Targeting a unique mRNA decapping enzyme for trypanosomatid infectious disease drug discovery, chemical biology and biotechnology applications

针对锥虫传染病药物发现、化学生物学和生物技术应用的独特 mRNA 脱帽酶

• 批准号: 515378686
• 负责人: Professorin Dr. Susanne Kramer
• 金额: --
• 依托单位: Narodowe Centrum Nauki
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/515378686?language=en
• 关键词: Targeting; unique; mRNA; decapping; enzyme

Kinetoplastids encode no homologues of canonical mRNA decapping enzymes. We have recently identified the ApaH-like phosphatase ALPH1 acting as the major or only mRNA decapping enzyme in the sleeping sickness causing Trypanosoma brucei and likely in all Kinetoplastids. mRNA decapping by an ApaH like phosphatase is unprecedented in the eukaryotic kingdom. In contrast to canonical decapping enzymes, ALPH1 cleaves its substrate between the beta and gamma Phosphate instead of between the alpha and beta phosphate. The aim of this proposal is the identification and characterisation of an ALPH1-specific inhibitor for three main reasons: First, we aim to understand this novel mechanism of mRNA decapping in more detail. A specific inhibitor offers the opportunity to study the pathway in all pathogenic Kinetoplastids, including parasites and stages that cannot be genetically manipulated and cultured and is also essential to control in vitro activity studies of the enzyme. Second, the fact that the enzyme is essential in Kinetoplastids combined with the absence of the entire enzyme family of ApaH-like phosphatases from mammalian systems renders ALPH1 attractive for target-based drug discovery to treat African Sleeping sickness, the Leishmaniases, Chagas disease and related animal diseases. Third, an ALPH1 inhibitor is required to employ ALPH1 for biotechnology applications, for example for the production of diphosphorylated mRNAs. We have assembled an international team combining expertise in trypanosome biology, first stage drug discovery and structural biology of mRNA interacting proteins. We have already purified active enzyme at sufficient amounts and we have developed a robust, high-throughput compatible assay. It is now feasible to identify specific inhibitors of ALPH1 activity by compound library screens followed by assessment of validated hit compounds for potency in cell culture and for decapping inhibition monitored in situ. Structural characterisation of ALPH1 with bound inhibitor will be leveraged for mechanistic insight and exploratory chemistry guiding rational design. Ultimately, we aim to develop these compounds into essential research tools, for biotechnology application (harnessing the unique ALPH1 enzymatic activity) and novel anti-trypanosomatid preclinical candidate drugs.

动质体不编码经典mRNA脱帽酶的同源物。我们最近发现ApaH样磷酸酶ALPH1作为主要的或唯一的mRNA脱帽酶在昏睡病引起布氏锥虫，并可能在所有动质体。在真核生物界中，通过ApaH样磷酸酶对mRNA进行去帽是前所未有的。与典型的去帽酶相反，ALPH1在β和γ磷酸盐之间而不是在α和β磷酸盐之间切割其底物。该提案的目的是识别和表征ALPH1特异性抑制剂，主要有三个原因：首先，我们的目标是更详细地了解这种mRNA去帽的新机制。特异性抑制剂提供了在所有致病动质体中研究该途径的机会，包括寄生虫和不能进行遗传操作和培养的阶段，并且对于控制酶的体外活性研究也是必不可少的。其次，该酶在动质体中是必需的，加上哺乳动物系统中缺乏ApaH样磷酸酶的整个酶家族，这一事实使得ALPH1对于治疗非洲昏睡病、利什曼病、恰加斯病和相关动物疾病的靶向药物发现具有吸引力。第三，ALPH1抑制剂需要将ALPH1用于生物技术应用，例如用于生产二磷酸化mRNA。我们组建了一支国际团队，结合了锥虫生物学，第一阶段药物发现和mRNA相互作用蛋白质的结构生物学方面的专业知识。我们已经以足够的量纯化了活性酶，并且我们已经开发了一种稳健的、高通量的相容性测定。现在可以通过化合物库筛选鉴定ALPH1活性的特异性抑制剂，然后评估经验证的命中化合物在细胞培养中的效力和原位监测的去帽抑制。结合抑制剂的ALPH1的结构表征将用于机制洞察和探索性化学指导合理设计。最终，我们的目标是将这些化合物开发成生物技术应用（利用独特的ALPH1酶活性）和新型抗锥虫临床前候选药物的基本研究工具。