Haemotoxic and cytotoxic snake venom metalloproteinases - production, enzymatic specificity, snakebite treatment, and biomedical use

Snake venoms are composed of a cocktail of many different (~20-100) toxins that cause very diverse effects. The snake venom metalloproteinases (SVMPs) are a family of toxins particularly abundant in viper venom, often with >12 of these enzymes found in a single venom and making up to 65% of the venom content. These SVMPs are responsible for causing destruction of tissue, systemic bleeding and blood clotting disorders in snakebite victims, which can result in death or lifelong disability. Some SVMPs act on multiple targets, while others are highly specific. The latter often contain additional protein domains (e.g. disintegrin and cysteine-rich) which contribute to target recognition. Well-known SVMP-targets include factor X, prothrombin and fibrinogen, all of which are responsible for controlling blood clotting, as well as various components of the walls of blood vessels or receptors on platelets. However, because SVMPs are difficult to isolate from one another, functional characterisation of these bioactive proteins is currently hampered by a lack of protocols on how to prepare them as recombinant proteins. Production of SVMPs is usually toxic to the producing cells, thereby preventing facile overexpression of these enzymes.

To overcome this bottleneck in production we will engineer the SVMP proteins and their domains and use a baculovirus insect cell expression system to produce them. We aim to produce inactive SVMP zymogens with a prodomain that can be removed to activate the metalloproteinase. We will also co-express chaperones that support the folding of these cysteinerich proteins and small inhibitory peptides, as well as neutralising antibodies, to facilitate expression and cell survival. As benchmarks and gold standards, we will also purify several representative SVMPs from venom. Successfully produced and purified SVMPs and disintegrin domains will be further characterised to determine their specific targets and cleavage sites using functional assays and mass spectrometry.

Next, we will use purified SVMPs as targets to select specific nanobodies (single-domain antibody fragments) as the basis for the development of new snakebite antivenom treatment. The World Health Organization declared snakebite envenoming as a neglected tropical disease with >100,000 deaths occurring annually. Provision of safe, efficient antivenom is key to life-saving treatment, yet current antivenoms are based on sera of hyper-immunised horses/sheep and have many shortcomings, including poor effectiveness and poor safety profiles. There is therefore a clear need for antivenom based on toxin-specific recombinant antibodies or antibody fragments, and SVMPs are the key toxin targets for neutralisation by these treatments. As the basis of new antivenom, we will select anti-SVMP nanobodies from a synthetic library using 'ribosome display in vitro selection and evolution' technology, and test them for their efficient neutralisation and cross-reactivity with a broad range of SVMP targets.

Finally, we will harness the biomedical potential and substrate specificity of SVMPs and disintegrin domains for the development of new anti-platelet drugs and clinical diagnostic tools for people suffering from blood clotting and bleeding disorders. Of the small number of SVMPs studied to date, several are used on a daily basis as standards for hospital blood clotting tests, while two disintegrins inspired the design of anti-platelet medications that are used for treating angina and heart attacks. We will use our recombinantly expressed, engineered and purified toxins, as well as crude snake venoms, to discover new anti-platelet toxins with desirable characteristics for drug development for treating thromboses, while simultaneously identifying toxins that activate blood clotting factors VIII and IX to enable the development of better hospital tests for identifying patients suffering from bleeding disorders like haemophilia and von Willebrand's disease.

Production and characterisation of snake venom metalloproteinase (SVMPs) toxins is highly challenging. Currently, robust protocols for overexpression of these cytotoxic and haemotoxic enzymes are lacking. However, the availability of isolated SVMPs and their disintegrin domains is vital for characterisation and evaluation of their potential biomedical application.
Here, we aim to establish production of SVMPs in baculovirus insect cells. We will use protein engineering to express the SVMPs as zymogens with an inhibitory prodomain which is cleaved off at a later step for enzyme activation. In addition we will co-express the chaperone protein disulfide isomerase, small inhibitory tripeptides and neutralising nanobodies to keep the SVMPs in an inactive state during expression.

Venom SVMPs cause extensive pathology in snakebite victims that is ineffectively treated by current antivenom therapy. Neutralising anti-toxin nanobodies (single-domain antibody fragments) will be generated using recombinant and native SVMPs and disintegrins as antigens. We will use a synthetic library with a size of ~10^12 members for ribosome display in vitro selections. The affinity and toxin-neutralisation capability of selected nanobodies will be determined, including assessment of breadth against diverse SVMP isoforms. Resulting nanobodies will be ready for future preclinical development as novel snakebite treatments.

SVMPs and disintegrins exhibit functional specificities desirable for biomedical purposes, as several are used as the basis for anti-platelet drugs or standards for the clinical diagnosis of bleeding disorders. We will use recombinant, native and engineered toxins to identify new platelet inhibitors that block specific platelet surface receptors that are known drug targets. We will also identify new activators of intrinsic pathway coagulation factors VIII and IX to address clinical laboratory testing needs associated with congenital and acquired bleeding disorders.