Development of a membrane-disruption assay using phospholipid vesicles as a proxy for the detection of cellular membrane degradation.

Snakebite envenoming is a global health issue that affects millions of people worldwide, and that causes morbidity rates surpassing 450,000 individuals annually. Patients suffering from snakebite morbidities may experience permanent disabilities such as pain, blindness and amputations. The (local) tissue damage that causes these life-long morbidities is the result of cell- and tissue-damaging toxins present in the venoms. These compounds belong to a variety of toxin classes and may affect cells in various ways, for example, by affecting the cell membrane. In this study, we have developed a high-throughput in vitro assay that can be used to study membrane disruption caused by snake venoms using phospholipid vesicles from egg yolk as a substrate. Resuspended chicken egg yolk was used to form these vesicles, which were fluorescently stained to allow monitoring of the degradation of egg yolk vesicles on a plate reader. The assay proved to be suitable for studying phospholipid vesicle degradation of crude venoms and was also tested for its applicability for neutralisation studies of varespladib, which is a PLA2 inhibitor. We additionally made an effort to identify the responsible toxins using liquid chromatography, followed by post-column bioassaying and protein identification using high-throughput venomics. We successfully identified various toxins in the venoms of C. rhodostoma and N. mossambica, which are likely to be involved in the observed vesicle-degrading effect. This indicates that the assay can be used for screening the membrane degrading activity of both crude and fractionated venoms as well as for neutralisation studies.

Tissue damaging toxins in snake venoms: mechanisms of action, pathophysiology and treatment strategies.

Snakebite envenoming is an important public health issue responsible for mortality and severe morbidity. Where mortality is mainly caused by venom toxins that induce cardiovascular disturbances, neurotoxicity, and acute kidney injury, morbidity is caused by toxins that directly or indirectly destroy cells and degrade the extracellular matrix. These are referred to as 'tissue-damaging toxins' and have previously been classified in various ways, most of which are based on the tissues being affected (e.g., cardiotoxins, myotoxins). This categorisation, however, is primarily phenomenological and not mechanistic. In this review, we propose an alternative way of classifying cytotoxins based on their mechanistic effects rather than using a description that is organ- or tissue-based. The mechanisms of toxin-induced tissue damage and their clinical implications are discussed. This review contributes to our understanding of fundamental biological processes associated with snakebite envenoming, which may pave the way for a knowledge-based search for novel therapeutic options.

African polyvalent antivenom can maintain pharmacological stability and ability to neutralise murine venom lethality for decades post-expiry: evidence for increasing antivenom shelf life to aid in alleviating chronic shortages.

INTRODUCTION: Antivenom is a lifesaving medicine for treating snakebite envenoming, yet there has been a crisis in antivenom supply for many decades. Despite this, substantial quantities of antivenom stocks expire before use. This study has investigated whether expired antivenoms retain preclinical quality and efficacy, with the rationale that they could be used in emergency situations when in-date antivenom is unavailable. METHODS: Using WHO guidelines and industry test requirements, we examined the in vitro stability and murine in vivo efficacy of eight batches of the sub-Saharan African antivenom, South African Institute for Medical Research polyvalent, that had expired at various times over a period of 30 years. RESULTS: We demonstrate modest declines in immunochemical stability, with antivenoms older than 25 years having high levels of turbidity. In vitro preclinical analysis demonstrated all expired antivenoms retained immunological recognition of venom antigens and the ability to inhibit key toxin families. All expired antivenoms retained comparable in vivo preclinical efficacy in preventing the lethal effects of envenoming in mice versus three regionally and medically important venoms. CONCLUSIONS: This study provides strong rationale for stakeholders, including manufacturers, regulators and health authorities, to explore the use of expired antivenom more broadly, to aid in alleviating critical shortages in antivenom supply in the short term and the extension of antivenom shelf life in the longer term.

High throughput identification of human monoclonal antibodies and heavy-chain-only antibodies to treat snakebite.

