In vivo HTS assay for novel modulators of Apolipoprotein B

载脂蛋白 B 新型调节剂的体内 HTS 测定

基本信息

批准号：
10398022
负责人：
STEVEN A FARBER
金额：
$ 47.97万
依托单位：
CARNEGIE INSTITUTION OF WASHINGTON, D.C.
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-30 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10398022
关键词：
Address Animals Apolipoproteins B Area Biological Assay Biological Markers Biological Models Biology Cardiovascular Diseases Cells Chemicals Chronic Clinical Collaborations Collection Communities Complement Coupled Cultured Cells Development Diabetes Mellitus Disease Disease Progression Disease model Drug Modelings Eligibility Determination Energy Metabolism FDA approved Fatty acid glycerol esters Fishes Funding Genome engineering Health Hepatic Human Image Individual Inflammation Insulin Resistance Investigation Laboratories Larva Lead Libraries Link Luciferases Mediator of activation protein Metabolic Metabolic Diseases Metabolic syndrome Metabolism Modeling Monitor Mus Organism Pharmaceutical Preparations Pharmacologic Substance Pharmacology Phenotype Physiology Probability Process Production Reporter Research Risk Factors Robotics Scientist Series Structure Study models System Techniques Testing Therapeutic Toxic effect Transgenic Organisms Translations Triglycerides United States National Institutes of Health Universities Validation Whole Organism Zebrafish assay development atherogenesis base combat dietary disease phenotype drug discovery efficacy evaluation endoplasmic reticulum stress experimental study fatty liver disease feeding high throughput screening imaging platform in vivo internal control lead candidate lipid metabolism metabolic phenotype novel novel therapeutics screening statistics success therapeutic development therapeutic evaluation tool

项目摘要

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.

载脂蛋白-B (ApoB) 既是生物标志物，也是许多代谢核心标志的因果介质疾病，包括胰岛素抵抗、脂肪肝、动脉粥样硬化、内质网应激和慢性炎症。因此，ApoB 可作为有用的表型读数来识别产生多种代谢益处的化合物。本提案将执行高通量筛选（HTS），使用自动化机器人平台识别新型 ApoB 降低化合物，该平台能够使用基因编码的化学发光报告基因对活斑马鱼幼虫进行筛选灵敏地检测个体鱼的 ApoB 水平。为了完成这项工作，我们组建了一个团队约翰霍普金斯大学的科学家和各自领域的领导者。法伯有建立斑马鱼作为脊椎动物脂质代谢研究的模型后，玛姆创造了一个强大的 HTS斑马鱼筛选平台，Ahima是哺乳动物能量代谢领域的世界领先者，Lectka是一位资深化学家为这项工作带来了屏幕点击优先级方面的重要专业知识。 HTS 将采取分两次迭代进行，第一次迭代筛选了约 3,000 种临床批准的化合物库化合物，以便可以快速重新调整其用途来治疗与 Apob 相关的一系列疾病扰动。第二次迭代筛选是更大的努力，以最大化化合物多样性（30,000 化合物）并发现潜在的全新治疗途径。来自两个屏幕的点击量将是受到高内容辅助屏幕的影响，该屏幕使用自动成像平台来监测对使用一组携带几种重要荧光报告基因的转基因斑马鱼研究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的点击进行分类和优先排序初步筛选并鉴定在活体脊椎动物中具有经过验证的代谢益处的化合物子集证明进一步研究和治疗开发合理的生物体。来自初级的有前途的化合物二次筛选也将在哺乳动物模型（包括小鼠和人类）中验证活性培养的细胞。这些努力的结果将是第一个针对 ApoB 修饰剂偶联的完整动物 HTS 与高内涵二级筛选一起将能够快速识别化合物改善许多代谢疾病表型，以及一系列用于开发新药物的热门产品对抗日益严重的全球代谢疾病负担的疗法。