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

描述（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年内致死。我们团队的成员已经确定了两个基因的突变，其突变导致显性遗传性家族性ALS（fALS）：Cu，Zn超氧化物歧化酶-1（SOD 1），第一个确定的ALS基因，和FUS/TLS。分别占fALS病例的20%和5%。目前，还不清楚突变体SOD 1或突变体FUS如何引起fALS;很可能前者涉及突变体SOD 1蛋白的不稳定性和异常蛋白质加工，而后者涉及RNA功能的扰动，如突变体FUS经常错误定位于细胞质的观察所示。该项目的目标是发现抑制SOD 1和FUS/TLS突变形式细胞毒性的小分子。我们的假设是：（1）将FUS从细胞质重定向到细胞核的因素将减弱ALS患者中FUS介导的毒性;（2）毒性MUSC-SOD 1蛋白负荷的减少将使ALS患者中FUS介导的毒性降低。 ALS患者的治疗效果。我们有大量的数据证明我们的筛选和验证试验是有效的，因此我们相信我们的高通量筛选有可能鉴定出改善FUS和SOD 1突变形式毒性的小分子和遗传因子。我们的研究有四个目标。目的1是使用FUS毒性的酵母模型（Aim 1A）、FUS错误定位的HEK 293细胞模型（Aim 1B）和可定量的SOD 1表达的HEK模型（Aim 1C）对减轻突变FUS和SOD 1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，其效力水平<5 μ M，毒性水平>25 μ M。目的2是使用果蝇和FUS毒性的原代神经元模型（目的2A）和人细胞系分析SOD 1基因表达，验证目的1的命中。目的3：优化先导化合物系列，建立构效关系。目标2和3的可交付物是来自两个筛选平台（总共六个）中的每一个的三种化合物，其功效<1 μ M且LD 50>50 μ M，然后将在FUS和ALS转基因小鼠中进行测试。目的4是在转基因FUS和SOD 1G 93 A小鼠中进行六种最佳验证化合物的试验（每种三种化合物）。这些研究将解决两个问题。首先，化合物命中是否在体内达到预期的靶效应（对于FUS：减少FUS介导的病理学;对于SOD 1：降低SOD 1 mRNA和蛋白质水平）（目的4A）？而且，这些打击是否改善了运动或行为异常或延长了生存期？(Aim 4B）。在我们看来，这一计划需要高度的创新，无论是在所采用的测定和迄今发现的化合物的试点集。我们还认为，这些研究具有相当大的意义;对ALS进行任何有意义的治疗干预的必要性是迫切的。此外，可以想象的是，在这些研究中发现的化合物将证明在除了单独的ALS之外的神经退行性疾病中有益。 公共卫生相关性：肌萎缩侧索硬化症（ALS）是一种无法治愈的疾病，它会杀死运动神经（控制运动），并在3-5年内导致人类死亡。我们提出的项目将集中在ALS的两个遗传原因：突变（或基因缺陷）在铜，锌超氧化物歧化酶-1（SOD 1）和融合在肉瘤/易位在脂肪肉瘤（FUS/TLS或FUS）。这些基因的突变占遗传性ALS病例的25 - 30%。虽然我们还不完全了解SOD 1或FUS的突变是如何导致ALS的，但我们知道减少运动神经元中的MUD-SOD 1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集中在细胞质中，其围绕细胞核。我们提出了高通量筛选药物样分子，可以降低SOD 1的水平，降低FUS的毒性，可能是通过将FUS恢复到细胞核。因此，这些药物样分子可能为ALS患者带来新的治疗方法。

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.