To address this therapeutic gap, we identified the aetiological venom toxins responsible for causing local dermonecrosis. While cytotoxic three-finger toxins were primarily responsible for causing spitting cobra cytotoxicity in cultured keratinocytes, their potentiation by phospholipases A2 toxins was essential to cause dermonecrosis in vivo. This evidence of probable toxin synergism suggests that a single toxin-family inhibiting drug could prevent local envenoming. We show that local injection with the repurposed phospholipase A2-inhibiting drug varespladib significantly prevents local tissue damage caused by several spitting cobra venoms in murine models of envenoming. Our findings therefore provide a new therapeutic strategy to more effectively prevent life-changing morbidity caused by snakebite in rural Africa. Significance StatementSpitting cobra venoms cause extensive local tissue damage surrounding the site of a snakebite. This damage cannot be effectively prevented with current antivenom treatments, and patients are often left with life-changing wounds. In this study we used cellular and mouse experiments to determine which toxins in African spitting cobra venom are responsible for causing tissue damage, revealing that a combination of two different types of toxins are required to cause pathology in vivo. We then showed that the repurposed drug, varespladib, which targets one of these toxin types, effectively prevents skin and muscle damage in mouse models of envenoming. Collectively these findings suggest that varespladib could be an effective new type of therapy for preventing snakebite morbidity in Africa.

pharmacology and toxicology↗

bioRxiv2023

Machine-learning guided Venom Induced Dermonecrosis Analysis tooL: VIDAL

Snakebite envenoming is a global public health issue that causes significant morbidity and mortality, particularly in low-income regions of the world. The clinical manifestations of envenomings vary depending on the snakes venom, with paralysis, haemorrhage, and necrosis being the most common and medically relevant effects. To assess the efficacy of antivenoms against dermonecrosis, a preclinical testing approach involves in vivo mouse models that mimic local tissue effects of cytotoxic snakebites in humans. However, current methods for assessing necrosis severity are time-consuming and susceptible to human error. To address this, we present the Venom Induced Dermonecrosis Analysis tool (VIDAL), a machine-learning-guided image-based solution that can automatically identify dermonecrotic lesions in mice, adjust for lighting biases, scale the image, extract lesion area and discolouration, and calculate the severity of dermonecrosis. We also introduce a new unit, the dermonecrotic unit (DnU), to better capture the complexity of dermonecrosis severity. Our tool is comparable to the performance of state-of-the-art histopathological analysis, making it an accessible, accurate, and reproducible method for assessing dermonecrosis. Given the urgent need to address the neglected tropical disease that is snakebite, high-throughput technologies such as VIDAL are crucial in developing and validating new and existing therapeutics for this debilitating disease.

pharmacology and toxicology↗

bioRxiv2023

An in vitro assay to investigate venom neurotoxin activity on muscle-type nicotinic acetylcholine receptor activation and for the discovery of toxin-inhibitory molecules

Snakebite envenoming is a neglected tropical disease that causes over 100,000 deaths annually. Envenomings result in variable pathologies, but systemic neurotoxicity is among the most serious and is currently only treated with difficult to access and variably efficacious commercial antivenoms. Venom-induced neurotoxicity is often caused by -neurotoxins antagonising the muscle-type nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel. Discovery of therapeutics targeting -neurotoxins is hampered by relying on binding assays that do not reveal restoration of receptor activity or more costly and/or lower throughput electrophysiology-based approaches. Here, we report the validation of a screening assay for nAChR activation using immortalised TE671 cells expressing the {gamma}-subunit containing muscle-type nAChR and a fluorescent dye that reports changes in cell membrane potential. Assay validation using traditional nAChR agonists and antagonists, which either activate or block ion fluxes, was consistent with previous studies. We then characterised antagonism of the nAChR by a variety of elapid snake venoms that cause muscle paralysis in snakebite victims, before defining the toxin-inhibiting activities of commercial antivenoms, and new types of snakebite therapeutic candidates, namely monoclonal antibodies, decoy receptors, and small molecules. Our findings show robust evidence of assay uniformity across 96-well plates and highlight the amenability of this approach for the future discovery of new snakebite therapeutics via screening campaigns. The described assay therefore represents a useful first-step approach for identifying -neurotoxins and their inhibitors in the context of snakebite envenoming, and it should provide wider value for studying modulators of nAChR activity from other sources.