Snakebite envenoming is a priority Neglected Tropical Disease that causes an estimated 81,000-135,000 fatalities each year. The development of a new generation of safer, affordable, and accessible antivenom therapies is urgently needed. With this goal in mind, rigorous characterisation of the specific toxins in snake venom is key to generating novel therapies for snakebite. Monoclonal antibodies directed against venom toxins are emerging as potentially strong candidates in the development of new snakebite diagnostics and treatment. Venoms comprise many different toxins of which several are responsible for their pathological effects. Due to the large variability of venoms within and between species, formulations of combinations of human antibodies are proposed as the next generation antivenoms. Here a high-throughput screening method employing antibody-based ligand fishing of venom toxins in 384 filter-well plate format has been developed to determine the antibody target/s The approach uses Protein G beads for antibody capture followed by exposure to a full venom or purified toxins to bind their respective ligand toxin(s). This is followed by a washing/centrifugation step to remove non-binding toxins and an in-well tryptic digest. Finally, peptides from each well are analysed by nanoLC-MS/MS and subsequent Mascot database searching to identify the bound toxin/s for each antibody under investigation. The approach was successfully validated to rapidly screen antibodies sourced from hybridomas, derived from venom-immunised mice expressing either regular human antibodies or heavy-chain-only human antibodies (HCAbs).

Synthetic development of a broadly neutralizing antibody against snake venom long-chain α-neurotoxins.

Snakebite envenoming is a major global public health concern for which improved therapies are urgently needed. The antigenic diversity present in snake venom toxins from various species presents a considerable challenge to the development of a universal antivenom. Here, we used a synthetic human antibody library to find and develop an antibody that neutralizes long-chain three-finger α-neurotoxins produced by numerous medically relevant snakes. Our antibody bound diverse toxin variants with high affinity, blocked toxin binding to the nicotinic acetylcholine receptor in vitro, and protected mice from lethal venom challenge. Structural analysis of the antibody-toxin complex revealed a binding mode that mimics the receptor-toxin interaction. The overall workflow presented is generalizable for the development of antibodies that target conserved epitopes among antigenically diverse targets, and it offers a promising framework for the creation of a monoclonal antibody-based universal antivenom to treat snakebite envenoming.

Snakebite prevalence and risk factors in a nomadic population in Samburu County, Kenya: A community-based survey.

INTRODUCTION: Snakebite is an important public health concern, especially in tropical areas, but the true burden remains unclear due to sub-optimal reporting and over-reliance on health facility-based data. METHODS: A community-based cross-sectional survey was conducted in Samburu County, Kenya from December 2019 to March 2020. Geospatial techniques were used to create a sampling frame of all households in Samburu County and a multistage cluster sampling strategy to select households and recruit study participants. Five year prevalence and mortality rates were estimated, the characteristics and circumstances of snakebite were described, and multilevel logistic regression models were built to identify independent risk factors for snakebite. RESULTS: We recruited 3,610 individuals living in 875 households from 30 clusters. The 5-year prevalence of snakebite was 2.2% (95% CI 1.4%-3.4%), and the 5-year mortality rate was 138 (95% CI 44-322) deaths per 100,000 inhabitants, resulting in an estimated 1,406 snakebites and 88 deaths from snakebites per year in Samburu County. Snakebite incidents often occurred at night between 9pm and 6 am (44%, n = 36), and the participants were mostly walking/playing outdoors (51%, n = 41) or sleeping (32%, n = 27) when they were bitten. Lower household socioeconomic status and smaller numbers of people per house were significant independent risk factors. CONCLUSION: Samburu County has a high snakebite burden and the most victims are bitten while sleeping or walking outdoors at night. Snakebite prevention and health promotion programs in Samburu County, and other endemic regions, need to be contextualised and consider the geographic, seasonal, and temporal specificities found in our study. Our findings also have implications for health care delivery, especially identification of the need for night-time staffing with expertise in snakebite management and antivenom availability to better manage patients and thereby improve outcomes.

[A global core outcome measurement set for snakebite clinical trials]. / Définition d'un jeu universel de critères de décision de base pour les essais cliniques sur les morsures de serpent.

