Miniaturized GPCR signaling studies in 1536-well format

G protein-coupled receptors (GPCRs) are involved in various physiological processes, such as behavior changes, mood alteration, and regulation of immune-system activity. Thus, GPCRs are popular targets in drug screening, and a well-designed assay can speed up the discovery of novel drug candidates. The Promega cAMP-Glo Assay is a homogenous bioluminescent assay to monitor changes in intracellular cyclic adenosine monophosphate (cAMP) concentrations in response to the effect of an agonist, antagonist, or test compound on GPCRs. Together with the Labcyte Echo 555 acoustic liquid handler and the Deerac Fluidics Equator HTS reagent dispenser, this setup can screen compounds in 96-, 384-, and 1536-well formats for their effects on GPCRs. Here, we describe our optimization of the cAMP-Glo assay in 1536-well format, validate the pharmacology, and assess the assay robustness for HTS. We have successfully demonstrated the use of the assay in primary screening applications of known agonist and antagonist compounds, and confirmed the primary hits via secondary screening. Implementing a high-throughput miniaturized GPCR assay as demonstrated here allows effective screening for potential drug candidates.