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.
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.
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