Background: Snakebite clinical trials have often used heterogeneous outcome measures and there is an urgent need for standardisation. Method: A globally representative group of key stakeholders came together to reach consensus on a globally relevant set of core outcome measurements. Outcome domains and outcome measurement instruments were identified through searching the literature and a systematic review of snakebite clinical trials. Outcome domains were shortlisted by use of a questionnaire and consensus was reached among stakeholders and the patient group through facilitated discussions and voting. Results: Five universal core outcome measures should be included in all future snakebite clinical trials: mortality, WHO disability assessment scale, patient-specific functional scale, acute allergic reaction by Brown criteria, and serum sickness by formal criteria. Additional syndrome-specific core outcome measures should be used depending on the biting species. Conclusion: This core outcome measurement set provides global standardisation, supports the priorities of patients and clinicians, enables meta-analysis, and is appropriate for use in low-income and middle-income settings.

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 snake's 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 in mice. 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.

ADDovenom: Thermostable Protein-Based ADDomer Nanoparticles as New Therapeutics for Snakebite Envenoming.

Snakebite envenoming can be a life-threatening medical emergency that requires prompt medical intervention to neutralise the effects of venom toxins. Each year up to 138,000 people die from snakebites and threefold more victims suffer life-altering disabilities. The current treatment of snakebite relies solely on antivenom-polyclonal antibodies isolated from the plasma of hyperimmunised animals-which is associated with numerous deficiencies. The ADDovenom project seeks to deliver a novel snakebite therapy, through the use of an innovative protein-based scaffold as a next-generation antivenom. The ADDomer is a megadalton-sized, thermostable synthetic nanoparticle derived from the adenovirus penton base protein; it has 60 high-avidity binding sites to neutralise venom toxins. Here, we outline our experimental strategies to achieve this goal using state-of-the-art protein engineering, expression technology and mass spectrometry, as well as in vitro and in vivo venom neutralisation assays. We anticipate that the approaches described here will produce antivenom with unparalleled efficacy, safety and affordability.

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 current antivenoms. Repurposed small molecule drugs that inhibit specific snake venom toxins show considerable promise for tackling this neglected tropical disease. Using human skin cell assays as an initial model for snakebite-induced dermonecrosis, we show that the drugs 2,3-dimercapto-1-propanesulfonic acid (DMPS), marimastat, and varespladib, alone or in combination, inhibit the cytotoxicity of a broad range of medically important snake venoms. Thereafter, using preclinical mouse models of dermonecrosis, we demonstrate that the dual therapeutic combinations of DMPS or marimastat with varespladib significantly inhibit the dermonecrotic activity of geographically distinct and medically important snake venoms, even when the drug combinations are delivered one hour after envenoming. These findings strongly support the future translation of repurposed drug combinations as broad-spectrum therapeutics for preventing morbidity caused by snakebite.

Snakebite epidemiology, outcomes and multi-cluster risk modelling in Eswatini.

BACKGROUND: Halving snakebite morbidity and mortality by 2030 requires countries to develop both prevention and treatment strategies. The paucity of data on the global incidence and severity of snakebite envenoming causes challenges in prioritizing and mobilising resources for snakebite prevention and treatment. In line with the World Health Organisation's 2019 Snakebite Strategy, this study sought to investigate Eswatini's snakebite epidemiology and outcomes, and identify the socio-geographical factors associated with snakebite risk. METHODOLOGY: Programmatic data from the Ministry of Health, Government of Eswatini 2019-2021, was used to assess the epidemiology and outcomes of snakebite in Eswatini. We developed a snake species richness map from the occurrence data of all venomous snakes of medical importance in Eswatini that was subjected to niche modelling. We formulated four risk indices using snake species richness, various geospatial datasets and reported snakebites. A multivariate cluster modelling approach using these indices was developed to estimate risk of snakebite and the outcomes of snakebite in Eswatini. PRINCIPAL FINDINGS: An average of 466 snakebites was recorded annually in Eswatini. Bites were recorded across the entire country and peaked in the evening during summer months. Two cluster risk maps indicated areas of the country with a high probability of snakebite and a high probability of poor snakebite outcomes. The areas with the highest rate of snakebite risk were primarily in the rural and agricultural regions of the country. SIGNIFICANCE: These models can be used to inform better snakebite prevention and treatment measures to enable Eswatini to meet the global goal of reducing snakebite morbidity and mortality by 50% by 2030. The supply chain challenges of antivenom affecting southern Africa and the high rates of snakebite identified in our study highlight the need for improved snakebite prevention and treatment tools that can be employed by health care workers stationed at rural, community clinics.

