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A Scalable Neuron-Based High-Throughput Screening Platform for the Discovery of Compounds that Restore Protein Expression Caused by Genetic Haploinsufficiency

一种可扩展的基于神经元的高通量筛选平台，用于发现可恢复由遗传单倍体不足引起的蛋白质表达的化合物

基本信息

批准号：
9370360
负责人：
GAVIN R RUMBAUGH
金额：
$ 68.73万
依托单位：
SCRIPPS FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-01 至 2020-03-31
项目状态：
已结题

项目摘要

PROJECT SUMMARY Drug discovery pipelines for neuropsychiatric disorders are dry. One approach to rejuvenating these pipelines would be to create assays based on relevant disease phenotypes in primary neurons, something that is currently lacking. However, a scalable assay development platform that is based on bona fide neurons, remains cost effective, and that can support industrial level HTS does not currently exist. Over the past five years, our collaborative group has created a flexible and scalable primary neuron assay development system that is compatible with industrial-level HTS. Here, our goal is to optimize these procedures and workflows to determine the limit of scalability of neuron-based HTS phenotypic assays so that they can easily support very large campaigns of >200K compounds. A substantial proportion of childhood brain disorders are caused by single autosomal dominant variants resulting in genetic haploinsufficiency. The rare genetic brain disorders that arise from these variants offer the greatest potential for discovery of robust therapeutics because the disease mechanism is often straight forward (i.e. low protein expression). Therefore, a rationale strategy to improve conditions in these patients would be to treat them with “magic bullet” compounds that raise expression of functional proteins from the remaining undamaged allele (e.g. “boosting compounds”). De novo nonsense variants that cause SYNGAP1 haploinsufficiency lead to a genetically-defined form of intellectual disability with autism and epilepsy (MRD5;; OMIM#603384) that may explain up to 1-2% of all ID cases. The accepted cause of this disorder is low functional protein expression in neurons caused most often by truncating SYNGAP1 nonsense variants. As a means to refine the neuron-based HTS system, and to advance treatment for ASD-related disorders, we are seeking to scale-up and implement an assay for SynGAP expression that is compatible with industrial-level robotics. In the first Aim, we will optimize an HTS-compatible and disease-relevant SynGAP expression assay. This assay is based on mouse primary neurons where tdTomato fluorescence reflects steady-state endogenous SynGAP protein levels. In the second aim, we will miniaturize the SynGAP expression assay to the 1536-well format. This miniaturization process would enable an HTS-scale screen of this, or any other related neuron-based phenotypic assay, of up to 400,000 culture wells using a standard screening budget. Finally, we will implement the SynGAP expression assay in a true uHTS environment and then validate lead compounds that emerge from a 20K compound pilot screen, including a 10K compound repurposing screen of known “safe in human” compounds. The impact of this project that we expect to develop procedures that will increase the scale of HTS campaigns in neurons by 10-fold or more relative to the current state-of-the-art in academic screening centers. We also expect to validate at least one lead compound that boosts SynGAP expression, hopefully from the repurposing library.

项目总结