Quantitative cell-based high-content screening for vasopressin receptor agonists using transfluor technology.
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.
Ghosh
Richik N
RN
Cellomics, Inc., Pittsburgh, PA 15219, USA. rghosh@cellomics.com
DeBiasio
Richard
R
Hudson
Christine C
CC
Ramer
Everett R
ER
Cowan
Conrad L
CL
Oakley
Robert H
RH
D000818:Animals; D001120:Arginine / Q000737:chemistry; D019390:Arrestins / Q000378:metabolism; D001562:Benzimidazoles / Q000494:pharmacology; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D011992:Endosomes / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008164:Luminescent Proteins / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D017483:Receptors, Vasopressin / Q000819:agonists*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013552:Swine; D014667:Vasopressins / Q000737:chemistry; D000071557:beta-Arrestins
D019390:Arrestins; D001562:Benzimidazoles; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D010455:Peptides; D017483:Receptors, Vasopressin; D000071557:beta-Arrestins; D014667:Vasopressins; D049452:Green Fluorescent Proteins; D001120:Arginine; C017807:bisbenzimide ethoxide trihydrochloride