Drug Therapies Identified through Modeling AT Deficiency in C Elegans

项目总结 α1抗胰蛋白酶缺乏症(ATD)是肝病最常见的遗传原因。的经典形式 ATD是由于一个错义突变(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有显著的差异。 在纯合子中。由于ATZ所致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引起的肝毒性的新化合物。这个 模型的价值将显著增加，因为它还具有遗传处理能力，以：1)阐明 组织损伤和内源性蛋白平衡途径的遗传修饰物 蛋白质错误折叠导致的损伤，以及2)精确定位哪些生化途径和/或分子是目标 通过新发现的化合物。我们表明，ATZ诱导的肝病在 线虫，线虫。表达野生型人类AT的转基因动物分泌了这种蛋白。相比之下， 表达ATZ的动物通过细胞内演示真实地再现了ATZ的ER转运缺陷 包涵体(扩张的ER池)，并变得不健康，表现为生长缓慢，雏鸟体积小， 寿命下降。使用这一模型，我们开发了一种自动化、活体动物、高含量筛选(HCS) 可与任何基于细胞的系统相媲美的检测方法。到目前为止，我们已经鉴定了大约30种热门化合物，其中包括几种 这通过增强自噬来减少ATZ的积累，自噬是一种已知的ATZ消除途径。使用 在修改我们的HCS战略的同时，我们还开发了一种半自动技术，可以减少劳动力 全基因组RNAi筛选的强度。我们确定了几个潜在的遗传修饰物/途径 ATZ累积量。总之，这些研究表明，这种转基因线虫模型是一种 强大的平台，启动了新型药物和基因的发现，以改变ATZ的肝脏毒性。这个 项目2的目标是发现更多用于治疗ATZ引起的疾病的HIT化合物 通过HCS和计算机辅助分子模拟，识别线虫的疾病修饰物的表型 主要和次要影响，并确定不同的突变疾病修饰物是否会改变对 治疗性化合物。

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.