Mechanism of Action of Prion Protein-Lowering Small Molecules

项目总结 Prion病是一种迅速致命的神经退行性疾病，由一种单一的蛋白质引起，这种蛋白质是天然的Prion 蛋白质(PrP)，它能够翻译后转换为自我模板和致命的构象 被称为普里恩。PrP在这一过程中的中心地位，作为疾病的基本底物，长期以来一直导致 脑内PrP减少的治疗假说。然而，尽管有强有力的遗传证据和 药理学证明使用反义寡核苷酸的概念，全脑PrP已被减少 用大的基因、生物甚至寡核苷酸疗法很难实现规模化。我们最近做了 发现了两个具有降低PrP特性的小分子，这两个小分子可能更易于治疗 实现这一目标的方法，同时阐明PrP生物学和基础调控的新方面。 在通过高通量免疫荧光表型筛查发现它们之后，我们现在有了 证实PrP在前两个下调的基因中既有效力又有相当大的蛋白质选择性 任何一种化合物处理后细胞中的蛋白质。我们现在寻求采取以下步骤来深入 了解这些分子与Pron生物学的相关性，既要通过识别 他们正在降低PrP，并通过探索他们在体内做到这一点的能力。1)靶向细胞生物学和遗传学 降低PrP化合物的后续行动。我们将使用分子生物学工具来探测PrP的变化 处理后的加工、定位、合成速率和降解率。我们将对药物进行评估 最小异位表达细胞系统的响应性。我们还将进行有针对性的击倒和 串联质量标签显示复合处理对非PrP蛋白过表达的影响 (TMT)蛋白质组学。2)不偏不倚地发现降低PrP的化合物的机理。AS 作为蛋白质组学的补充，我们将对复合处理和未处理的细胞进行RNA测序。至 为了完善我们的药物活性蛋白质组图谱的特异性，我们将在一系列范例中进行TMT 利用先导化合物的非活性类似物，具有相同注释机制的非活性化合物 作用、PrP基因敲除细胞和多个治疗时间点。最后，我们将进行免疫荧光- 基于全基因组的CRISPR筛选，以发现药物介导的PrP的效应者和敏感者 还原。3)体内药代动力学和靶点结合。利用强大的药代动力学 我们的先导化合物的概况，我们将给小鼠剂量，以实现在已知PrP表达组织中的蓄积， 包括表达PrP的外周组织，以对抗低大脑摄取的可能性。我们会 然后评估药物累积与PrP水平之间的关系。总而言之，这项工作将揭示 一条或多条能够调节PrP水平的生物途径，并提供急需的新方向 针对这种致命且目前无法治愈的疾病的翻译努力，可能为援助提供线索 第一个降低PrP的小分子药物的开发。

PROJECT SUMMARY Prion disease is a rapidly fatal neurodegenerative disease that arises from a single protein, the native prion protein (PrP), which is capable of post-translational conversion into a self-templating and deadly conformer termed a prion. The centrality of PrP to this process, as the essential substrate for disease, has long led to the therapeutic hypothesis of PrP reduction in the brain. However, despite strong genetic evidence and pharmacological proof of concept using antisense oligonucleotides, whole-brain PrP reduction has been difficult to achieve at scale with large gene, biologic or even oligonucleotide therapeutics. We have recently identified two small molecules with PrP-lowering properties that may point towards more therapeutically facile ways to achieve this end, while simultaneously illuminating new facets of PrP’s biology and basal regulation. Following their discovery through a high-throughput immunofluorence-based phenotypic screen, we have now confirmed both potency and considerable proteomic selectivity with PrP among the top two downregulated proteins in cells following treatment with either compound. We now seek to take the following steps to deeply understand the relevance of these molecules to prion biology, both by identifying the mechanisms by which they are lowering PrP and by exploring their ability to do so in vivo. 1) Targeted cell biological and genetic follow-up of PrP-lowering compounds. We will use molecular biology tools to probe changes to PrP’s processing, localization, synthesis rate and degradation rate following treatment. We will assess the drug responsiveness of a minimal ectopic expression cellular system. We will also perform targeted knockdown and overexpression of the non-PrP proteins most effected by compound treatment according to tandem mass tag (TMT) proteomics. 2) Unbiased approaches to discover the mechanism of PrP-lowering compounds. As a complement to proteomics, we will perform RNA sequencing on compound treated and untreated cells. To refine the specificity of our proteomic profiles of drug activity, we will perform TMT in a range of paradigms leveraging inactive analogs of lead compounds, inactive compounds with the same annotated mechanism of action, PrP knockout cells, and multiple treatment timepoints. Finally, we will perform an immunofluorescence- based, pooled genome-wide CRISPR screen to discover effectors and sensitizers of drug-mediated PrP reduction. 3) In vivo pharmacokinetics and target engagement. Leveraging the strong pharmacokinetic profiles of our lead compounds, we will dose mice to achieve accumulation in known PrP-expressing tissues, including PrP-expressing peripheral tissues for robustness against the possibility of low brain uptake. We will then assess the relationship between drug accumulation and PrP levels. Altogether, this work will both uncover one or more biological pathways capable of regulating PrP levels and provide much-needed new directions for translational efforts against this fatal and currently untreatable disease, potentially offering clues to aid development of the first PrP-lowering small molecule drug.