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PubMed · 16093557

Quantitative cell-based high-content screening for vasopressin receptor agonists using transfluor technology.

Richik N Ghosh·Richard DeBiasio·Christine C Hudson·Everett R Ramer·Conrad L Cowan·Robert H Oakley
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Abstract

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.

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AnimalsArginineArrestinsBenzimidazolesCell Line, TumorCloning, MolecularDose-Response Relationship, DrugDrug Evaluation, PreclinicalEndosomesFluorescent DyesGene LibraryGreen Fluorescent ProteinsHumansImage Processing, Computer-AssistedLuminescent ProteinsPeptidesReceptors, VasopressinSpectrometry, FluorescenceSwineVasopressinsbeta-ArrestinsJournal Article

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Richik N Ghosh, Richard DeBiasio, Christine C Hudson, Everett R Ramer, Conrad L Cowan, Robert H Oakley. 2005. Quantitative cell-based high-content screening for vasopressin receptor agonists using transfluor technology.. https://doi.org/10.1177/1087057105274896

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