In vivo HTS assay for novel modulators of Apolipoprotein B

载脂蛋白-B（ApoB）是代谢紊乱的许多中心标志的生物标志物和因果介导物。 疾病，包括胰岛素抵抗、脂肪肝疾病、动脉粥样硬化形成、内质网应激，以及 慢性炎症因此，ApoB作为一个有用的表型读数，用于鉴定 产生多种代谢益处的化合物。本提案将执行高通量 使用自动化机器人平台筛选（HTS）以鉴定新型降低ApoB的化合物， 使用遗传编码的荧光报告基因在活的斑马鱼幼虫中进行筛选， 灵敏地检测个体鱼中的ApoB水平。为了实现这一目标，我们组建了一个团队， 科学家都位于约翰霍普金斯大学和领导人在各自的领域。法伯有 建立了斑马鱼作为脊椎动物脂质代谢研究的模型，Mumm创造了一个强大的 HTS斑马鱼筛选平台，Ahima是哺乳动物能量代谢的世界领导者，Lectka是 一个成熟的化学家带来了屏幕点击优先级的重要专业知识的努力。HTS将采取 分两次迭代，第一次迭代筛选约3，000个临床批准的化合物库， 这些化合物可以迅速用于治疗与Apob相关的许多疾病， 扰动第二次迭代筛选是更大的努力，以最大化复合多样性（30，000 化合物），并发现潜在的全新治疗途径。两个屏幕的点击量将 接受高内容的二次筛选，该筛选使用自动成像平台来监测 使用一组携带几种重要的荧光报告基因的转基因斑马鱼进行疾病进展 代谢性疾病的危险因素。该辅助屏幕将有效地对来自 初步筛选和鉴定在活脊椎动物中具有经验证的代谢益处的化合物子集 微生物，值得进一步研究和治疗开发。有前途的化合物从主要 还将在哺乳动物模型（包括小鼠和人）中验证二级筛选的活性 培养细胞这些努力的结果将是有史以来第一个完整的动物HTS的载脂蛋白B修饰剂耦合 与高含量的二次筛选一起，将能够快速鉴定化合物， 改善许多代谢疾病表型，以及一系列用于开发新的 应对全球日益增长的代谢疾病负担的疗法。

Apolipoprotein-B (ApoB) is both a biomarker and a causal mediator of many central hallmarks of metabolic disease, including insulin resistance, fatty liver disease, atherogenesis, endoplasmic reticulum stress, and chronic inflammation. ApoB therefore serves as a useful phenotypic readout for the identification of compounds that engender diverse metabolic benefits. The present proposal will perform a high-throughput screen (HTS) to identify novel ApoB-lowering compounds using an automated robotics platform that enables screening to take place in live larval zebrafish using a genetically encoded chemiluminescent reporter to sensitively detect ApoB levels in individual fish. To accomplish this effort, we have brought together a team of scientists all located at Johns Hopkins University and leaders in their respective fields. Farber has established the zebrafish as a model for studies of vertebrate lipid metabolism, Mumm has created a powerful HTS zebrafish screening platform, Ahima is a world leader in mammalian energy metabolism and Lectka is an established chemist bringing significant expertise in screen hit prioritization to the effort. The HTS will take place in two iterations, with the first iteration screening a ~3,000 compound library of clinically approved compounds so that hits can be rapidly repurposed to treat a host of disease associated with Apob perturbations. The second iteration screening is much larger effort to maximize compound diversity (30,000 compounds) and discover potentially entirely new avenues for treatment. Hits from the both screens will be subjected to a high-content secondary screen that uses an automated imaging platform to monitor effects on disease progression using a panel of transgenic zebrafish carrying fluorescent reporters of several important metabolic disease risk factors. This secondary screen will efficiently classify and prioritize hits from the primary screen and identify the subset of compounds with validated metabolic benefits in live vertebrate organisms that justify further investigation and therapeutic development. Promising compounds from primary and secondary screening will also be validated for activity in mammalian models, including mouse and human cultured cells. The results of these efforts will be the first ever whole animal HTS for ApoB modifiers coupled with a high-content secondary screen that together will enable the rapid identification of compounds to ameliorate many metabolic disease phenotypes, as well as a collection of hits for the development of novel therapies to combat the growing global burden of metabolic disease.