A novel cell-based high-throughput assay for ALS

描述（由申请人提供）：超氧化物歧化酶-1 （SOD1）突变导致家族性肌萎缩侧索硬化症（ALS）。嵌合或条件突变小鼠的研究表明，非神经元细胞在als相关的神经变性中起重要作用。我们发现，用突变SOD1星形胶质细胞培养的培养基在体外杀死野生型胚胎干细胞衍生的运动神经元（ES-MNs）。这项为期2年的临床研究旨在鉴定用于治疗ALS的神经保护剂。我们已经成功地实现了ALS ES-MNs细胞模型的小型化，并获得了所有必要的专业知识、设备和化学文库，从而开展了高通量筛选（HTS）工作。我们将首先完成我们的分析的优化和验证。然后，我们将分四个步骤执行HTS。首先，为了微调检测参数，我们将筛选2000种生物活性化合物，以确定它们在SOD1星形细胞条件培养基中提高ES-MN存活的能力。其次，在单一浓度（10 5M）和单一参数（ES-MN存活）下，逐一筛选约80,000种化合物。第三，命中将通过以下方式进行验证：(a)在多个重复中重复测试；(b)再合成/回购后的复验；(c)在初始筛选中使用的浓度范围内绘制剂量-反应曲线。第四，我们收集的与结构相关的化合物将与验证的命中同时进行测试，以确认每一类化合物的生物活性，并选择最有效的化合物。铅将被定义为具有可重复的神经保护活性的化合物，效力为EC50 d 55m，最大效果为阳性对照的75%。与本项目公告的意图一致，我们的提案提供了一套研究，旨在开发一种用于治疗筛选的检测方法。我们预计，到这个项目结束时，我们的整个小分子库将被测试，大约100个命中将被选中，直接进入ALS的治疗开发项目。后者将包括基于体外二次筛选的临床前研究，一组体内毒性和药代动力学研究，以及在表达突变SOD1的转基因小鼠中测试主要候选药物。

DESCRIPTION (provided by applicant): Mutations in superoxide dismutase-1 (SOD1) cause familial amyotrophic lateral sclerosis (ALS). Studies in chimeric or conditional mutant mice indicate that non- neuronal cells play an important role in ALS-related neurodegeneration. We have found that culture medium conditioned with mutant SOD1 astrocytes kills wild- type embryonic stem cell-derived motor neurons (ES-MNs) in vitro. This 2-year translational R21 aims at identifying neuroprotective agents for the treatment of ALS. We have succeeded in miniaturizing our ES-MNs cell model of ALS and have secured all of the necessary expertise, equipment, and chemical libraries to embark on a high-throughput screening (HTS) effort. We will begin by completing the optimization and validation of our assay. We will then perform our HTS in four steps. First, to fine-tune the assay parameters, we will screen a small collection of 2,000 biologically active compounds for their ability to enhance ES-MN survival in the presence of SOD1 astrocyte-conditioned medium. Second, a collection of ~80,000 compounds will then be screened one-by-one at a single concentration (10 5M) and with one parameter (ES-MN survival). Third, hits will be validated by: (a) repeat testing in multiple replicates; (b) retesting after resynthesis/repurchase; and (c) performing dose-response curves spanning the concentrations used in the initial screening. Fourth, structurally-related compounds from our collection will be tested in parallel with validated hits to confirm the bioactivity of each class of compounds, and to select the most potent. Leads will be defined as compounds with a reproducible neuroprotective activity, a potency of EC50 d 5 5M and a maximum effect e 75% of that of the positive control. In agreement with the intent of this program announcement, our proposal offers a set of investigations geared toward developing an assay for the screening of therapeutics. We anticipate that, by the end of this project, our entire library of small molecules will have been tested and that ~100 hits will have been selected, leading directly into a therapy development project for ALS. The latter will consist of preclinical studies based on in vitro secondary screens, a set of in vivo toxicity and pharmacokinetic investigations, and testing of the lead candidates in transgenic mice expressing mutant SOD1. Public Health Relevance: Amyotrophic lateral sclerosis (ALS) is a common fatal paralytic disorder affecting adults with no effective treatment. We have recently identified and validated a means of reproducing ALS in a laboratory dish - a breakthrough which has led to the development of functional cell models of this disease. By using these new cell models in conjunction with high- throughput technologies, we will screen large numbers of small molecules in a brief period of time with the ultimate goal of discovering compounds with neuroprotective activities against ALS.