High Throughput Screening for Compounds to Mitigate Toxicity of FUS/TLS & SOD1

高通量筛选化合物以减轻 FUS/TLS 的毒性

• 批准号: 8348533
• 负责人: Robert H. Brown
• 金额: $ 53.91万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8348533
• 关键词: Accounting; Address; Age-Months; Amyotrophic Lateral Sclerosis; Attenuated; Behavioral; Biological Assay; Brain; Cause of Death; Cell Nucleus; Cell model; Cells; Cuprozinc Superoxide Dismutase; Cytoplasm; Cytosol; Data; Defect; Disease; Drosophila genus; Drug Kinetics; Familial Amyotrophic Lateral Sclerosis; Gene Expression; Genes; Genetic; Gliosis; Goals; Human; Human Cell Line; In Vitro; Inflammation; Inherited; Investigation; Lead; Lethal Dose 50; Link; Location; Mammalian Cell; Mediating; Messenger RNA; Metabolic; Modeling; Motor; Motor Neurons; Movement; Mus; Mutate; Mutation; Nerve; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Paralysed; Pathology; Patients; Pharmaceutical Chemistry; Pharmaceutical Preparations; Preclinical Drug Evaluation; Prions; Process; Proteins; RNA; RNA-Binding Protein FUS; Screening procedure; Series; Spinal Cord; Structure-Activity Relationship; Testing; Therapeutic Effect; Therapeutic Intervention; Toxic effect; Transgenes; Transgenic Mice; Transgenic Organisms; Validation; Variant; Wild Type Mouse; Yeast Model System; Yeasts; cytotoxicity; experience; gene discovery; high throughput screening; human disease; in vivo; innovation; killings; liposarcoma; member; motor neuron degeneration; mouse model; mutant; physical property; programs; promoter; protein expression; research study; sarcoma; small molecule; stable cell line; superoxide dismutase 1; therapy development

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is an untreatable, paralytic neurodegenerative disorder that is uniformly lethal, usually within 3-5 years. Members of our team have identified mutations in two genes whose mutations cause dominantly inherited familial ALS (fALS): Cu,Zn superoxide dismutase-1 (SOD1), the first identified ALS gene, and FUS/TLS. Respectively, these account for 20% and 5% of fALS cases. Presently, it is not clear how mutant SOD1 or mutant FUS cause fALS; it is likely that the former involves instability of the mutant SOD1 protein and aberrant protein processing, while the latter implicates perturbations of RNA function, as indicated in part by the observation that mutant FUS is often mislocalized to the cytosol. The goal of this proposed project is to discover small molecules that inhibit the cytotoxicity of mutant forms of SOD1 and FUS/TLS. Our hypotheses are that (1) factors that re-direct FUS from the cytoplasm to the nucleus will attenuate FUS- mediated toxicity in ALS patients; and (2) a reduction in the load of toxic mutant-SOD1 proteins will have a therapeutic effect in ALS patients. We have substantial data documenting that our screening and validation assays are functional and thus are confident that our high-throughput screening has the potential to identify small molecules and genetic factors that ameliorate toxicity of mutant forms of both FUS and SOD1. Our study has four aims. Aim 1 is to conduct high throughput screening for compounds that mitigate toxicity of mutant FUS and SOD1 using a yeast model of FUS toxicity (Aim 1A), an HEK293 cell model of FUS mislocalization (Aim 1B) and an HEK model of quantifiable SOD1 expression (Aim 1C). The deliverable from Aim 1 is at least five compounds with efficacy at levels <5 ¿M and toxicity at levels >25 ¿M. Aim 2 is to validate hits from Aim 1, using Drosophila and primary neuronal models of FUS toxicity (Aim 2A) and human cell lines for analysis of SOD1 gene expression. Aim 3 is to optimize the lead compound series and establish structure-activity relationships. The deliverable from Aims 2 and 3 are three compounds from each of the two screening platforms (six total) with efficacy a <1 ¿M and LD50 >50 ¿M that will then be tested in FUS and ALS transgenic mice. Aim 4 is to conduct trials of the six best validated compounds in the transgenic FUS and SOD1G93A mice (three compounds in each). These studies will address two questions. First, do the compound hits achieve anticipated target effects in vivo (for FUS: reduction of FUS-mediated pathology; for SOD1: reduction of SOD1 mRNA and protein levels) (Aim 4A)? And, do the hits ameliorate motor or behavioral abnormalities or prolong survival? (Aim 4B). In our view, this program entails a high degree of innovation both in the assays employed and in the pilot set of compounds discovered so far. We also believe that these investigations hold considerable significance; the need for any meaningful therapeutic intervention in ALS is compelling. Moreover, it is conceivable that the compounds discovered in these studies will prove beneficial in neurodegenerative conditions other than ALS alone. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS) is an untreatable disease that kills motor nerves (which control movement) and causes death in humans within 3-5 years. Our proposed project will focus on two genetic causes of ALS: mutations (or gene defects) in both Cu,Zn superoxide dismutase-1 (SOD1) and Fused in sarcoma/Translocated in liposarcoma (FUS/TLS or FUS). Mutations in these genes account for 25 30% of inherited ALS cases. While we do not fully understand how mutations in SOD1 or FUS cause ALS, we know that reducing the amount of mutant-SOD1 in motor neurons protects these cells from dying, and that the location of FUS in the cell correlates with its toxicity. Normally FUS is concentrated in the nucleus. However mutated FUS is concentrated in the cytoplasm, which surrounds the nucleus. We propose high-through put screens for drug-like molecules that can reduce levels of SOD1 and reduce the toxicity of FUS, possibly by restoring FUS to the nucleus. These drug-like molecules may therefore lead to new treatments for ALS patients.

