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This version was published on August 1, 2005
Journal of Biomolecular Screening, Vol. 10, No. 5, 476-484 (2005)
DOI: 10.1177/1087057105274896

Quantitative Cell-Based High-Content Screening for Vasopressin Receptor Agonists Using Transfluor®Technology

Richik N. Ghosh

Richard DeBiasio

Cellomics, Inc., Pittsburgh, PA.

Christine C. Hudson

Xsira Pharmaceuticals, Inc. (formerly Norak Biosciences), Morrisville, NC.

Everett R. Ramer

Cellomics, Inc., Pittsburgh, PA.

Conrad L. Cowan

Robert H. Oakley

Xsira Pharmaceuticals, Inc. (formerly Norak Biosciences), Morrisville, NC.

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 ß-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 30min. Agonist stimulation causes V2R internalization into endosomes. GFP-ß-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 platformand 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.

Key Words: high-content screening • cell-based assays • vasopressin • ß-arrestin • G-protein-coupled receptors • Transfluor ®


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