Quantitative Cell-Based High-Content Screening for Vasopressin Receptor Agonists Using Transfluor®Technology
| Autor: | Richard Debiasio, Conrad L. Cowan, Richik N. Ghosh, Everett R. Ramer, Christine C. Hudson, Robert H. Oakley |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
0301 basic medicine
Agonist Receptors Vasopressin Arrestins Swine Vasopressins Endosome medicine.drug_class Green Fluorescent Proteins Drug Evaluation Preclinical Endosomes Biology Arginine 01 natural sciences Biochemistry Analytical Chemistry Green fluorescent protein 03 medical and health sciences Cell Line Tumor Arginine vasopressin receptor 2 Image Processing Computer-Assisted medicine Animals Humans Cloning Molecular beta-Arrestins Fluorescent Dyes Gene Library Vasopressin receptor G protein-coupled receptor Dose-Response Relationship Drug Beta-Arrestins 0104 chemical sciences Cell biology Luminescent Proteins 010404 medicinal & biomolecular chemistry Spectrometry Fluorescence 030104 developmental biology High-content screening Molecular Medicine Benzimidazoles Peptides Biotechnology |
| Zdroj: | SLAS Discovery. 10:476-484 |
| ISSN: | 2472-5552 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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