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（由适用提供）：肌萎缩性侧索硬化症（ALS）是一种不可治疗的麻痹神经退行性疾病，通常在3 - 5年内均匀致命。我们团队的成员已经确定了两个基因的突变，它们的突变引起了主要遗传的家族ALS（FALS）：CU，Zn超氧化物歧化酶1（SOD1），第一个鉴定出的ALS基因和FUS/TLS。这些分别占伪造病例的20％和5％。目前，尚不清楚突变的SOD1或突变型FUS如何引起伪造。前者很可能涉及突变体SOD1蛋白和异常蛋白质加工的不稳定性，而后者则实现RNA功能的扰动，这在某种程度上表明了突变型FUS通常将突变型FUS定位在细胞质上。该提出的项目的目的是发现抑制SOD1和FUS/TLS突变形式的细胞毒性的小分子。我们的假设是（1）将FUS从细胞质重新引导到细胞核的因素会衰减ALS患者的FUS介导的毒性； （2）减少有毒突变体-SOD1蛋白的负载将具有 ALS患者的治疗作用。我们有大量的数据文档，即我们的筛选和验证测定具有功能性，因此有信心我们的高通量筛选有可能鉴定小分子和遗传因素，从而改善FUS和SOD1突变形式的毒性。我们的研究有四个目标。 AIM 1是使用FUS毒性的酵母模型（AIM 1A），FUS错误定位的HEK293细胞模型（AIM 1B）和可量化SOD1表达的HEK模型（AIM 1C）进行高吞吐量筛选，以减轻突变型FUS和SOD1的毒性（AIM 1A），HEK293 HEK293细胞模型（AIM 1C）。 AIM 1的可传递是至少五种化合物，其水平<5？m的效率和> 25»M的毒性。AIM2是使用果蝇和FUS毒性的原发性神经元模型（AIM 2A）和人类细胞系和人类细胞系进行SOD1基因表达的分析，从而验证AIM 1的命中。目标3是优化铅复合系列并建立结构活性关系。 AIMS 2和3的可传递是来自两个筛选平台中的每个化合物（总计六个），效率A <1？m和LD50> 50»M，然后将在FUS和ALS转基因小鼠中进行测试。 AIM 4是进行转基因FUS和SOD1G93A小鼠中六种最佳验证化合物的试验（每种化合物中的三种化合物）。这些研究将解决两个问题。首先，化合物命中是否在体内实现了预期的靶标作用（用于FUS：降低FUS介导的病理学；对于SOD1：SOD1 mRNA和蛋白质水平的降低）（AIM 4A）？而且，命中是否可以改善运动或行为异常或延长生存期？ （AIM 4B）。我们认为，该计划在迄今为止发现的主张和试点组合中都需要高度的创新。我们还认为，这些投资具有重要意义；对ALS的任何有意义的治疗干预的需求令人信服。此外，可以想象，这些研究中发现的化合物将在ALS以外的其他神经退行性疾病中被证明是有益的。 公共卫生相关性：肌萎缩性侧索硬化症（ALS）是一种无法治疗的疾病，可杀死运动神经（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介绍ALS的两个遗传原因：Cu，Cu，Zn超氧化物歧化酶1（SOD1）中的突变（或基因缺陷），并在肉瘤/liposarcoma（fus/tls或fus）中融合。这些基因中的突变占遗传ALS病例的2530％。虽然我们不完全了解SOD1或FUS中的突变是如何引起ALS的，但我们知道，减少运动神经元中突变体-SOD1的量会保护这些细胞垂死，并且FUS在细胞中的位置与其毒性相关。通常，FUS集中在核中。然而，突变的Fus集中在细胞质中，周围核周围。我们建议通过恢复FUS对核us的FUS来降低SOD1的水平并降低FUS的毒性，以降低SOD1的水平并降低FUS的毒性。因此，这些药物样分子可能会导致ALS患者的新治疗方法。