Activation of the Orphan G Protein–Coupled Receptor GPR27 by Surrogate Ligands Promotes β-Arrestin 2 Recruitment

G protein-coupled receptors are the most important drug targets for human diseases. An important number of them remain devoid of confirmed ligands. GPR27 is one of these orphan receptors, characterized by a high level of conservation among vertebrates and a predominant expression in the central nervous system. In addition, it has recently been linked to insulin secretion. However, the absence of endogenous or surrogate ligands for GPR27 complicates the examination of its biologic function. Our aim was to validate GPR27 signaling pathways, and therefore we sought to screen a diversity-oriented synthesis library to identify GPR27-specific surrogate agonists. To select an optimal screening assay, we investigated GPR27 ligand-independent activity. Both in G protein-mediated pathways and in β -arrestin 2 recruitment, no ligand-independent activity could be measured. However, we observed a recruitment of β -arrestin 2 to a GPR27V 2 chimera in the presence of membrane-anchored G protein-coupled receptor kinase-2. Therefore, we optimized a firefly luciferase complementation assay to screen against this chimeric receptor. We identified two compounds [ N -[4-(anilinocarbonyl)phenyl]-2,4-dichlorobenzamide (ChemBridge, San Diego, CA; ID5128535) and 2,4-dichloro- N -{4-[(1,3-thiazol-2-ylamino)sulfonyl]phenyl}benzamide (ChemBridge ID5217941)] sharing a N -phenyl-2,4-dichlorobenzamide scaffold, which were selective for GPR27 over its closely related family members GPR85 and GPR173. The specificity of the activity was confirmed with a NanoLuc Binary Technology β -arrestin 2 assay, imaging of green fluorescent protein-tagged β -arrestin 2, and PathHunter β -arrestin 2 assay. Interestingly, no G protein activation was detected upon activation of GPR27 by these compounds. Our study provides the first selective surrogate agonists for the orphan GPR27.