High-content screening for TPI Deficiency therapeutics

TPI 缺乏疗法的高内涵筛选

基本信息

批准号：
10662471
负责人：
Michael John Palladino
金额：
$ 54.33万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10662471
关键词：
Acute Amino Acid Substitution Anemia Automation Biochemical Biological Assay Brain Injuries Cell Culture Techniques Cell Line Cell model Cells Cellular Assay Cessation of life Chemicals Childhood Chronic Clinical Clinical Trials Collection Data Defect Deficiency Diseases Development Diagnosis Disease Dose Drosophila genus Enzymes Equipment Exhibits Flow Cytometry Foundations Functional disorder Genetic Genetic Models Genetic Screening Heat-Shock Proteins 70 Heat-Shock Proteins 90 Heterozygote Human Image Impairment In Vitro Inpatients Intervention Isomerase Laboratories Lead Libraries Library Collection Development Longevity Mammals Mediating Metabolic Diseases Metabolism Methodology Missense Mutation Modeling Motor Mus Mutation Neurologic Optics Paralysed Pathogenesis Pathogenicity Pathway interactions Patients Pharmacotherapy Property Proteasome Inhibitor Proteins Quality Control Severity of illness Structure-Activity Relationship System Testing Therapeutic Toxic effect Toxicology Triose-Phosphate Isomerase Ubiquitin United States National Institutes of Health Universities Validation Work combinatorial chemistry early childhood efficacy testing fly genome wide screen in vivo in vivo evaluation member mouse model multicatalytic endopeptidase complex mutant neuromuscular novel pharmacologic premature protein degradation protein function reduce symptoms response screening small molecule therapeutic development therapeutic target therapy development

项目摘要

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.

摘要/项目摘要： TPI DF是一种毁灭性的不可治疗的儿童代谢疾病，导致贫血，麻痹，不可逆的脑损伤和过早死亡。许多微妙的氨基酸三氧磷酸异构酶（TPI）中的取代是致病性的，导致迅速进展的多系统疾病。重要的是，所有已知的致病性TPI DF突变结果在保留功能和发病机理的蛋白质中，已知是由于增加而导致的通过蛋白质质量控制途径（PQC）对功能蛋白的营业额。我们有基于“常见” E104D的细胞模型开发了人类细胞TPI DF分析突变并实现用于高素质，高通量成像。我们已经使用了试点屏幕中的模型，并验证了其效用以识别调节的新型化合物人类细胞中突变的TPI蛋白水平。我们已经开发了完整HTS的测定法标准和建议将225,000名成员NIH MLSMR复合库筛选为鉴定命中率对铅化合物，以发展为TPI DF小分子疗法。我们将在TPI DF患者细胞中验证TPI稳定性和活性的次要测定中的点击率优先考虑它们在体外毒理学和代谢测定中，检查结构活动铅化合物的关系（SARS），并使用体内实质上验证它们建立的果蝇和小鼠模型，反映了TPI DF疾病的整个范围严重性。总体而言，该项目将发现开发和测试的初始疗法我们的铅化合物在TPI DF模型中的功效，包括新验证的鼠标模型。