pharmacology and toxicology↗

bioRxiv2022

Exploring the utility of ssDNA aptamers directed against snake venom toxins as new therapeutics for tropical snakebite envenoming

Snakebite is a neglected tropical disease that causes considerable death and disability in the tropical world. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Antivenoms are the mainstay therapy for treating the toxic effects of snakebite, but despite saving thousands of lives annually, these therapies are associated with limited cross-snake species efficacy due to venom variation, which ultimately restricts their therapeutic utility to particular geographical regions. In this study, we sought to explore the potential of ssDNA aptamers as toxin-specific inhibitory alternatives to antibodies. As a proof of principle model, we selected snake venom serine protease toxins, which are responsible for contributing to venom-induced coagulopathy following snakebite envenoming, as our target. Using SELEX technology, we selected ssDNA aptamers against recombinantly expressed versions of the fibrinogenolytic SVSPs Ancrod from the venom of Calloselasma rhodostoma and Batroxobin from Bothrops atrox. From the resulting pool of specific ssDNA aptamers directed against each target, we identified candidates that exhibited low nanomolar binding affinities to their targets. Downstream ALISA, fibrinogenolysis, and coagulation profiling experiments demonstrated that the candidate aptamers were able to recognise native and recombinant SVSP toxins and inhibit toxin- and venom-induced prolongation of plasma clotting times and consumption of fibrinogen, with inhibitory potencies highly comparable to commercial polyvalent antivenoms. Our findings demonstrate that rationally selected toxin-specific aptamers can exhibit broad in vitro cross-reactivity against toxins found in different snake venoms and are capable of inhibiting toxins in pathologically relevant in vitro and ex vivo models of venom activity. These data highlight the potential utility of ssDNA aptamers as novel toxin-inhibiting therapeutics of value for tackling snakebite envenoming.

pharmacology and toxicology↗

bioRxiv2022

Repurposed drugs and their combinations prevent morbidity-inducing dermonecrosis caused by diverse cytotoxic snake venoms

Morbidity from snakebite envenoming affects approximately 400,000 people annually. Tissue damage at the bite-site often leaves victims with catastrophic life-long injuries and is largely untreatable by currently available antivenoms. Repurposing small molecule drugs that inhibit specific snake venom toxins offers a potential new treatment strategy for tackling this neglected tropical disease. Using human skin cell assays as an initial model for snakebite-induced dermonecrosis, we show that the repurposed drugs 2,3-dimercapto-1-propanesulfonic acid (DMPS), marimastat, and varespladib, alone or in combination, reduce the cytotoxic potency of a broad range of medically important snake venoms up to 5.7-fold. Thereafter, using a preclinical murine model of dermonecrosis, we demonstrate that the dual therapeutic combinations of DMPS or marimastat with varespladib potently inhibit the dermonecrotic activity of three geographically distinct and medically important snake venoms. These findings strongly support the future translation of repurposed drug combinations as broad-spectrum therapeutics for preventing morbidity caused by snakebite.

pharmacology and toxicology↗

bioRxiv2022

Two snakebite antivenoms have potential to reduce Eswatini's dependency upon a single, increasingly unavailable product: results of preclinical efficacy testing

