High-content screening for TPI Deficiency therapeutics

摘要/项目摘要： TPI Df 是一种毁灭性的、无法治愈的儿童代谢疾病，会导致贫血， 瘫痪、不可逆转的脑损伤和过早死亡。多种细微氨基酸 磷酸三糖异构酶 (TPI) 中的取代具有致病性，并会迅速导致 进行性多系统疾病。重要的是，所有已知的致病性 TPI Df 突变都会导致 已知保留功能和发病机制的蛋白质是由于增加的 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 开发了基于“常见”E104D 细胞模型的人类细胞 TPI Df 测定 突变并将其用于高内涵、高通量成像。我们已经用过这个 在试点筛选中建立模型，并验证了其在识别调节新化合物方面的实用性 人类细胞中突变的 TPI 蛋白水平。我们开发了完整 HTS 的检测方法 标准，并建议筛选 225,000 名 NIH MLSMR 化合物库成员，以 识别命中先导化合物以开发为 TPI Df 小分子疗法。我们会 验证 TPI Df 患者细胞中 TPI 稳定性和活性的二次测定中的命中，确定优先级 对它们进行一组体外毒理学和代谢测定，检查结构活性 先导化合物的关系（SAR）并使用以下方法在体内对其进行实质性验证 建立了反映整个 TPI Df 疾病范围的果蝇和小鼠模型 严重程度。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（包括新验证的小鼠模型）中的功效。

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.