Inhibition of NF1 Protein Degradation as a Treatment for NF1 Haploinsufficiency

抑制 NF1 蛋白降解作为 NF1 单倍体不足的治疗方法

• 批准号: 10490386
• 负责人: Michelle Mattson-Hoss
• 金额: $ 14.38万
• 依托单位: INFIXION BIOSCIENCE, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-20 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10490386
• 关键词: Address; Age; Alleles; American; Animals; Biological Assay; Cell Line; Cell Proliferation; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cutaneous; Cyclic AMP; DNA Sequence Alteration; Data; Detection; Development; Disease Progression; Dominant Genetic Conditions; Dose; ELK1 gene; Engineering; Environment; Enzyme-Linked Immunosorbent Assay; Evaluation; FRAP1 gene; Fibroblasts; Gene Mutation; Genes; Genetic; Genetic Diseases; Genetic Screening; Genome; Genotype; Goals; Growth Factor; HCN1 gene; Half-Life; Heterozygote; Human; Hypoxia; In Vitro; Individual; Investigation; Libraries; Link; Luciferases; MEK inhibition; MEKs; Modification; Mutate; Mutation; NF1 gene; Neurofibromatoses; Neurofibromatosis 1; Orphan; Other Genetics; Pathology; Pathway interactions; Patients; Persons; Pharmaceutical Preparations; Phase; Phenotype; Plant Roots; Play; Preventive treatment; Proteins; Proteomics; Proto-Oncogene Proteins c-akt; Publishing; Ras Signaling Pathway; Reaction Time; Reporter; Research; Research Institute; Research Personnel; Risk; Role; Safety; Schwann Cells; Serum; Signal Transduction; Small Business Innovation Research Grant; Small Interfering RNA; Supravalvular aortic stenosis; Symptoms; Therapeutic; Tissues; Transcend; Transfection; Ubiquitin-Activating Enzymes; Ubiquitin-Conjugating Enzymes; Ubiquitination; Williams Syndrome; Yang; associated symptom; base; brain pathway; cell type; commercialization; drug candidate; drug discovery; drug repurposing; gene therapy; high throughput screening; improved; in vivo; in vivo Model; induced pluripotent stem cell; inhibitor; innovation; knock-down; medication safety; neurofibroma; novel; novel therapeutics; programs; protein degradation; protein expression; screening; small molecule; small molecule libraries; success; tool; transcription factor; tumor; tumor progression; ubiquitin ligase

PROJECT ABSTRACT. Haploinsufficiency plays a crucial role in Neurofibromatosis (NF1), an autosomal dominant genetic disorder impacting over 120,000 Americans. Current therapeutic approaches target downstream components of NF1 signaling, for example MEK inhibition in tumors, thus failing to address the broad range of signaling and symptoms associated with NF1 mutations. Given that NF1 is characterized by both autosomal dominance and haploinsufficiency (lack of normal protein), inhibiting NF1 protein degradation, causing a net increase in NF1 protein, has the potential to alleviate a broad range of NF1 symptoms and halt overall disease progression. Infixion proposes to identify and validate genes involved in NF1 protein ubiquitination and degradation, and to build a protein-tagged reporter assay to screen the impact of known drugs on NF1 protein levels, with a focus on modulators of NF1 protein degradation. By identifying candidate drugs inhibiting NF1 ubiquitination, we target an increase in overall NF1 protein, and thus a normalizing of Ras (and other) pathway signaling in individuals with NF1. We propose this as a novel path of NF1 drug discovery, with potential impact on a broad range of NF1 patients and symptoms, in a preventative manner, and applicable to the very wide spectrum of unique NF1 genetic mutations. Research Background. Increasing NF1 expression via transfection reverses abnormal Ras activation resulting from NF1 loss (Wallis, 2018; Mellert, 2018). Increased protein expression in other genetic conditions such as Willams-Beuren Syndrome, and Supravalvular Aortic Stenosis compensates for haploinsufficiency (Giordano, et al. 2012). Lastly, overcoming haploinsufficiency in other autosomal dominant conditions (Sim1; Pax6 genes) have shown an ability in vivo to correct symptoms. (Matharu, et. al. 2019; Rabiee, et. al. 2020). Specific Aims. 1) Identify regulators of NF1 protein stability using siRNA libraries as a genetic (knock-down) screen in NF1-relevant cell types. Identifying regulators of NF1 ubiquitination and subsequent degradation will allow for rational selection of additional libraries to screen for compounds to increase NF1 protein. 2) Construct an assay, engineering the endogenous NF1 gene to tag the NF1 protein, in a well characterized, publicly available (ATCC), immortalized NF1 +/- Schwann cell line. Validate assay using compounds already verified by Infixion to increase NF1 protein levels. 3) Deploy NF1 protein stabilization assay to screen a 13,000+ compound repurposing library of known drugs available from Scripps Research Institute (known as ReFrame), and other targeted libraries. The top hits from these screens will be evaluated utilizing immortalized Schwann, primary fibroblast and iPSC derived NF1+/- cells, for the following: a) ability to induce NF1 protein expression using Westerns/ELISA, b) impact on Ras signaling (pERK, ELK-1, AKT, etc.) utilizing a targeted quantitative mass spec proteomics assay, c) impact on cell proliferation, and d) safety profile based on published data. Program goal is to prioritize 3-5 candidate compounds for an SBIR Phase 2 pre-IND evaluation.

