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-抗抗抑制蛋白（AT）缺乏症是不断扩大的构象疾病的原型，其特征是错误折叠/聚集的蛋白质的组织损伤。 AT缺乏的经典形式涉及突变，从而增强突变蛋白ATZ的自聚合和聚集。 ATZ分泌不足，并且在肝细胞的内质网（ER）内积聚。在肺中降低蛋白酶抑制剂功能，肺气肿的循环减少。相比之下，肝细胞中ATZ的积累导致有毒的功能收益，如肝衰竭和癌所证明。缺乏症是化学预防的有吸引力的靶标，因为该疾病主要涉及ER易位缺陷。尽管最少分泌，但ATZ仍然保留了其一些抗假酶活性。因此，增加ATZ分泌的小分子可以从理论上预防肺和肝脏的组织损伤。同样，ATZ诱导的组织损伤的严重程度受遗传和环境改性剂的影响，这些遗传和环境修饰符调节内源性质量控制机制以处理错误折叠的蛋白质。可以在患者中使用增强这些降解过程的化合物，以防止肝脏损伤与旨在防止肺损伤的策略结合使用。该疾病的可探讨遗传模型将大大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缺乏症的有毒ER易位缺陷可以在秀丽隐杆线虫中建模。表达野生型的动物分泌蛋白质。相比之下，表达ATZ的动物会出现细胞内夹杂物，并显示出缓慢的生长和幼体停滞。此外，我们与匹兹堡U的药物发现研究所合作，我们使用该模型可以轻松地适用于自动化的高吞吐量筛查的测定法的开发。该项目的目标包括在秀丽隐杆线虫中使用全基因组向前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的存活的分子途径，从而为设计基于机制的疗法提供了框架。秀丽隐杆线虫模型还将适应对药物库的高通量筛查，从而为缺乏症状提供了一种无偏见的肝脏和肺部疾病的化学预防方法。具体目的是：1）提供对秀丽隐杆线虫模型的详细表征和验证，2）阐明修改ATZ在体内的细胞命运和致病作用的基础遗传程序，以及3）识别预防ATZ内细胞内积累或消除其毒性效应其毒性的药物。与公共卫生有关：该提案的主要目标是发现新药物的疾病，这是童年时期肝病的最常见遗传原因，也是最常见的遗传性肝病，需要肝移植。