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High Throughput Genetic and Drug Screens for Alph-1-Antitrypsin Deficiency

针对 Alph-1-抗胰蛋白酶缺乏症的高通量遗传和药物筛选

基本信息

批准号：
8013391
负责人：
GARY ARTHUR SILVERMAN
金额：
$ 9.2万
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：
2010
资助国家：
美国
起止时间：
2010-02-22 至 2012-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8013391
关键词：
Affect Alzheimer&apos s Disease Animals Biological Assay Caenorhabditis elegans Carcinoma Cells Characteristics Chemoprophylaxis Childhood Chronic Obstructive Airway Disease Collaborations Connective Tissue Defect Degenerative Disorder Development Diagnostic Disease Elastases Endoplasmic Reticulum Extracellular Fluid Funding Future Gene Mutation Genetic Genetic Models Genetic Programming Genetic Screening Goals Growth Hepatocyte Huntington Disease Injury Institutes Lead Leukocyte Elastase Libraries Liver Liver Failure Liver diseases Longevity Lung Lung diseases Methods Modeling Molecular Mutagenesis Mutation Pathogenicity Pathway interactions Patients Pharmaceutical Preparations Phenotype Play Point Mutation Polymers Preclinical Drug Evaluation Predisposition Process Protease Inhibitor Protein C Inhibitor Proteins Public Health Pulmonary Emphysema Quality Control RNA Interference Research Personnel Role Serine Severities System Therapeutic Tissues Toxic effect Universities Validation assay development base carcinogenesis cell type chemical genetics design drug discovery gain of function genome-wide high throughput screening in vivo liver transplantation loss of function lung injury mutant polymerization positional cloning prevent programs protein aggregate protein misfolding prototype small molecule small molecule libraries

项目摘要

DESCRIPTION (provided by applicant): ?-1-antitrypsin (AT) deficiency is prototypic of an expanding number of conformational diseases characterized by tissue damage from misfolded/aggregated proteins. The classical form of AT deficiency involves a mutation that enhances self-polymerization and aggregation of the mutant protein, ATZ. ATZ is poorly secreted and accumulates within the endoplasmic reticulum (ER) of liver cells. Decreased circulating AT leads to a loss of protease inhibitor function in the lung and predisposition to emphysema. In contrast, accumulation of ATZ in the ER of liver cells leads to a toxic gain-of-function as evidenced by liver failure and carcinoma. AT deficiency is an attractive target for chemoprophylaxis as the disease predominantly involves an ER translocation defect. Although minimally secreted, ATZ still retains some of its anti-elastase activity. Thus, small molecules that increase ATZ secretion could theoretically prevent tissue damage in both lung and liver. Also, the severity of ATZ-induced tissue injury is influenced by genetic and environmental modifiers that regulate endogenous quality control mechanisms for disposal of misfolded proteins. Compounds that enhance these degradative processes, could be used in patients to prevent liver damage in combination with strategies designed to prevent lung injury. A tractable genetic model of this disease would greatly enhance our ability to elucidate the mechanism of tissue damage and the endogenous mechanisms that protect against protein misfolding. Our preliminary results show that the toxic ER translocation defect of AT deficiency can be modeled in C. elegans. Animals expressing wild-type AT secrete the protein. In contrast, animals expressing ATZ develop intracellular inclusions, and show slow growth and larval arrest. Further, in collaboration with the Drug Discovery Institute of the U of Pittsburgh, we show the development of an assay using this model that can easily be adapted to automated high throughput screening. The goals of this project include using the power of genome-wide forward and reverse genetic screens in C. elegans to identify molecular pathways that modulate ATZ aggregation and alter the survival of affected animals, therein providing a framework for designing mechanism-based therapeutics. The C. elegans model will also be adapted for high-throughput screening of drug libraries, thereby providing an unbiased method for chemoprophylaxis of liver and lung disease in AT deficiency. The specific aims are: 1) provide detailed characterization and validation of the C. elegans model of AT deficiency, 2) elucidate the underlying genetic program(s) that modify the cellular fate and pathogenic effects of ATZ in vivo, and 3) identify drugs that prevent intracellular accumulation of ATZ or eliminate its toxic effects in vivo. Relevance to public health: The major goal of this proposal is to discover new drugs for AT deficiency, the most common genetic cause of liver disease in childhood and the most frequent genetic liver disease necessitating liver transplantation.

描述(由申请人提供)：？-1-抗胰蛋白酶(AT)缺乏是越来越多的构象疾病的典型，其特征是错误折叠/聚集的蛋白质造成组织损伤。经典形式的AT缺乏症涉及到一种突变，它增强了突变蛋白ATZ的自我聚合和聚集。ATZ分泌不良，聚集在肝细胞的内质网(ER)内。循环AT减少会导致肺内蛋白水解酶抑制功能丧失，易患上肺气肿。相反，肝细胞内质网中ATZ的积聚会导致毒性的功能获得，这是肝功能衰竭和癌症的证据。AT缺乏症是化学预防的一个有吸引力的靶点，因为该病主要涉及内质网易位缺陷。虽然ATZ的分泌量很小，但它仍然保留了一些抗弹性蛋白酶的活性。因此，从理论上讲，增加ATZ分泌的小分子可以防止肺和肝脏的组织损伤。此外，ATZ诱导的组织损伤的严重程度受到遗传和环境修饰物的影响，这些遗传和环境修饰物调节错误折叠蛋白质的处置的内源性质量控制机制。增强这些降解过程的化合物，可以与预防肺损伤的策略结合使用，用于患者预防肝脏损伤。一种易于处理的这种疾病的遗传模型将极大地提高我们阐明组织损伤机制和防止蛋白质错误折叠的内源性机制的能力。我们的初步结果表明，AT缺乏症的毒性内质网易位缺陷可以在线虫中模拟。表达野生型AT的动物分泌这种蛋白质。相比之下，表达ATZ的动物会产生细胞内的包涵体，并表现出缓慢的生长和幼虫停滞。此外，我们与匹兹堡大学药物发现研究所合作，展示了一种使用该模型的分析方法的开发，该模型可以很容易地适应自动化高通量筛选。该项目的目标包括利用线虫全基因组正向和反向遗传筛选的力量来识别调节ATZ聚集并改变受影响动物生存的分子途径，从而为设计基于机制的治疗提供一个框架。线虫模型还将适用于高通量筛选药库，从而为AT缺乏症的肝、肺疾病的化学预防提供一种公正的方法。具体目的是：1)提供线虫AT缺乏症模型的详细描述和验证；2)阐明在体内改变ATZ细胞命运和致病效应的潜在遗传程序(S)；以及3)寻找防止ATZ在细胞内积累或消除其体内毒性效应的药物。与公众健康相关：这项提案的主要目标是发现治疗AT缺乏症的新药，AT缺乏症是儿童肝病最常见的遗传原因，也是需要肝移植的最常见的遗传性肝病。