Mechanism of Action of Prion Protein-Lowering Small Molecules

降低朊病毒蛋白小分子的作用机制

• 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
• 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

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.

项目摘要 朊病毒病是一种迅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（PrP），能够翻译后转化为自模板和致命的构象异构体 称为朊病毒。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 PrP在大脑中减少的治疗假设。然而，尽管有强有力的遗传证据和 使用反义寡核苷酸的药理学概念证明，全脑PrP减少已经被证实是有效的。 难以用大的基因、生物或甚至寡核苷酸疗法大规模实现。我们最近 发现了两种具有降低PrP特性的小分子， 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。 在通过高通量的基于免疫荧光的表型筛选发现它们之后，我们现在 证实了效力和相当大的蛋白质组学选择性，PrP在前两个下调中 用任一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， 了解这些分子与朊病毒生物学的相关性，无论是通过识别机制， 他们正在降低PrP，并探索他们在体内这样做的能力。1)靶细胞生物学和遗传学 降低PrP化合物的随访。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、合成速率和降解速率。我们会评估药物 最小异位表达细胞系统的反应性。我们还将进行有针对性的击倒， 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)发现降低PrP化合物机制的无偏见方法。作为 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。到 为了完善我们的药物活性蛋白质组学特征的特异性，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敲除细胞和多个治疗时间点。最后，我们会做一个免疫荧光- 基于混合全基因组CRISPR筛选，以发现药物介导的PrP的效应子和敏化剂 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，以实现在已知的PrP表达组织中的积累， 包括表达PrP的外周组织，以抵抗低脑摄取的可能性。我们将 然后评估药物蓄积和PrP水平之间的关系。总之，这项工作将揭示 一个或多个生物学途径能够调节PrP水平，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转化努力，可能为帮助 第一个降低PrP的小分子药物的开发。