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：
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
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/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

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.

项目摘要 α1-抗胰蛋白酶（AT）缺乏症（ATD）是肝病最常见的遗传原因。古典形式的形式 ATD是由于单个错义突变（Z）导致突变蛋白（ATZ）变形并积累在肝细胞的内质网中，作为有毒的低聚物，聚合物或聚集体。但是，到期对于遗传和环境修饰符，肝病的事件和严重程度有明显的变化在纯合子中。由于严重ATZ引起的肝损伤的唯一治疗方法是肝移植，因此我们开发了一种动物模型，可适应于临床前的高通量药物筛查技术，大力有助于发现新化合物，以减少或消除ATZ诱导的肝毒性。该模型的价值将显着增加，因为它还具有：1）阐明组织损伤的遗传修饰符和防止蛋白质错误折叠诱导的损伤，以及2）针对哪种生化途径和/或分子的靶向由新发现的化合物。我们表明，ATZ引起的肝病是忠实地建模的线虫，秀丽隐杆线虫。表达野生型人类的转基因动物分泌了蛋白质。相比之下，表达ATZ的动物忠实地概括了ATZ的ER贩运缺陷，通过证明细胞内夹杂物（扩张的Er cisterna），并变得不健康，如生长缓慢，小育雏尺寸和寿命降低。使用此模型，我们开发了一种自动化的，现场的，高内心筛选（HCS）测定将任何基于细胞的系统的障碍。到目前为止，我们已经确定了约30种命中化合物，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的积累。使用修改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符/途径 ATZ积累。综上所述，这些研究表明，这种转基因秀丽隐杆线虫模型是强大的平台来启动发现ATZ肝毒性的新型药物和基因。这项目2的目的是发现用于治疗ATZ诱导疾病的其他命中化合物 HCS和计算机辅助分子建模中的秀丽隐杆线虫的表型，鉴定重大和次要效应，并确定不同的突变疾病修饰符是否改变了对治疗化合物。