Analytical Size Exclusion Chromatography Coupled with Mass Spectrometry in Parallel with High-Throughput Venomics and Bioassaying for Venom Profiling.

Modern analytical size exclusion chromatography (SEC) is a suitable technique to separate venom toxin families according to their size characteristics. In this study, a method was developed to separate intact venom toxins from Bungarus multicinctus and Daboia russelii venoms via analytical SEC using volatile, non-salt-containing eluents for post-column mass spectrometry, coagulation bioassaying and high-throughput venomics. Two venoms were used to demonstrate the method developed. While the venom of Bungaurs multicinctus is known to exert anticoagulant effects on plasma, in this study, we showed the existence of both procoagulant toxins and anticoagulant toxins. For Daboia russelii venom, the method revealed characteristic procoagulant effects, with a 90 kDa mass toxin detected and matched with the Factor X-activating procoagulant heterotrimeric glycoprotein named RVV-X. The strong procoagulant effects for this toxin show that it was most likely eluted from size exclusion chromatography non-denatured. In conclusion, the separation of snake venom by size gave the opportunity to separate some specific toxin families from each other non-denatured, test these for functional bioactivities, detect the eluting mass on-line via mass spectrometry and identify the eluted toxins using high-throughput venomics.

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 α-neurotoxins antagonising the muscle-type nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel. Discovery of therapeutics targeting α-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 γ-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 α-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.

Development of a high-throughput in vitro screening method for the assessment of cell-damaging activities of snake venoms.

Snakebite envenoming is a globally important public health issue that has devastating consequences on human health and well-being, with annual mortality rates between 81,000 and 138,000. Snake venoms may cause different pathological effects by altering normal physiological processes such as nervous transfer and blood coagulation. In addition, snake venoms can cause severe (local) tissue damage that may result in life-long morbidities, with current estimates pointing towards an additional 450,000 individuals that suffer from permanent disabilities such as amputations, contractions and blindness. Despite such high morbidity rates, research to date has been mainly focusing on neurotoxic and haemotoxic effects of snake venoms and considerably less on venom-induced tissue damage. The molecular mechanisms underlaying this pathology include membrane disruption and extracellular matrix degradation. This research describes methods used to study the (molecular) mechanisms underlaying venom-induced cell- and tissue damage. A selection of cellular bioassays and fluorescent microscopy were used to study cell-damaging activities of snake venoms in multi-well plates, using both crude and fractionated venoms. A panel of 10 representative medically relevant snake species was used, which cover a large part of the geographical regions most heavily affected by snakebite. The study comprises both morphological data as well as quantitative data on cell metabolism and viability, which were measured over time. Based on this data, a distinction could be made in the ways by which viper and elapid venoms exert their effects on cells. We further made an effort to characterise the bioactive compounds causing these effects, using a combination of liquid chromatography methods followed by bioassaying and protein identification using proteomics. The outcomes of this study might prove valuable for better understanding venom-induced cell- and tissue-damaging pathologies and could be used in the process of developing and improving snakebite treatments.

A long-term observational study of paediatric snakebite in Kilifi County, south-east Kenya.

INTRODUCTION: Estimates suggest that one-third of snakebite cases in sub-Saharan Africa affect children. Despite children being at a greater risk of disability and death, there are limited published data. This study has determined the: population-incidence and mortality rate of hospital-attended paediatric snakebite; clinical syndromes of snakebite envenoming; and predictors of severe local tissue damage. METHODS: All children presenting to Kilifi County Hospital, Kenya with snakebite were identified through the Kilifi Health and Demographic Surveillance System (KHDSS). Cases were prospectively registered, admitted for at least 24-hours, and managed on a paediatric high dependency unit (HDU). Households within the KHDSS study area have been included in 4-monthly surveillance and verbal autopsy, enabling calculation of population-incidence and mortality. Predictors of severe local tissue damage were identified using a multivariate logistic regression analysis. RESULTS: Between 2003 and 2021, there were 19,606 admissions to the paediatric HDU, of which 584 were due to snakebite. Amongst young children (≤5-years age) the population-incidence of hospital-attended snakebite was 11.3/100,000 person-years; for children aged 6-12 years this was 29.1/100,000 person-years. Incidence remained consistent over the study period despite the population size increasing (98,967 person-years in 2006; and 153,453 person-years in 2021). Most cases had local envenoming alone, but there were five snakebite associated deaths. Low haemoglobin; raised white blood cell count; low serum sodium; high systolic blood pressure; and an upper limb bite-site were independently associated with the development of severe local tissue damage. CONCLUSION: There is a substantial burden of disease due to paediatric snakebite, and the annual number of cases has increased in-line with population growth. The mortality rate was low, which may reflect the species causing snakebite in this region. The identification of independent predictors of severe local tissue damage can help to inform future research to better understand the pathophysiology of this important complication.

