qHTS to Identify Modulators of Lipid Storage

qHTS 识别脂质储存调节剂

• 批准号: 7628150
• 负责人: Mathias Beller
• 金额: $ 2.5万
• 依托单位: MAX PLANCK INSTITUTE FOR BIOPHYS CHEM
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-09 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7628150
• 关键词: Affect; Area; Atherosclerosis; Biological Assay; Biology; Cell Line; Cells; Characteristics; Chemicals; Cytometry; Cytoplasm; Deposition; Detection; Diabetes Mellitus; Disease; Drosophila genus; Drosophila melanogaster; Embryo; Endoplasmic Reticulum; Environment; Event; Fatty Acids; Genetic Epistasis; Genomics; Goals; Hepatocyte; In Vitro; Lasers; Lead; Libraries; Lipase; Lipids; Lipodystrophy; Lipolysis; Measures; Mediating; Metabolic Diseases; Metabolism; Methods; Microscopic; Miniaturization; Molecular Bank; Mus; Nonesterified Fatty Acids; Obesity; Organelles; Organism; Pathway interactions; Pharmaceutical Preparations; Process; Proteins; RNA Interference; Regulation; Regulatory Pathway; Scanning; Screening procedure; Series; Staining method; Stains; Testing; Titrations; Triglycerides; United States National Institutes of Health; assay development; base; candidate validation; combinatorial; gene function; genome-wide; high throughput screening; human disease; insight; lipid biosynthesis; lipid metabolism; loss of function; novel; public health relevance; repository; research study; response; small molecule; therapeutic development; tool; trafficking

DESCRIPTION (provided by applicant): The primary goal of this project is to identify chemical probes that either increase or decrease the lipid content of cells. Most, and probably all cells are capable of storing energy rich lipids (mainly triacylglycerols), which are generated on basis of de novo synthesized fatty acids or non-esterified free fatty acids (NEFA) taken up from the environment. Storage lipids are deposited in specialized organelles, the so-called lipid droplets (LDs). LDs are thought to arise at the endoplasmic reticulum in a budding-like process. Once released into the cytoplasm, LD volume increases by targeted lipogenesis or fusion of existing droplets, suggesting extensive trafficking events. Remobilization of LD stores involves enzymatic breakdown of the storage lipids by lipases. Lipogenesis and lipolysis needs to be delicately fine-tuned, as diminished or increased lipid stores have dramatic consequences for the organism, as demonstrated in human diseases including lipodystrophy, atherosclerosis or obesity. There are only few drugs for treating metabolic diseases and a very limited number of chemical probes to study lipid storage in vitro. We found that also embryonic Drosophila S3 and Kc167 cells are capable of depositing LDs and developed an assay to measure lipid storage amounts by fluorescent staining of LDs and cells and microscopic or laser-scanning microplate cytometry based detection. In a genome-wide RNA interference (RNAi) screen, the assay identified about 500 gene-functions regulating lipid stores. The same assay was further optimized for small compound screening in S3 cells. Assay profiling confirmed its precision, robust response to known small-molecule modulators of lipid storage and suitability for miniaturization and HTS. Screening to date of 8,874 compounds has yielded a small number of actives, some of which are connected with protein targets previously associated with organismic lipid storage regulation. Further screening will increase the chance that potent compounds can be found with the potential of mechanistic insights and therapeutic development. Validation of candidate chemical probes during assay development in mouse AML12 hepatocytes demonstrated evolutionary conservation of our findings. Orthologous testing with RNAi-mediated knockdown experiments additionally provided epistasis information suitable to further characterize identified compounds. PUBLIC HEALTH RELEVANCE: The chemical probes yielded by the project should be useful tools in providing a better understanding of cellular and organismic lipid storage on a functional and evolutionary level. Furthermore, active substances might result in therapies treating emerging lipid storage associated diseases including atherosclerosis, diabetes or obesity. As well, the present assay will establish a profile of compounds within the MLSMR that modulate this ubiquitous area of biology.

描述（由申请人提供）：本项目的主要目标是确定增加或减少细胞脂质含量的化学探针。大多数甚至可能所有细胞都能够储存富含能量的脂质（主要是三酰甘油），这些脂质是基于从头合成的脂肪酸或从环境中吸收的非酯化游离脂肪酸（NEFA）产生的。储存脂质沉积在专门的细胞器中，即所谓的脂滴（LD）。LD被认为是在内质网中以出芽样过程出现的。一旦释放到细胞质中，LD体积通过靶向脂肪生成或现有液滴的融合而增加，表明广泛的运输事件。LD贮存的再动员涉及通过脂肪酶对贮存脂质的酶促分解。脂肪生成和脂肪分解需要精细地微调，因为减少或增加的脂质储存对生物体具有显著的后果，如在包括脂肪营养不良、动脉粥样硬化或肥胖症的人类疾病中所证明的。目前用于治疗代谢性疾病的药物很少，用于体外研究脂质储存的化学探针也非常有限。我们发现胚胎果蝇S3和Kc167细胞也能够沉积LD，并开发了一种通过LD和细胞的荧光染色和显微镜或激光扫描微孔板细胞术检测来测量脂质储存量的测定法。在全基因组RNA干扰（RNAi）筛选中，该试验确定了约500个调节脂质储存的基因功能。进一步优化相同的测定法用于S3细胞中的小化合物筛选。测定分析证实了其精密度、对已知的脂质储存小分子调节剂的稳健响应以及小型化和HTS的适用性。迄今为止，对8，874种化合物的筛选产生了少量活性物质，其中一些与先前与有机物脂质储存调节相关的蛋白质靶点有关。进一步的筛选将增加发现具有机制见解和治疗开发潜力的有效化合物的机会。在小鼠AML 12肝细胞中检测开发过程中对候选化学探针的验证证明了我们的发现的进化保守性。用RNAi介导的敲低实验进行的正向测试另外提供了适合于进一步表征所鉴定的化合物的上位性信息。公共卫生关系：该项目产生的化学探针应该是在功能和进化水平上更好地了解细胞和器官脂质储存的有用工具。此外，活性物质可能导致治疗新出现的脂质储存相关疾病的疗法，包括动脉粥样硬化、糖尿病或肥胖症。同样，本试验将建立MLSMR中调节这一普遍存在的生物学领域的化合物谱。