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

血液毒性和细胞毒性蛇毒金属蛋白酶 - 生产、酶特异性、蛇咬伤治疗和生物医学用途

• 批准号: BB/Y007581/1
• 负责人: Christiane Berger-Schaffitzel
• 金额: $ 170万
• 依托单位: University of Bristol
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FY007581%2F1
• 关键词: Haemotoxic; cytotoxic; snake; venom; metalloproteinases

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.

蛇毒由许多不同（约20-100次）毒素的鸡尾酒组成，这些毒素会引起非常多样化的作用。蛇毒金属蛋白酶（SVMP）是毒素中特别丰富的毒素家族，通常在单个毒液中发现了12种这些酶，最多可占毒液含量的65％。这些SVMP负责造成蛇咬受害者的组织，全身性出血和血液凝结障碍的破坏，这可能导致死亡或终生残疾。一些SVMP在多个目标上起作用，而另一些则是高度具体的。后者通常包含其他有助于靶识别的蛋白质结构域（例如崩解蛋白和半胱氨酸）。众所周知的SVMP目标包括因子X，凝血酶原和纤维蛋白原，所有这些因素均负责控制血液凝结，以及血小板上血管或受体的各种成分。但是，由于SVMP难以彼此分离，因此这些生物活性蛋白的功能表征目前受到缺乏有关如何使它们作为重组蛋白的方案的障碍。 SVMP的产生通常对生产细胞有毒，从而防止这些酶的易于过表达。为了克服生产中的这种瓶颈，我们将设计SVMP蛋白及其结构域，并使用杆状病毒昆虫细胞表达系统生产它们。我们旨在用可以去除的prodomain生产非活性SVMP zymogen，以激活金属蛋白酶。我们还将共同表达伴侣，以支持这些Cysteinerich蛋白和小抑制性肽以及中和抗体的折叠，以促进表达和细胞存活。作为基准和金标准，我们还将净化毒液的几个代表性SVMP。成功产生和纯化的SVMP和解体蛋白结构域将进一步表征，以使用功能测定和质谱法确定其特定靶标和切割位点。接下来，我们将使用纯化的SVMP作为靶标，以选择特定的纳米化（单域抗体片段）作为开发新的蛇咬抗蛇毒物治疗的基础。世界卫生组织宣布蛇咬为一种被忽视的热带疾病，每年发生> 100,000例死亡。提供安全，有效的抗蛇毒是挽救生命治疗的关键，但是当前的抗蛇毒基于超免疫的马/绵羊的血清，并且存在许多缺点，包括效力差和安全性差。因此，显然需要基于毒素特异性重组抗体或抗体片段的抗蛇毒，而SVMP是这些治疗方法中和的关键毒素靶标。作为新抗蛇毒的基础，我们将使用“体外选择和进化”技术从合成库中选择抗SVMP纳米生物，并测试其有效的中和和交叉反应与广泛的SVMP目标。最后，我们将利用SVMP和解体蛋白结构域的生物医学潜力和底物特异性来开发新的抗血小板药物，以及针对患有血液凝结和出血疾病的人们的临床诊断工具。在迄今为止研究的少量SVMP中，每天使用几个SVMP作为医院血液凝血测试的标准，而两种崩解蛋白启发了用于治疗心绞痛和心脏病发作的抗血小板药物的设计。 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威勒布兰氏病。