Next-Generation Sequencing for Venomics: Application of Multi-Enzymatic Limited Digestion for Inventorying the Snake Venom Arsenal.

To improve the characterization of snake venom protein profiles, we report the application of a new generation of proteomic methodology to deeply characterize complex protein mixtures. The new approach, combining a synergic multi-enzymatic and a time-limited digestion (MELD), is a versatile and straightforward protocol previously developed by our group. The higher number of overlapping peptides generated during MELD increases the quality of downstream peptide sequencing and of protein identification. In this context, this work aims at applying the MELD strategy to a venomics purpose for the first time, and especially for the characterization of snake venoms. We used four venoms as the test models for this proof of concept: two Elapidae (Dendroaspis polylepis and Naja naja) and two Viperidae (Bitis arietans and Echis ocellatus). Each venom was reduced and alkylated before being submitted to two different protocols: the classical bottom-up proteomics strategy including a digestion step with trypsin only, or MELD, which combines the activities of trypsin, Glu-C and chymotrypsin with a limited digestion approach. The resulting samples were then injected on an M-Class chromatographic system, and hyphenated to a Q-Exactive Mass Spectrometer. Toxins and protein identification were performed by Peaks Studio X+. The results show that MELD considerably improves the number of sequenced (de novo) peptides and identified peptides from protein databases, leading to the unambiguous identification of a greater number of toxins and proteins. For each venom, MELD was successful, not only in terms of the identification of the major toxins (increasing of sequence coverage), but also concerning the less abundant cellular components (identification of new groups of proteins). In light of these results, MELD represents a credible methodology to be applied as the next generation of proteomics approaches dedicated to venomic analysis. It may open new perspectives for the sequencing and inventorying of the venom arsenal and should expand global knowledge about venom composition.

Diagnostic characteristics of the 20-minute whole blood clotting test in detecting venom-induced consumptive coagulopathy following carpet viper envenoming.

INTRODUCTION: Envenoming by Echis spp. (carpet or saw-scaled vipers) causes haemorrhage and coagulopathy and represents a significant proportion of snakebites in the savannah regions of West Africa. Early diagnosis of envenoming is crucial in the management of these patients and there is limited evidence on the utility of the 20-minute whole blood clotting test (20WBCT) in diagnosing venom-induced consumptive coagulopathy (VICC) following envenoming by Echis ocellatus. METHODS: A prospective observational cohort study was conducted at the Kaltungo General Hospital in North-eastern Nigeria from September 2019 to September 2021. Standardised 20WBCTs were conducted by trained hospital staff and citrated plasma samples were collected at numerous timepoints. Prothrombin time (PT) and international normalised ratio (INR) were determined using a semi-automated analyser and INR values were calculated using international sensitivity indices (ISI). The sensitivity, specificity, positive predictive values (PPV), negative predictive values (NPV), and likelihood ratios of the 20WBCT compared to an INR ≥ 1.4 were calculated, alongside 95% confidence intervals. RESULTS: We enrolled 121 patients into our study, with a median age of 26 (18.0-35.0) years and a male predominance (75.2%). The 20WBCT was positive (abnormal) in 101 out of 121 patients at timepoint 0h, of which 95 had an INR ≥ 1.4, giving a sensitivity of 87.2% (95%CI 79.4-92.8). Among patients with a negative 20WBCT (normal), six had an INR < 1.4 giving a specificity of 50% (95%CI 21.1-78.9). The positive and negative likelihood ratios were 1.7 (95%CI 1.6-1.9) and 0.3 (95%CI 0.1-0.4) respectively. CONCLUSION: The 20WBCT is a simple, cheap, and easily accessible bedside test with a high sensitivity for the detection of patients with venom induced consumptive coagulopathy (VICC) following envenoming by E. ocellatus, although false positives do occur. Repeated 20WBCTs can identify patients with new, persistent, and rebound coagulopathy.

