Ultra-High-Throughput Screening for Antagonists of A Gi-Coupled Receptor in A 2.2-μl 3,456-Well Plate Format CyclicAMP Assay

Abstract: 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 β-galactosidase enzyme, is compatible with uHTS requirements of a 2.2-μl 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 statist...