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

基本信息

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
项目类别：
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

项目摘要

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。单倍不足易在神经纤维瘤病（NF1）（常染色体）中起着至关重要的作用影响超过120,000美国人的主要遗传疾病。当前的治疗方法目标 NF1信号传导的下游成分，例如MEK抑制肿瘤，因此无法解决与NF1突变相关的广泛信号传导和症状。鉴于NF1的特征是常染色体优势和单倍症不足（缺乏正常蛋白），抑制NF1蛋白降解，引起NF1蛋白的净增加，有可能减轻广泛的NF1症状并停止总体疾病进展。 Infixion建议识别和验证参与NF1蛋白的基因泛素化和降解，并建立一个蛋白质标签的记者测定法以筛选已知的影响 NF1蛋白水平上的药物，重点是NF1蛋白降解的调节剂。通过识别候选人抑制NF1泛素化的药物，我们靶向总体NF1蛋白的增加，从而归一化RAS （和其他）NF1个体的途径信号传导。我们将其作为NF1药物发现的新道路，对广泛的NF1患者和症状的潜在影响，以预防性的方式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的表达会逆转异常的RAS激活由NF1损失造成（Wallis，2018； Mellert，2018）。在其他遗传条件下蛋白质表达增加例如Willams-Beuren综合征和上主动脉狭窄弥补单倍症（Giordano等人，2012年）。最后，在其他常染色体显性疾病中克服单倍体不足（SIM1； PAX6基因）表现出体内能够纠正症状的能力。（Matharu等人，2019年； Rabiee等2020年）。具体目标。 1）使用siRNA文库确定NF1蛋白稳定性的调节剂在NF1相关的单元格类型中进行屏幕。识别NF1泛素化和随后退化的调节剂将允许合理选择其他文库来筛选化合物以增加NF1蛋白质。 2）构造一种测定，以良好的公开特征，为内源性NF1基因设计标记NF1蛋白可用（ATCC），永生的NF1 +/- Schwann细胞系。使用已经证实的化合物进行验证测定促进NF1蛋白水平的脱节。 3）部署NF1蛋白稳定测定法以筛选13,000多从Scripps Research Institute（称为缩影）获得已知药物的复合库，和其他有针对性的库。这些屏幕的最高命中将通过不朽的Schwann评估，原代成纤维细胞和IPSC衍生的NF1 +/-细胞，以下内容：a）诱导NF1蛋白表达的能力使用西方/ELISA，b）利用目标定量的对RAS信号传导（PERK，ELK-1，AKT等）的影响质量规格蛋白质组学测定，c）对细胞增殖的影响，d）基于已发布的数据的安全性。程序目标是优先考虑SBIR 2阶段预先评估的3-5种候选化合物。