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、瘫痪的神经退行性疾病，通常在3-5年内致命。我们团队的成员已经发现了两个基因的突变，它们的突变主要导致遗传性家族性ALS(FALS)：铜，锌超氧化物歧化酶-1(SOD1)，第一个被发现的ALS基因，以及FUS/TLS。这两类个案分别占法援个案的20%和5%。目前，突变的SOD1或突变的FUS是如何导致FALS的尚不清楚；前者可能涉及突变的SOD1蛋白的不稳定和异常的蛋白质加工，而后者涉及RNA功能的扰动，部分原因是观察到突变的FUS经常错误地定位于细胞质。这项拟议项目的目标是发现能够抑制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从胞浆重定向到细胞核的因子将减弱FUS介导的ALS患者的毒性；(2)有毒突变的SOD1蛋白负荷的减少将有 肌萎缩侧索硬化症患者的疗效。我们有大量数据证明，我们的筛选和验证分析是有效的，因此我们相信，我们的高通量筛选有潜力识别小分子和遗传因素，从而改善FUS和SOD1突变形式的毒性。我们的研究有四个目标。目标1是使用FUS毒性的酵母模型(Aim 1A)、FUS错位的HEK293细胞模型(Aim 1B)和可量化SOD1表达的HEK模型(Aim 1C)，高通量筛选减轻突变的FUS和SOD1毒性的化合物。目标1的释放物是至少五种化合物，其效力水平为5？M，毒性水平为25？M。目标2是验证目标1的HITS，使用果蝇和FUS毒性的初级神经元模型(目标2A)和用于分析SOD1基因表达的人类细胞系。目标3是优化先导化合物系列，建立构效关系。AIMS 2和AIMS 3的释放物分别来自两个筛选平台(总共六个)中的三个化合物，其有效性分别为1？M和LD50？M，然后将在FUS和ALS转基因小鼠身上进行测试。目的4是在转基因FUS和SOD1G93A小鼠身上进行6种最有效的化合物的试验(每种化合物3种)。这些研究将解决两个问题。首先，化合物HITS在体内是否达到预期的靶效应(对于FUS：减少FUS介导的病理；对于SOD1：降低SOD1mRNA和蛋白水平)(目标4A)？而且，这些运动是否改善了运动或行为异常，或者延长了生存时间？(目标4B)。在我们看来，这一计划需要在使用的分析和到目前为止发现的化合物试验集方面进行高度创新。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，在这些研究中发现的化合物将被证明对神经退行性疾病有益，而不仅仅是ALS。

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.