Drug Therapies Identified through Modeling AT Deficiency in C Elegans

通过线虫 AT 缺陷建模确定药物治疗

• 批准号: 10630352
• 负责人: GARY ARTHUR SILVERMAN
• 金额: $ 39.71万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-24 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10630352
• 关键词: Affect; Affinity Chromatography; Animal Model; Animals; Autophagocytosis; Binding; Biochemical Pathway; Biological Assay; CRISPR/Cas technology; Caenorhabditis elegans; Cell Line; Cells; Chemicals; Childhood; Chimeric Proteins; Computer Assisted; Defect; Disease; Disease Outcome; Double-Stranded RNA; Drug Combinations; Drug Targeting; Endoplasmic Reticulum; Ethylnitrosourea; Fibrosis; Gene Modified; Genes; Genetic; Genetic Screening; Goals; Green Fluorescent Proteins; Growth; Hepatocyte; Hepatotoxicity; Homologous Gene; Homozygote; Human; Human Cell Line; Human Genetics; Incidence; Individual; Injury; Integral Membrane Protein; Laboratories; Libraries; Live Birth; Liver; Liver Cirrhosis; Liver Fibrosis; Liver diseases; Longevity; Mammalian Cell; Methods; Minor; Missense Mutation; Modeling; Modification; Molecular; Molecular Genetics; Mouse Cell Line; Mus; Mutagenesis; Mutation; Nematoda; Organism; Other Genetics; Pathway interactions; Patients; Personal Satisfaction; Pharmaceutical Preparations; Pharmacotherapy; Phenocopy; Phenotype; Polymers; Predisposition; Preparation; Primary carcinoma of the liver cells; Protein Secretion; Proteins; RNA Interference; RNA interference screen; Recommendation; Reproducibility; Scanning; Severities; System; Technology; Testing; Therapeutic; Tissues; Toxic effect; Transgenic Animals; Transgenic Mice; Transgenic Organisms; Trypsin; Variant; alpha 1-Antitrypsin Deficiency; disease phenotype; drug discovery; effective therapy; exome sequencing; gain of function; gene replacement; genetic variant; genome sequencing; genome-wide; high-throughput drug screening; in silico; insulin signaling; knock-down; liver development; liver injury; liver transplantation; loss of function; misfolded protein; molecular modeling; mouse model; mutant; novel; novel therapeutics; pre-clinical; preclinical development; protective pathway; protein misfolding; proteostasis; screening; small molecule; trafficking; validation studies; variant of unknown significance; whole genome

PROJECT SUMMARY α1-antitrypsin (AT) deficiency (ATD) is the most common genetic cause of liver disease. The classical form of ATD is due to a single missense mutation (Z) that causes the mutant protein (ATZ) to misfold and accumulate within the endoplasmic reticulum (ER) of liver cells as toxic oligomers, polymers or aggregates. However, due to genetic and environmental modifiers, there is marked variation in the incidence and severity of liver disease among homozygotes. Since the only treatment for severe ATZ-induced hepatic injury is liver transplantation, we developed an animal model amenable to pre-clinical, high-throughput drug screening technologies that greatly assists in the discovery of new compounds that reduce or eliminate ATZ-induced hepatotoxicity. The value of the model would be markedly increased because it also possesses genetic tractability to: 1) elucidate the genetic modifiers of both tissue damage and the endogenous proteostasis pathways that protect against protein misfolding-induced injury, and 2) pinpoint which biochemical pathways and/or molecules are targeted by newly discovered compounds. We show that ATZ-induced liver disease is modeled faithfully in the nematode, C. elegans. Transgenic animals expressing wild-type human AT secreted the protein. In contrast, animals expressing ATZ faithfully recapitulated the ER-trafficking defect of ATZ by demonstrating intracellular inclusions (dilated ER cisterna), and becoming unhealthy as shown by slow growth, small brood sizes and decreased longevity. Using this model we developed an automated, live-animal, high-content screening (HCS) assay that rivals that of any cell-based system. So far we have identified ~30 hit compounds, including several that reduced ATZ accumulation by enhancing autophagy, a known pathway of ATZ elimination. Using a modification of our HCS strategy, we also developed a semi-automated technology that reduces the labor intensiveness of genome-wide RNAi screens. We identified several potential genetic modifiers/pathways of ATZ accumulation. Taken together, these studies demonstrated that this transgenic C. elegans model is a powerful platform to initiate the discovery of both novel drugs and genes that modify ATZ hepatotoxicity. The aims of Project 2 are to discover additional hit compounds for the treatment of ATZ-induced disease phenotypes in C. elegans by both HCS and computer-aided molecular modeling, identify disease modifiers of major and minor effect, and to determine whether different mutant disease modifiers alter responsiveness to therapeutic compounds.

项目摘要 α1-抗胰蛋白酶（AT）缺乏症（ATD）是肝脏疾病最常见的遗传原因。古典形式的 ATD是由于单个错义突变（Z）导致突变蛋白（ATZ）错误折叠和积累 在肝细胞的内质网（ER）内作为毒性低聚物、聚合物或聚集体。但由于 由于遗传和环境因素的影响，肝病的发病率和严重程度存在显著差异。 在纯合子中。由于严重ATZ诱导的肝损伤的唯一治疗方法是肝移植， 我们开发了一种适合临床前高通量药物筛选技术的动物模型， 大大有助于发现减少或消除ATZ诱导的肝毒性的新化合物。的 该模型的价值将显着增加，因为它也具有遗传易处理性：1）阐明 组织损伤和内源性蛋白酶抑制途径的遗传修饰剂， 蛋白质错误折叠诱导的损伤，以及2）查明靶向哪些生化途径和/或分子 新发现的化合物。我们表明，ATZ诱导的肝脏疾病是忠实地模仿在 nematode，C.优美的表达野生型人AT的转基因动物分泌该蛋白。与此相反， 表达ATZ的动物如实地再现了ATZ的ER运输缺陷， 内含物（扩张的ER池），并变得不健康，表现为生长缓慢，育雏量小， 寿命缩短。使用这个模型，我们开发了一个自动化，活动物，高内容筛选（HCS） 与任何基于细胞的系统相媲美的分析。到目前为止，我们已经确定了约30种命中化合物，包括几种 这通过增强自噬（一种已知的ATZ消除途径）来减少ATZ积累。使用 为了改进我们的HCS战略，我们还开发了一种半自动化技术， 全基因组RNAi筛选的强度。我们确定了几种潜在的遗传修饰剂/途径， ATZ累积。综上所述，这些研究表明，这种转基因C。elegans模型是一个 这是一个强大的平台，可以启动发现新的药物和基因，改变ATZ的肝毒性。的 项目2的目标是发现用于治疗ATZ诱导疾病的其他命中化合物 C.通过HCS和计算机辅助分子建模，确定 主要和次要作用，并确定不同的突变疾病修饰剂是否改变对 治疗化合物。