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

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原型，其特征是错误折叠/聚集蛋白质造成的组织损伤。经典的AT缺乏症包括一种突变，这种突变增强了突变蛋白ATZ的自聚合和聚集。ATZ分泌不良，并在肝细胞内质网（ER）内积聚。减少循环AT导致肺蛋白酶抑制剂功能的丧失和肺气肿的易感性。相反，肝细胞内质网中ATZ的积累导致毒性功能获得，肝衰竭和肝癌证明了这一点。由于该疾病主要涉及内质网易位缺陷，因此AT缺乏症是化学预防的一个有吸引力的目标。虽然ATZ的分泌量很少，但它仍然保留了一些抗弹性酶活性。因此，增加ATZ分泌的小分子理论上可以防止肺和肝的组织损伤。此外，atz诱导的组织损伤的严重程度受到遗传和环境修饰因子的影响，这些修饰因子调节内源性质量控制机制，以处理错误折叠的蛋白质。增强这些降解过程的化合物可用于患者预防肝损伤，并结合旨在预防肺损伤的策略。这种疾病的一个可处理的遗传模型将极大地增强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缺乏的毒性内质网易位缺陷可以在秀丽隐杆线虫中模拟。表达野生型AT的动物分泌该蛋白。相反，表达ATZ的动物发育胞内包涵体，表现出生长缓慢和幼虫停滞。此外，在与匹兹堡大学药物发现研究所的合作中，我们展示了使用该模型开发的一种检测方法，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秀丽隐杆线虫全基因组正向和反向遗传筛选的力量来识别调节ATZ聚集和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方法提供框架。秀丽隐杆线虫模型也将适用于药物文库的高通量筛选，从而为AT缺乏的肝脏和肺部疾病的化学预防提供一种公正的方法。具体目标是：1)提供详细的表征和验证秀丽隐杆线虫AT缺乏模型，2)阐明体内改变ATZ细胞命运和致病作用的潜在遗传程序，以及3)鉴定防止ATZ细胞内积聚或消除其体内毒性作用的药物。与公共卫生相关：本提案的主要目标是发现治疗AT缺乏症的新药，AT缺乏症是儿童肝脏疾病最常见的遗传原因，也是最常见的需要肝移植的遗传性肝病。

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.