Animal-derived antivenoms are the only specific therapies currently available for the treatment of snake envenoming, but these products have a number of limitations associated with their efficacy, safety and affordability for use in tropical snakebite victims. Small molecule drugs and drug candidates are regarded as promising alternatives for filling the critical therapeutic gap between snake envenoming and effective treatment. In this study, by using an advanced analytical technique that combines chromatography, mass spectrometry and bioassaying, we investigated the effect of several small molecule inhibitors that target phospholipase A₂ (varespladib) and snake venom metalloproteinase (marimastat, dimercaprol and DMPS) toxin families on inhibiting the activities of coagulopathic toxins found in Viperinae snake venoms. The venoms of Echis carinatus, Echis ocellatus, Daboia russelii and Bitis arietans, which are known for their potent haemotoxicities, were fractionated in high resolution onto 384-well plates using liquid chromatography followed by coagulopathic bioassaying of the obtained fractions. Bioassay activities were correlated to parallel recorded mass spectrometric and proteomics data to assign the venom toxins responsible for coagulopathic activity and assess which of these toxins could be neutralized by the inhibitors under investigation. Our results showed that the phospholipase A₂-inhibitor varespladib neutralized the vast majority of anticoagulation activities found across all of the tested snake venoms. Of the snake venom metalloproteinase inhibitors, marimastat demonstrated impressive neutralization of the procoagulation activities detected in all of the tested venoms, whereas dimercaprol and DMPS could only partially neutralize these activities at the doses tested. Our results provide additional support for the concept that combinations of small molecules, particularly the combination of varespladib with marimastat, serve as a drug-repurposing opportunity to develop new broad-spectrum inhibitor-based therapies for snakebite envenoming.

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.

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.

Snakebite envenomation is an important public health issue that has long had tremendous implications for human health and well-being. An estimated 1.8-5.4 million people per year fall victim to snakebites, with mortality rates surpassing 100,000 individuals. Although most life-threatening pathologies from venomous snakebites result from neurotoxic and haemotoxic effects, tissue-damaging activities are a significant cause of life-long disabilities. These morbidities include tissue necrosis, amputations, renal damage and blindness. Despite the fact that venom-induced tissue damage affects over 450,000 people annually, research efforts on this pathology are limited compared to neurotoxicity or haemotoxicity. The research in this work, therefore, focuses on elucidating the mechanisms of venom-induced cell- and tissue damage. The relevance of further research on venom-induced cell- and tissue damage is clarified in Chapter 1. This chapter provides an introduction to venomous snakes, discussing their historical and cultural significance and unique characteristics. The first section of this chapter further discusses the taxonomy of venomous snakes. It characterises the morphology of venom delivery systems, highlighting the variations between the various families of venomous snakes. The same chapter describes the global impact of snakebite envenoming, which affects at least 1.8 million people per year, especially in certain regions in Latin America, sub-Saharan Africa, and Southeast Asia. The main focus of Chapter 1 is on snake venom composition and the diverse pathological effects. It categorises these effects into neurotoxicity, haemotoxicity, and tissue-damaging effects and emphasises venom-induced tissue damage. The next section of this chapter provides an overview of the various assays that exist to study tissue-damaging venoms, both in vivo and in vitro. These include assays focusing on cell viability, membrane degradation, and molecular mechanisms underlying cell damage. The chapter then discusses the relevance of elucidating venom composition and points out why separating venoms into their individual components (i.e., their toxins) is essential. This section further provides an overview of the various LC techniques used to separate venoms into their components and stresses the importance of ‘nanofractionation analytics’ and ‘high-throughput (HT) venomics’. By combining bioactivity profiling and mass spectrometry (MS), the toxins of interest (i.e., the bioactive toxins measured with the bioassay used) can directly be identified from the MS data (i.e., LC-MS and/or toxin proteomics data) that is collected in parallel.