项目摘要。单倍体不足在神经纤维瘤病 (NF1) 中起着至关重要的作用，神经纤维瘤病是一种常染色体 显性遗传疾病影响超过 120,000 名美国人。目前的治疗方法目标 NF1信号传导的下游成分，例如肿瘤中的MEK抑制，因此未能解决 与 NF1 突变相关的广泛信号传导和症状。鉴于 NF1 的特点是 常染色体显性和单倍体不足（缺乏正常蛋白质），抑制 NF1 蛋白质降解， 导致 NF1 蛋白净增加，有可能减轻广泛的 NF1 症状并阻止 总体疾病进展。 Infixion 提议鉴定和验证与 NF1 蛋白相关的基因 泛素化和降解，并建立蛋白质标记的报告测定法来筛选已知的影响 影响 NF1 蛋白水平的药物，重点是 NF1 蛋白降解的调节剂。通过确定候选人 抑制 NF1 泛素化的药物，我们的目标是增加整体 NF1 蛋白，从而使 Ras 正常化 （和其他）NF1 个体的通路信号传导。我们建议将此作为 NF1 药物发现的新途径， 以预防的方式对广泛的 NF1 患者和症状产生潜在影响，以及 适用于非常广泛的独特 NF1 基因突变。 研究背景。通过转染增加 NF1 表达可逆转 Ras 异常激活 由 NF1 损失引起（Wallis，2018；Mellert，2018）。其他遗传条件下蛋白质表达增加 例如 Willams-Beuren 综合征和瓣膜上主动脉瓣狭窄可补偿单倍体不足 （佐丹奴等人，2012 年）。最后，克服其他常染色体显性遗传条件下的单倍体不足（Sim1； Pax6 基因）已显示出体内纠正症状的能力。 （Matharu 等人，2019 年；Rabiee 等人，2020 年）。 具体目标。 1) 使用 siRNA 文库作为遗传（敲低）来识别 NF1 蛋白稳定性的调节因子 筛选 NF1 相关细胞类型。识别 NF1 泛素化和随后降解的调节因子将 允许合理选择额外的文库来筛选增加 NF1 蛋白的化合物。 2）构建 一种分析方法，工程化内源性 NF1 基因以标记 NF1 蛋白，以充分表征、公开的方式进行 可用（ATCC），永生化 NF1 +/- Schwann 细胞系。使用已验证的化合物验证测定 固定可增加 NF1 蛋白水平。 3) 部署 NF1 蛋白稳定测定来筛选 13,000 多个 斯克里普斯研究所提供的已知药物的化合物再利用库（称为 ReFrame）， 和其他目标库。这些屏幕上的热门歌曲将利用不朽的施万进行评估， 原代成纤维细胞和 iPSC 衍生的 NF1+/- 细胞，用于：a) 诱导 NF1 蛋白表达的能力 使用 Westerns/ELISA，b) 利用靶向定量对 Ras 信号传导（pERK、ELK-1、AKT 等）的影响 质谱蛋白质组学测定，c) 对细胞增殖的影响，以及 d) 基于已发表数据的安全性概况。 项目目标是优先考虑 3-5 个候选化合物进行 SBIR 第 2 阶段 IND 前评估。