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

动质体编码的不是典型的信使核糖核酸解离酶的同源物。我们最近发现APAH样磷酸酶ALPH1在引起布鲁氏锥虫的昏睡病中是主要的或唯一的mRNA解离酶，可能在所有的动质体内也是如此。在真核生物王国中，APAH型磷酸酶对mRNA的降解是前所未有的。与经典的脱壳酶不同，ALPH1在贝塔和伽马磷酸之间切割底物，而不是在阿尔法和贝塔磷酸之间。这项建议的目的是鉴定和表征一种ALPH1特异性的抑制剂，主要有三个原因：第一，我们的目标是更详细地了解这一新的信使核糖核酸解离机制。一种特定的抑制剂提供了在所有致病动体中研究该途径的机会，包括寄生虫和不能通过遗传操作和培养的阶段，也是控制该酶的体外活性研究的关键。其次，这种酶在动体中是必不可少的，再加上哺乳动物系统中缺乏APAH样磷酸酶的整个酶家族，这使得ALPH1对于治疗非洲睡眠病、利什曼病、恰加斯病和相关动物疾病的靶向药物开发具有吸引力。第三，ALPH1抑制剂需要在生物技术应用中使用ALPH1，例如用于生产二磷酸化的mRNAs。我们组建了一个国际团队，结合了锥虫生物学、第一阶段药物发现和信使核糖核酸相互作用蛋白结构生物学的专业知识。我们已经提纯了足够数量的活性酶，并开发了一种强大的、高通量兼容的分析方法。现在，可以通过化合物文库筛选来鉴定ALPH1活性的特定抑制剂，然后评估验证的命中化合物在细胞培养中的效力，并在现场监测其对脱壳抑制的抑制作用。结合抑制剂的ALPH1的结构特征将被用于机械洞察和探索性化学指导合理的设计。最终，我们的目标是将这些化合物开发成基本的研究工具，用于生物技术应用(利用独特的ALPH1酶活性)和新型抗锥虫临床前候选药物。

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.