Monitoring Snake Venom-Induced Extracellular Matrix Degradation and Identifying Proteolytically Active Venom Toxins Using Fluorescently Labeled Substrates.

Snakebite envenoming is an important public health issue with devastating consequences and annual mortality rates that range between 81,000 and 138,000. Snake venoms may cause a range of pathophysiological effects affecting the nervous system and the cardiovascular system. Moreover, snake venom may have tissue-damaging activities that result in lifelong morbidities such as amputations, muscle degeneration, and organ malfunctioning. The tissue-damaging components in snake venoms comprise multiple toxin classes with various molecular targets including cellular membranes and the extracellular matrix (ECM). In this study, we present multiple assay formats that enable investigation of snake venom-induced ECM degradation using a variety of (dye-quenched) fluorescently labeled ECM components. Using a combinatorial approach, we were able to characterise different proteolytic profiles for different medically relevant snake venoms, followed by identification of the responsible components within the snake venoms. This workflow could provide valuable insights into the key mechanisms by which proteolytic venom components exert their effects and could therefore prove useful for the development of effective snakebite treatments against this severe pathology.

Application of an Extracellular Matrix-Mimicking Fluorescent Polymer for the Detection of Proteolytic Venom Toxins.

The cytotoxicity caused by snake venoms is a serious medical problem that greatly contributes to the morbidity observed in snakebite patients. The cytotoxic components found in snake venoms belong to a variety of toxin classes and may cause cytotoxic effects by targeting a range of molecular structures, including cellular membranes, the extracellular matrix (ECM) and the cytoskeleton. Here, we present a high-throughput assay (384-well plate) that monitors ECM degradation by snake venom toxins via the application of fluorescent versions of model ECM substrates, specifically gelatin and collagen type I. Both crude venoms and fractionated toxins of a selection of medically relevant viperid and elapid species, separated via size-exclusion chromatography, were studied using the self-quenching, fluorescently labelled ECM-polymer substrates. The viperid venoms showed significantly higher proteolytic degradation when compared to elapid venoms, although the venoms with higher snake venom metalloproteinase content did not necessarily exhibit stronger substrate degradation than those with a lower one. Gelatin was generally more readily cleaved than collagen type I. In the viperid venoms, which were subjected to fractionation by SEC, two (B. jararaca and C. rhodostoma, respectively) or three (E. ocellatus) active proteases were identified. Therefore, the assay allows the study of proteolytic activity towards the ECM in vitro for crude and fractionated venoms.

High-Throughput Venomics.

In this study, we present high-throughput (HT) venomics, a novel analytical strategy capable of performing a full proteomic analysis of a snake venom within 3 days. This methodology comprises a combination of RP-HPLC-nanofractionation analytics, mass spectrometry analysis, automated in-solution tryptic digestion, and high-throughput proteomics. In-house written scripts were developed to process all the obtained proteomics data by first compiling all Mascot search results for a single venom into a single Excel sheet. Then, a second script plots each of the identified toxins in so-called Protein Score Chromatograms (PSCs). For this, for each toxin, identified protein scores are plotted on the y-axis versus retention times of adjacent series of wells in which a toxin was fractionated on the x-axis. These PSCs allow correlation with parallel acquired intact toxin MS data. This same script integrates the PSC peaks from these chromatograms for semiquantitation purposes. This new HT venomics strategy was performed on venoms from diverse medically important biting species; Calloselasma rhodostoma, Echis ocellatus, Naja pallida, Bothrops asper, Bungarus multicinctus, Crotalus atrox, Daboia russelii, Naja naja, Naja nigricollis, Naja mossambica, and Ophiophagus hannah. Our data suggest that high-throughput venomics represents a valuable new analytical tool for increasing the throughput by which we can define venom variation and should greatly aid in the future development of new snakebite treatments by defining toxin composition.