Inhibition of NF1 Protein Degradation as a Treatment for NF1 Haploinsufficiency

项目摘要。单倍体功能不全在常染色体神经纤维瘤病(NF1)中起着关键作用 影响超过12万美国人的显性遗传病。当前治疗方法的靶向 NF1信号的下游成分，例如肿瘤中的MEK抑制，因此未能解决 与NF1突变相关的广泛的信号和症状。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蛋白的降解， 导致NF1蛋白的净增加，有可能缓解广泛的NF1症状并停止 总的疾病进展。Infix建议识别和验证与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实验来筛选已知的影响 药物对NF1蛋白水平的影响，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，我们的目标是增加总体NF1蛋白，从而使RAS正常化 NF1患者的(和其他)途径信号。我们提出这是NF1药物发现的一条新途径， 以预防性的方式，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的NF1基因突变。 研究背景。通过转基因提高NF1的表达逆转RAS的异常激活 由NF1的损失造成(Wallis，2018；Mellert，2018)。在其他遗传条件下蛋白质表达增加 如Willams-Beuren综合征和瓣膜上动脉狭窄补偿单倍体功能不全 (Giordano，et al.2012年)。最后，克服其他常染色体显性条件下的单倍体不足(Sim1； Pax6基因)在体内显示出纠正症状的能力。Matharu等人艾尔2019年；et Rabiee艾尔2020)。 明确的目标。1)使用siRNA文库识别NF1蛋白稳定性的调节因子作为基因(敲除) 筛选出与NF1相关的细胞类型。确定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以筛选增加NF1蛋白的化合物。2)构造 一种试验，设计内源性nf1基因来标记nf1蛋白，在一种具有良好特征的公开 可获得(ATCC)永生化的NF1/-雪旺细胞系。使用已由以下机构验证的化合物进行验证 固定可提高NF1蛋白水平。3)部署NF1蛋白稳定试验，筛选出13,000 从斯克里普斯研究所获得的已知药物的化合物再用途文库(称为ReFrame)， 和其他目标库。来自这些屏幕的最高点击量将使用不朽的施万恩进行评估， 原代成纤维细胞和IPSC来源的NF1/-细胞，用于以下方面：a)诱导NF1蛋白表达的能力 使用Westns/ELISA，b)对RAS信号的影响(PERK、ELK-1、AKT等)利用有针对性的数量 质谱学蛋白质组学分析，c)对细胞增殖的影响，d)基于已公布数据的安全性概况。 该计划的目标是为SBIR第二阶段Pre-IND评估确定3-5个候选化合物的优先级。

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.