Miniaturization of whole live cell-based GPCR assays using microdispensing and detection systems.
Cell-based beta-lactamase reporter gene assays designed to measure the functional responses of G-protein-coupled receptors (GPCRs) were miniaturized to less than 2 microL total assay volume in a 3456-well microplate. Studies were done to evaluate both receptor agonists and antagonists. The pharmacology of agonists and antagonists for target GPCRs originally developed in a 96-well format was recapitulated in a 3456-well microplate format without compromising data quality or EC(50)/IC(50) precision. These assays were employed in high-throughput screening campaigns, allowing the testing of more than 150,000 compounds in 8 h. The instrumentation used and practical aspects of the assay development are discussed.
Kornienko
Oleg
O
Merck Research Laboratories, Department of Automated Biotechnology, North Wales, PA 19454, USA. Oleg_Kornienko@merck.com
Lacson
Raul
R
Kunapuli
Priya
P
Schneeweis
Jonathan
J
Hoffman
Ira
I
Smith
Todd
T
Alberts
Melissa
M
Inglese
James
J
Strulovici
Berta
B
D000818:Animals; D001681:Biological Assay / Q000295:instrumentation* / Q000379:methods*; D020650:Combinatorial Chemistry Techniques; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods*; D017930:Genes, Reporter; D006801:Humans; D020128:Inhibitory Concentration 50; D036103:Nanotechnology / Q000379:methods; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000037:antagonists & inhibitors*; D018028:Receptors, Neurotensin / Q000187:drug effects / Q000235:genetics; D018045:Receptors, Oxytocin / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D001618:beta-Lactamases / Q000235:genetics
D043562:Receptors, G-Protein-Coupled; D018028:Receptors, Neurotensin; D018045:Receptors, Oxytocin; D001618:beta-Lactamases