描述（由申请人提供）：肌萎缩侧索硬化症 (ALS) 是一种无法治疗的麻痹性神经退行性疾病，通常在 3-5 年内致命。我们团队的成员已经确定了两个基因的突变，这两个基因的突变导致显性遗传家族性 ALS (fALS)：Cu,Zn 超氧化物歧化酶-1 (SOD1)（第一个确定的 ALS 基因）和 FUS/TLS。这些分别占 fALS 病例的 20% 和 5%。目前尚不清楚突变型SOD1或突变型FUS如何引起fALS；前者很可能涉及突变型 SOD1 蛋白的不稳定性和异常的蛋白加工，而后者则涉及 RNA 功能的扰动，这一点通过观察突变型 FUS 经常错误定位到细胞质中就可以看出。该项目的目标是发现抑制 SOD1 和 FUS/TLS 突变体细胞毒性的小分子。我们的假设是：(1) 将 FUS 从细胞质重新引导至细胞核的因素将减轻 ALS 患者中 FUS 介导的毒性； (2) 有毒突变体 SOD1 蛋白负载的减少将产生 ALS 患者的治疗效果。我们有大量数据证明我们的筛选和验证测定是有效的，因此我们相信我们的高通量筛选有潜力识别出改善 FUS 和 SOD1 突变形式毒性的小分子和遗传因素。我们的研究有四个目标。目标 1 是使用 FUS 毒性酵母模型（目标 1A）、FUS 错误定位 HEK293 细胞模型（目标 1B）和可量化 SOD1 表达的 HEK 模型（目标 1C）对减轻突变 FUS 和 SOD1 毒性的化合物进行高通量筛选。目标 1 的可交付成果是至少五种化合物，其功效水平 <5 µM，毒性水平 >25 µM。目标 2 是使用果蝇和 FUS 毒性的初级神经元模型 (目标 2A) 以及用于分析 SOD1 基因表达的人类细胞系来验证目标 1 的命中。目标 3 是优化先导化合物系列并建立构效关系。目标 2 和 3 的成果是来自两个筛选平台（总共六种）的三种化合物，其功效 <1 µM 且 LD50 >50 µM，然后将在 FUS 和 ALS 转基因小鼠中进行测试。目标 4 是在转基因 FUS 和 SOD1G93A 小鼠中对六种经过验证的最佳化合物进行试验（每种化合物三种）。这些研究将解决两个问题。首先，化合物命中是否在体内达到了预期的目标效果（对于 FUS：减少 FUS 介导的病理；对于 SOD1：减少 SOD1 mRNA 和蛋白质水平）（目标 4A）？而且，这些打击是否会改善运动或行为异常或延长生存期？ （目标 4B）。我们认为，该计划在所采用的检测方法和迄今为止发现的试点化合物方面都需要高度创新。我们也相信这些调查具有相当大的意义； ALS 迫切需要任何有意义的治疗干预。此外，可以想象的是，这些研究中发现的化合物将被证明对除 ALS 之外的神经退行性疾病有益。 公众健康相关性：肌萎缩侧索硬化症 (ALS) 是一种无法治愈的疾病，会杀死运动神经（控制运动）并在 3-5 年内导致人类死亡。我们提出的项目将重点关注 ALS 的两个遗传原因：Cu,Zn 超氧化物歧化酶-1 (SOD1) 和肉瘤融合/脂肪肉瘤易位（FUS/TLS 或 FUS）的突变（或基因缺陷）。这些基因突变占遗传性 ALS 病例的 25-30%。虽然我们不完全了解 SOD1 或 FUS 突变如何导致 ALS，但我们知道减少运动神经元中突变型 SOD1 的数量可以保护这些细胞免于死亡，并且 FUS 在细胞中的位置与其毒性相关。通常 FUS 集中在细胞核中。然而，突变的 FUS 集中在细胞核周围的细胞质中。我们建议对药物样分子进行高通量筛选，这些分子可以降低 SOD1 水平并降低 FUS 的毒性，可能是通过将 FUS 恢复到细胞核中来实现的。因此，这些类药物分子可能会为 ALS 患者带来新的治疗方法。