High-content screening for TPI Deficiency therapeutics

摘要/项目摘要： TPI Df是一种毁灭性的不可治疗的儿童代谢性疾病，可导致贫血， 瘫痪、不可逆转的脑损伤和过早死亡。多种微量氨基酸 磷酸丙糖异构酶（TPI）中的取代是致病性的， 进行性多系统疾病重要的是，所有已知的致病性TPI Df突变导致 在一个蛋白质，保留功能和发病机制是已知的结果，从增加 通过蛋白质质量控制途径（PQC）进行功能蛋白质的周转。我们有 开发了一种基于“常见”E104 D细胞模型的人细胞TPI Df试验 突变并将其用于高内容，高通量成像。我们已经用这种 模型中的中试筛选，并验证其效用，以确定新的化合物，调节 人细胞中突变TPI蛋白水平。我们已将检测方法开发至全HTS 标准，并建议筛选225，000名成员的NIH MLSMR化合物库， 鉴定靶向先导化合物，开发成TPI Df小分子疗法。我们将 在TPI Df患者细胞中验证TPI稳定性和活性的二级试验中的命中， 他们在一个小组的体外毒理学和代谢测定，检查结构活性 研究了先导化合物的SAR关系，并使用 建立了反映TPI Df疾病整个范围的果蝇和小鼠模型 严重性总的来说，该项目将发现用于开发和测试的初始疗法。 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.