Mechanism of Action of Prion Protein-Lowering Small Molecules

降低朊病毒蛋白小分子的作用机制

基本信息

批准号：
10637745
负责人：
Sonia Minikel Vallabh
金额：
$ 39.5万
依托单位：
BROAD INSTITUTE, INC.
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-01 至 2028-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10637745
关键词：
Antibodies Antisense Oligonucleotides Biological Biological Assay Biological Availability Biology Brain CRISPR screen Cell Separation Cell surface Cells Collaborations Complement Development Diabetes Mellitus Disease Distal Dose Drug Kinetics Ectopic Expression Enzyme-Linked Immunosorbent Assay Epitopes Exposure to Genes Genetic Genomics Goals Guide RNA Immunofluorescence Immunologic Infection Interphase Cell Knock-out Lead Libraries Lysosomes Malignant Neoplasms Mediating Methods Molecular Biology Mus Neurodegenerative Disorders Oligonucleotides Organ Pathogenicity Pathway interactions Penetration Peripheral Pharmaceutical Preparations Phenotype Population PrP Prion Diseases Prions Process Property Protein Biosynthesis Proteins Proteolytic Processing Proteome Proteomics Regulation Resistance Series Specificity System Testing Therapeutic Time Tissues Transfection Western Blotting Work analog conformer drug testing follow-up genome-wide in vivo knock-down mouse genome multicatalytic endopeptidase complex non-prion novel overexpression pharmacologic preclinical development protein expression proteomic signature response small molecule tool transcriptome transcriptome sequencing transcriptomics uptake

项目摘要

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.

项目概要朊病毒病是一种快速致命的神经退行性疾病，由单一蛋白质（天然朊病毒）引起蛋白质 (PrP)，能够在翻译后转化为自模板和致命的构象异构体 PrP 作为疾病的重要底物，在这一过程中发挥着中心作用，长期以来导致了这种疾病的发生。然而，尽管有强有力的遗传证据和证据，但治疗性大脑中 PrP 减少的假设。使用反义寡核苷酸的药理学概念证明，全脑 PrP 减少已被证实最近，我们很难通过大基因、生物甚至寡核苷酸疗法来大规模实现这一目标。确定了两种具有降低 PrP 特性的小分子，这可能有助于治疗更容易实现这一目标的方法，同时阐明 PrP 生物学和基础调节的新方面。继他们通过基于高通量免疫荧光的表型筛选发现之后，我们现在有了证实了 PrP 的效力和相当大的蛋白质组选择性，是前两个下调的我们现在寻求采取以下步骤来深入研究细胞中的蛋白质。通过确定这些分子与朊病毒生物学的相关性他们正在降低 PrP，并探索其在体内的能力 1) 靶向细胞生物学和遗传。我们将使用分子生物学工具来探究 PrP 的变化。我们将评估治疗后的加工、定位、合成率和降解率。我们还将进行靶向敲低和最小异位表达细胞系统的反应性。根据串联质量标签，受化合物处理影响最大的非 PrP 蛋白的过表达 (TMT) 蛋白质组学 2) 发现 As 降低 PrP 的机制的公正方法。作为蛋白质组学的补充，我们将对化合物处理和未处理的细胞进行 RNA 测序。完善药物活性蛋白质组学特征的特异性，我们将在一系列范式中进行 TMT 利用先导化合物的无活性类似物、具有相同注释机制的无活性化合物最后，我们将进行免疫荧光检测。基于汇集全基因组 CRISPR 筛选，以发现药物介导的 PrP 的效应子和敏化子 3) 体内药代动力学和靶点参与。我们的先导化合物的概况，我们将对小鼠进行给药以在已知的表达 PrP 的组织中实现积累，包括表达 PrP 的外周组织，以抵抗大脑摄取低的可能性。然后评估药物蓄积和 PrP 水平之间的关系，这项工作将共同揭示这一点。一种或多种能够调节 PrP 水平的生物途径，并为以下方面提供急需的新方向：针对这种致命且目前无法治愈的疾病的转化努力，可能为援助提供线索开发第一个降低 PrP 的小分子药物。