Inhibition of NF1 Protein Degradation as a Treatment for NF1 Haploinsufficiency

项目摘要。单倍不足在神经纤维瘤病（NF 1）中起着至关重要的作用， 显性遗传疾病影响了超过12万美国人目前的治疗方法靶向 NF 1信号传导的下游组分，例如肿瘤中的MEK抑制，因此未能解决 与NF 1突变相关的广泛信号传导和症状。假设NF 1的特征在于： 常染色体显性和单倍不足（缺乏正常蛋白），抑制NF 1蛋白降解， 导致NF 1蛋白的净增加，有可能减轻广泛的NF 1症状， 总体疾病进展。Infixion提出鉴定和验证NF 1蛋白相关基因 泛素化和降解，并建立一个蛋白质标记的报告分析，以筛选已知的 药物对NF 1蛋白水平的影响，重点是NF 1蛋白降解的调节剂。通过确定候选人 抑制NF 1泛素化的药物，我们的目标是增加总体NF 1蛋白，从而使Ras正常化。 (and其他）途径信号转导与NF 1的个体。我们提出这是NF 1药物发现的新途径， 以预防的方式对广泛的NF 1患者和症状产生潜在影响， 适用于非常广泛的独特NF 1基因突变。 研究背景。通过转染增加NF 1表达逆转异常Ras活化 由NF 1损失引起（沃利斯，2018; Mellert，2018）。其他遗传条件下蛋白质表达增加 如Willams-Beuren综合征，以及主动脉瓣上狭窄补偿了单倍功能不全 （Giordano等人，2012年）。最后，克服其他常染色体显性疾病中的单倍不足（Sim 1; Pax 6基因）已经显示出在体内纠正症状的能力。（Matharu，et. 2019; Rabiee，et. 2020）。 具体目标。1)使用SiRNA库作为遗传（敲除）来识别NF 1蛋白稳定性的调节因子 筛选NF 1相关的细胞类型。确定NF 1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以筛选增加NF 1蛋白的化合物。2)构建体 一种分析，工程化的内源性NF 1基因标记NF 1蛋白，在一个良好的表征，公开 可获得的（ATCC），永生化NF 1 +/-许旺细胞系。使用已验证的化合物进行重复测定 增加NF 1蛋白水平。3)部署NF 1蛋白稳定化检测，筛选13，000 + 可从Scripps研究所获得的已知药物的化合物再利用库（称为ReFrame）， 和其他目标图书馆。这些屏幕上的热门歌曲将使用永生的施旺进行评估， 原代成纤维细胞和iPSC衍生的NF 1 +/-细胞，用于以下目的：a）诱导NF 1蛋白表达的能力 使用Western/ELISA，B）对Ras信号传导（pERK、ELK-1、AKT等）的影响利用目标定量 质谱蛋白质组学分析，c）对细胞增殖的影响，和d）基于公开数据的安全性特征。 计划目标是优先考虑3-5种候选化合物进行SBIR 2期IND前评价。

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.