BackgroundSnakebite is a major public health concern in Eswatini, where treatment relies upon one antivenom - SAIMR Polyvalent. Although effective in treating snakebite, SAIMR Polyvalent is difficult to source outside its manufacturing country (South Africa) and is dauntingly expensive. We compared the preclinical venom-neutralising efficacy of two alternative antivenoms and SAIMR Polyvalent against the lethal and tissue-destructive effects of venoms from five species of medically important snakes using in vivo murine assays. The test antivenoms were Panafrican manufactured by Instituto Clodomiro Picado and PANAF manufactured by Premium Serums & Vaccines. Principal FindingsIn vivo murine preclinical studies identified both test antivenoms were equally or more effective than SAIMR Polyvalent at neutralising lethal and tissue-destructive effects of Naja mossambica venom. Both test antivenoms were less effective than SAIMR Polyvalent at neutralising the lethal effects of Bitis arietans, Dendroaspis polylepis, Hemachatus haemachatus and Naja annulifera venoms, but similarly effective at neutralising tissue damage induced by B. arietans and H. haemachatus venoms. In vitro immunological assays identified that IgG titres and toxin-specificities of the test antivenoms were comparable to SAIMR Polyvalent. Plasma clotting disturbances by H. haemachatus and N. mossambica were effectively neutralised by the test antivenoms, whereas SAIMR Polyvalent failed to neutralise this bioactivity of N. mossambica venom. The SVMP activity of B. arietans venom was equally reduced by all three antivenoms. The PLA2 activities of H. haemachatus and N. mossambica were effectively neutralised by all three antivenoms. ConclusionsPanafrican outperformed PANAF, though both were less poly-specifically effective than SAIMR Polyvalent. The efficacy of these antivenoms against the lethal and tissue-destructive effects of N. mossambica venom, the most common biting species in Eswatini, identify that Panafrican and PANAF antivenoms offer effective alternatives to SAIMR for the treatment of snakebite in Eswatini, and potentially for neighbouring countries. Author SummaryTreatment of snakebite in Eswatini is reliant upon a single antivenom (SAIMR Polyvalent) manufactured in South Africa. This highly effective product is increasingly difficult to source and is expensive - alternative/additional antivenoms are urgently required to improve patient outcomes following snake envenoming. Using murine preclinical venom toxicity and antivenom efficacy assays, we identified two alternative antivenoms whose venom-neutralising characteristics, while less poly-specifically effective than SAIMR Polyvalent, were as effective against the lethal and tissue-destructive effects of the most common biting snake in Eswatini - Naja mossambica. This murine data, already shared with the Eswatini Ministry of Health, supports and justifies human testing of these two antivenoms in Eswatini because increasing the availability of effective and affordable treatments could resolve the current medical dependency of Eswatini snakebite patients upon a single, rarely available and expensive product.

pharmacology and toxicology↗

bioRxiv2022

Exploring the utility of recombinantly expressed snake venom serine protease toxins as immunogens for generating experimental snakebite antivenoms

Snakebite is a neglected tropical disease that causes high rates of global mortality and morbidity. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Despite polyclonal antibody-based antivenoms being the mainstay life-saving therapy for snakebite, they are associated with limited cross-snake species efficacy, as there is often extensive toxin variation between snake venoms, including those used as immunogens for antivenom production. This restricts the therapeutic utility of any antivenom to certain geographical regions. In this study, we explored the feasibility of using recombinantly expressed toxins as immunogens to stimulate focused, pathology-specific, antibodies to broadly counteract specific toxins associated with snakebite envenoming. Three snake venom serine proteases (SVSP) toxins, sourced from geographically diverse and medically important viper snake venoms were successfully expressed in HEK293F mammalian cells and used for murine immunisation. Analyses of the resulting antibody responses revealed that ancrod and RVV-V stimulated the strongest immune responses, and that experimental antivenoms directed against these recombinant SVSP toxins, and a mixture of the three different immunogens, extensively recognised and exhibited immunological binding towards a variety of native snake venoms. While the experimental antivenoms showed some reduction in abnormal clotting parameters stimulated by the toxin immunogens and crude venom, specifically reducing the depletion of fibrinogen levels and prolongation of prothrombin times, fibrinogen degradation experiments revealed they broadly protected against venom- and toxin-induced fibrinogenolytic functional activities. Overall, our findings further strengthen the case for the use of recombinant venom toxins as supplemental immunogens to stimulate focused and desirable antibody responses capable of neutralising venom-induced pathological effects, and therefore potentially circumventing some of the limitations associated with current snakebite therapies.

immunology↗