A Bioluminescent-Based,HTS-Compatible Assay to Monitor G-Protein-Coupled Receptor Modulation of Cellular Cyclic AMP

We have developed a novel assay for monitoring changes in intracellular cyclic AMP (cAMP) concentration with high sensitivity (30 ± 5 fmol [mean ± standard error of the mean] of cAMP per well) and reproducibility (Z′ of > 0.8). The assay is of format amenable to high throughput screening (HTS) in 96-, 384-, and 1,536-well plates, and as a bioluminescent assay is potentially less prone to interferences originating from fluorescent compounds. Because of its high sensitivity, fewer numbers of cells (1,000 cells per well) in low-volume 384-well plates are required to screen for changes in cAMP concentrations. The assay does not rely on the use of antibodies, and thus it does not suffer from changes in the affinity or quality of the antibodies. The assay is based on the fact that cAMP is a potent activator of cAMP-dependent protein kinase (PKA), and activation of PKA can be monitored by measuring ATP utilization in a kinase reaction. The amount of ATP consumed can be measured using a luciferase/luciferin luminescent reaction. Since the amount of relative luminescence units (RLU) generated is a measure of the remaining ATP, a reciprocal relationship between RLU and both the activity of PKA and the intracellular concentration of cAMP is observed. Thus, the functional activity of agents that modulate the activity of Gα _s or Gα _i forms of G-protein-coupled receptors (GPCRs), which cause change in intracellular cAMP, can be monitored by the change in the activity of PKA and the amount of RLU readout. The assay can be performed in two steps and requires only 30 min after cell lysis for completion. The assay has been successfully used to generate 50% effective concentration (EC₅₀) values for forskolin, a known direct activator of cellular adenylate cyclases, and EC₅₀ values for agonists and 50% inhibitory concentration values for antagonists modulating GPCRs that alter adenylate cyclase activity (Gα _s and Gα _i). Finally, adherent, suspension, and frozen cells have been successfully used in this assay, thus offering flexibility and convenience for many HTS applications.