Ultra-high-throughput screening for antagonists of a Gi-coupled receptor in a 2.2-microl 3,456-well plate format cyclicAMP assay.
3',5'-Cyclic adenosine monophosphate (cAMP) is a common intracellular second messenger that enables cells to respond to external stimuli. Measurement of intracellular cAMP concentrations is thus widely used for studying guanosine triphosphate binding protein-coupled receptors (GPCRs), which make up a large class of pharmaceutical drug targets. Although several assay technologies exist to measure cAMP, most are not suitable for ultra-high-throughput screening (uHTS), as is often required for screening large (greater than 1 million) chemical libraries for the identification of suitable leads for drug development. Here we report that the enzyme fragment complementation assay, a homogeneous gain of signal assay based on complementation of two fragments of a beta-galactosidase enzyme, is compatible with uHTS requirements of a 2.2-microl total assay volume in 3,456-well plate format. We describe the miniaturization of this assay into 3,456-well plate format exhibiting comparable sensitivity and plate statistics to those of a 384-well assay and the application of this assay in uHTS for the identification of antagonists of a Gi-coupled receptor.
Weber
Michael
M
Department of Automated Biotechnology, Merck Research Laboratories, North Wales, PA 19454, USA. michael_weber@merck.com
Muthusubramaniam
Lalitha
L
Murray
Justin
J
Hudak
Ed
E
Kornienko
Oleg
O
Johnson
Eric N
EN
Strulovici
Berta
B
Kunapuli
Priya
P
D001681:Biological Assay / Q000379:methods*; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D008832:Microchemistry / Q000379:methods*; D012371:Robotics / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D019204:GTP-Binding Proteins