High-content screening for TPI Deficiency therapeutics

摘要/项目摘要： TPI DF是一种毁灭性的无法治疗的儿童代谢性疾病，会导致贫血， 瘫痪，不可逆转的脑损伤和过早死亡。众多微妙的氨基酸 磷酸丙糖异构酶(TPI)中的替换是致病的，并迅速导致 进展中的多系统疾病。重要的是，所有已知的致病TPI DF突变都会导致 在一种保留功能和致病机制的蛋白质中，已知是由于 通过蛋白质质量控制途径(PQC)实现功能蛋白质的周转。我们有 基于“常见”E104D的细胞模型建立了一种人细胞TPI DF检测方法 突变，并将其用于高内容、高通量成像。我们用过这个 在试点屏幕上建立模型，并验证其有效性，以识别调节 突变的TPI蛋白在人类细胞中的水平。我们已经开发出了完全HTS的化验方法 标准，并建议筛选225,000个成员的NIH MLSMR化合物文库以 识别Hit-to-Lead化合物，以开发成TPI-DF小分子疗法。我们会 在二次检测中验证TPI稳定性和TPI DF患者细胞中的活动，确定优先顺序 在体外毒理学和新陈代谢测试中，检查结构活性 先导化合物的相互关系(SARS)，并在体内基本上验证它们使用 建立了反映整个TPI DF疾病范围的果蝇和小鼠模型 严肃性。总体而言，该项目将发现用于开发和测试的初步疗法 我们的先导化合物在TPI DF模型中的有效性，包括新验证的小鼠模型。

Abstract/Project summary: TPI Df is a devastating untreatable childhood metabolic disease resulting in anemia, paralysis, irreversible brain damage and premature death. Numerous subtle amino acid substitutions in Triosephosphate Isomerase (TPI) are pathogenic and result in rapidly progressing multisystem disease. Importantly, all known pathogenic TPI Df mutations result in a protein that retains function and pathogenesis is known to result from increased turnover of the functioning protein by Protein Quality Control pathways (PQC). We have developed a human cellular TPI Df assay based on a cellular model of the “common” E104D mutation and implemented it for high-content, high-throughput imaging. We have used this model in a pilot screen and validated its utility to identify novel compounds that modulate mutant TPI protein levels in human cells. We have developed the assay to full HTS standards, and propose to screen the 225,000 member NIH MLSMR compound library to identify hit-to-lead compounds to develop into TPI Df small molecule therapies. We will validate hits in secondary assays for TPI stability and activity in TPI Df patient cells, prioritize them in a panel of in vitro toxicology and metabolism assays, examine structure activity relationships (SARs) of the lead compounds and substantially validate them in vivo using established Drosophila and mouse models that reflect the entire range of TPI Df disease severities. Overall, this project will discover initial therapies for development and test efficacy of our lead compounds in TPI Df models, including a newly validated mouse model.