Quantitative cell-based high-content screening for vasopressin receptor agonists using Transfluoro® technology
Quantitative cell-based high-content screening for vasopressin receptor agonists using Transfluoro® technology
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DOI：
10.1177/1087057105274896
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发表时间：
2005-08-01
影响因子：
--
通讯作者：
Oakley, RH
中科院分区：
文献类型：
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作者：
Ghosh, RN;DeBiasio, R;Oakley, RH
作者演示了使用一个简单的，通用的g蛋白偶联受体（GPCR）测定筛选激动剂为特定的GPCR。稳定表达绿色荧光蛋白（GFP）标记的β -抑制素融合蛋白和血管加压素V2受体（V2R）的细胞用于高含量筛选（HCS）试验，以筛选V2R激动剂的小肽库。用终浓度为500 nM的肽处理细胞30分钟。激动剂刺激导致V2R内化到核内体。gfp - β -阻滞蛋白仍然与内体中的V2R相关，导致细胞内斑点的荧光模式。使用HCS成像平台和快速交钥匙图像分析应用程序对检测受体激活和细胞内斑点进行自动成像和定量分析。进一步评估击球的效果。结合独特的生物学，自动化的高含量分析，和一个强大的手段验证命中结果更好的线索。
The authors demonstrate the use of a simple, universal G-protein-coupled receptor (GPCR) assay to screen for agonists for a specific GPCR. Cells stably expressing a green fluorescent protein (GFP)-labeled beta-arrestin fusion protein and the vasopressin V2 receptor (V2R) were used in a high-content screening (HCS) assay to screen a small peptide library for V2R agonists. Cells were treated with the peptides at a final concentration of 500 nM for 30 min. Agonist stimulation causes V2R internalization into endosomes. GFP-beta-arrestin remains associated with the V2R in endosomes, resulting in a fluorescent pattern of intracellular spots. Assay plates were automatically imaged and quantitatively analyzed using an HCS imaging platform and a fast turnkey image analysis application optimized for detection of receptor activation and intracellular spots. Hits were further evaluated to determine their potency. The combination of unique biology, automated high-content analysis, and a powerful means of validating hits results in better leads.