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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

基本信息

批准号：
10441252
负责人：
GARY ARTHUR SILVERMAN
金额：
$ 40.35万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
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/10441252
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 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 Lead Libraries Live Birth Liver Liver Cirrhosis Liver Fibrosis Liver diseases Longevity Mammalian Cell Methods Minor Missense Mutation Modeling Modification Molecular Molecular Genetics 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 Proteins RNA Interference RNA interference screen Reproducibility Scanning Severities System Technology Testing Therapeutic Tissues Toxic effect Transgenic Animals Transgenic Mice Transgenic Organisms Variant alpha 1-Antitrypsin Deficiency base 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 protein misfolding proteostasis screening small molecule trafficking validation studies variant of unknown significance whole genome

项目摘要

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.

项目概要 α1-抗胰蛋白酶 (AT) 缺乏症 (ATD) 是肝病最常见的遗传原因。的经典形式 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，以有毒低聚物、聚合物或聚集体的形式存在。然而，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着差异纯合子之间。由于 ATZ 引起的严重肝损伤的唯一治疗方法是肝移植，我们开发了一种适用于临床前、高通量药物筛选技术的动物模型，极大地有助于发现减少或消除 ATZ 诱导的肝毒性的新化合物。这该模型的价值将显着增加，因为它还具有遗传易处理性：1）阐明组织损伤和内源性蛋白质稳态途径的遗传修饰剂可防止蛋白质错误折叠引起的损伤，2) 查明哪些生化途径和/或分子是目标由新发现的化合物。我们证明 ATZ 诱导的肝病在线虫，线虫。表达野生型人类 AT 的转基因动物分泌该蛋白质。相比之下，表达 ATZ 的动物通过证明细胞内的内含物（扩张的内质网池），并且变得不健康，表现为生长缓慢、巢体尺寸小和寿命缩短。使用该模型，我们开发了一种自动化的活体动物高内涵筛查 (HCS) 与任何基于细胞的系统相媲美的测定。到目前为止，我们已经鉴定出约 30 种热门化合物，其中包括几种通过增强自噬（一种已知的 ATZ 消除途径）来减少 ATZ 积累。使用修改我们的 HCS 策略，我们还开发了一种半自动化技术，可以减少劳动力全基因组 RNAi 筛选的强度。我们确定了几种潜在的遗传修饰剂/途径 ATZ积累。综上所述，这些研究表明，这种转基因线虫模型是一种启动发现改变 ATZ 肝毒性的新药和基因的强大平台。这项目 2 的目标是发现用于治疗 ATZ 引起的疾病的其他热门化合物通过 HCS 和计算机辅助分子建模确定秀丽隐杆线虫的表型，确定疾病修饰因子主要和次要影响，并确定不同突变疾病修饰剂是否改变对治疗化合物。