Developing a cocktail of enzyme inhibitors to universally treat haemotoxicity caused by snakebite Background  Snakebite is a neglected tropical disease that kills ~100,000 people each, year and causes long-term morbidity in 3-5 times that number. Venom variation between snake species severely undermines the efficacy of existing snakebite therapies, known as antivenoms. These intravenously delivered treatments work poorly against bites caused by snake species that were not used to make the antivenom. Therefore many different antivenoms have to be manufactured for each region of the world. In addition, existing antivenoms have (i) poor specificity, (ii) poor safety and (iii) high treatment costs. There is therefore an urgent and compelling need to dramatically improve therapy for the rural impoverished people of the tropics suffering the greatest burden of snakebite.  Rationale  The most common lethal snakebite pathology is haemotoxicity, which can present clinically as haemorrhage, coagulopathy and/or hypotension, and is primarily caused by three enzymatic venom toxin families. In this project, we seek to develop a new type of therapy for treating haemotoxicity, consisting of repurposed enzyme inhibitors (small molecules). Our preliminary data shows that various enzyme inhibitors are capable of neutralising the enzymatic activity of haemotoxic venom protein families irrespective of the snake species involved, and at lower therapeutic doses than antivenom. We have also demonstrated that inhibitory molecules are capable of preventing snake venom induced lethality in vivo in pre-clinical animal studies. We therefore anticipate that a rationally selected mixture of different enzyme inhibitors will result in a low-dose therapy that (i) neutralises venom lethality irrespective of the snake species, and thus will exert worldwide efficacy, (ii) exhibits a significantly improved safety profile, and (iii) is substantially more affordable than antivenom.  Approach  We will use a variety of enzyme inhibitors previously approved for human use and test their in vitro and in vivo neutralising capability against ten medically-important, haemotoxic, snake venoms. 1. First, we will use a variety of enzymatic in vitro assays to characterise the haemorrhagic, coagulopathic and hypotensive action of each venom. Subsequently, we will repeat these assays with each venom mixed with individual enzyme inhibitors to identify those inhibitors that confer venom neutralisation. 2. Our preliminary data suggests that combinations of different inhibitors will be required for 'generic' (i.e. across lots of different snake species) neutralisation of venom lethality in vivo, due to the complexity of toxic venom components, and that combinations of enzyme inhibitors can reduce the therapeutic dose required for neutralisation. Consequently, we will repeat our in vitro assays using rational combinations of the different inhibitors identified (those that exhibited neutralising capabilities in 1) at different dose combinations. 3. Lastly, we will demonstrate the superior efficacy of the two inhibitor combinations exhibiting the highest neutralising capability in vitro, in in vivo neutralisation of venom lethality studies. We will use murine pre-clinical efficacy studies (survival and survival times) and subsequent biomarker analyses (e.g. markers of haemorrhage, coagulopathy, hypotension) to determine whether the enzyme inhibitor mixtures exhibit superior venom neutralisation than existing commercial antivenoms.  Implications  We anticipate that our optimal enzyme inhibitory mixture will exert superior venom neutralisation characteristics (lower therapeutic doses, generic cross-species neutralisation) than existing "gold-standard" antivenoms. Consequently, this project has the potential to revolutionise snakebite therapy via the generation of a pre-clinically validated "generic", affordable, treatment for the millions of impoverished snakebite victims who suffer from venom-induced haemotoxicity each year. The Global Challenges Research Fund (GCRF) supports cutting-edge research to address challenges faced by developing countries. The fund addresses the UN sustainable development goals. It aims to maximise the impact of research and innovation to improve lives and opportunity in the developing world.