While G-protein-coupled receptors (GPCRs) represent the largest class of cell surface proteins, there are ≥100 orphan GPCRs whose endogenous ligands are unknown. Accordingly, these could prove to be potential therapeutic targets for the pharmaceutical intervention of various diseases. Constitutively active orphan GPCRs are activated without ligands; thus, inverse agonists may be very useful pharmacological tools for inhibiting constitutive activity. However, in general, inverse agonist screening is considered more difficult to perform with high quality than antagonist screening, particularly due to the narrow assay window. We developed a high-throughput screening (HTS)-compatible assay to identify inverse agonists of GPR3. GPR3 is expressed in the central nervous system (CNS) and is known to be related to Alzheimer's disease and other CNS diseases. The GPR3 inducible cell line was established using T-REx 293 cells that stably expressed the tetracycline repressor protein, and the cAMP biosensor, GloSensor, was stably co-expressed. After optimization of the induction level of GPR3 and assay conditions, the GloSensor assay showed an approximately 20-fold signal-to-background ratio and high sensitivity. Using the HTS method, we successfully screened a library of hundreds of thousands of compounds for the inhibition of constitutive activity with good quality and excellent reproducibility. Finally, 35 compounds were identified as GPR3 selective inverse agonists. This inverse agonist screening approach using GloSensor in combination with the inducible expression of orphan GPCR indicates universal applicability to the search for inverse agonists of constitutively active orphan GPCRs.
Keywords: GPR3; GloSensor; HTS; constitutively active orphan GPCR; inverse agonist.