Activation of the Orphan G Protein-Coupled Receptor GPR27 by Surrogate Ligands Promotesβ-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 inβ-arrestin 2 recruitment, no ligand-independent activity could be measured. However, we observed a recruitment ofβ-arrestin 2 to a GPR27V2chimera 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 aN-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 Technologyβ-arrestin 2 assay, imaging of green fluorescent protein-taggedβ-arrestin 2, and PathHunterβ-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.
Laboratory of Molecular Pharmacology, GIGA-Molecular Biology of Diseases (N.D., C.L., J.G., P.G., A.S., J.H.), Laboratory of Medicinal Chemistry, Center for Interdisciplinary Research on Medicines (N.D., B.P., J.H.), Neuroendocrinology Unit, GIGA-Neurosciences (D.F., A.-S.P.), Laboratory of Protein Signaling and Interactions, GIGA-Molecular Biology of Diseases (J.-C.T.), University of Liège, Liège, Belgium; and Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, Luxembourg (M.S., A.C.).
Nadine
N
Dupuis
Laboratory of Molecular Pharmacology, GIGA-Molecular Biology of Diseases (N.D., C.L., J.G., P.G., A.S., J.H.), Laboratory of Medicinal Chemistry, Center for Interdisciplinary Research on Medicines (N.D., B.P., J.H.), Neuroendocrinology Unit, GIGA-Neurosciences (D.F., A.-S.P.), Laboratory of Protein Signaling and Interactions, GIGA-Molecular Biology of Diseases (J.-C.T.), University of Liège, Liège, Belgium; and Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, Luxembourg (M.S., A.C.).
Céline
C
Laschet
Laboratory of Molecular Pharmacology, GIGA-Molecular Biology of Diseases (N.D., C.L., J.G., P.G., A.S., J.H.), Laboratory of Medicinal Chemistry, Center for Interdisciplinary Research on Medicines (N.D., B.P., J.H.), Neuroendocrinology Unit, GIGA-Neurosciences (D.F., A.-S.P.), Laboratory of Protein Signaling and Interactions, GIGA-Molecular Biology of Diseases (J.-C.T.), University of Liège, Liège, Belgium; and Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, Luxembourg (M.S., A.C.).
Delphine
D
Franssen
Laboratory of Molecular Pharmacology, GIGA-Molecular Biology of Diseases (N.D., C.L., J.G., P.G., A.S., J.H.), Laboratory of Medicinal Chemistry, Center for Interdisciplinary Research on Medicines (N.D., B.P., J.H.), Neuroendocrinology Unit, GIGA-Neurosciences (D.F., A.-S.P.), Laboratory of Protein Signaling and Interactions, GIGA-Molecular Biology of Diseases (J.-C.T.), University of Liège, Liège, Belgium; and Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, Luxembourg (M.S., A.C.).
Martyna
M
Szpakowska
Laboratory of Molecular Pharmacology, GIGA-Molecular Biology of Diseases (N.D., C.L., J.G., P.G., A.S., J.H.), Laboratory of Medicinal Chemistry, Center for Interdisciplinary Research on Medicines (N.D., B.P., J.H.), Neuroendocrinology Unit, GIGA-Neurosciences (D.F., A.-S.P.), Laboratory of Protein Signaling and Interactions, GIGA-Molecular Biology of Diseases (J.-C.T.), University of Liège, Liège, Belgium; and Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, Luxembourg (M.S., A.C.).
Julie
J
Gilissen
Laboratory of Molecular Pharmacology, GIGA-Molecular Biology of Diseases (N.D., C.L., J.G., P.G., A.S., J.H.), Laboratory of Medicinal Chemistry, Center for Interdisciplinary Research on Medicines (N.D., B.P., J.H.), Neuroendocrinology Unit, GIGA-Neurosciences (D.F., A.-S.P.), Laboratory of Protein Signaling and Interactions, GIGA-Molecular Biology of Diseases (J.-C.T.), University of Liège, Liège, Belgium; and Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, Luxembourg (M.S., A.C.).
Pierre
P
Geubelle
Laboratory of Molecular Pharmacology, GIGA-Molecular Biology of Diseases (N.D., C.L., J.G., P.G., A.S., J.H.), Laboratory of Medicinal Chemistry, Center for Interdisciplinary Research on Medicines (N.D., B.P., J.H.), Neuroendocrinology Unit, GIGA-Neurosciences (D.F., A.-S.P.), Laboratory of Protein Signaling and Interactions, GIGA-Molecular Biology of Diseases (J.-C.T.), University of Liège, Liège, Belgium; and Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, Luxembourg (M.S., A.C.).
Arvind
A
Soni
Laboratory of Molecular Pharmacology, GIGA-Molecular Biology of Diseases (N.D., C.L., J.G., P.G., A.S., J.H.), Laboratory of Medicinal Chemistry, Center for Interdisciplinary Research on Medicines (N.D., B.P., J.H.), Neuroendocrinology Unit, GIGA-Neurosciences (D.F., A.-S.P.), Laboratory of Protein Signaling and Interactions, GIGA-Molecular Biology of Diseases (J.-C.T.), University of Liège, Liège, Belgium; and Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, Luxembourg (M.S., A.C.).
Anne-Simone
AS
Parent
Laboratory of Molecular Pharmacology, GIGA-Molecular Biology of Diseases (N.D., C.L., J.G., P.G., A.S., J.H.), Laboratory of Medicinal Chemistry, Center for Interdisciplinary Research on Medicines (N.D., B.P., J.H.), Neuroendocrinology Unit, GIGA-Neurosciences (D.F., A.-S.P.), Laboratory of Protein Signaling and Interactions, GIGA-Molecular Biology of Diseases (J.-C.T.), University of Liège, Liège, Belgium; and Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, Luxembourg (M.S., A.C.).
Bernard
B
Pirotte
Laboratory of Molecular Pharmacology, GIGA-Molecular Biology of Diseases (N.D., C.L., J.G., P.G., A.S., J.H.), Laboratory of Medicinal Chemistry, Center for Interdisciplinary Research on Medicines (N.D., B.P., J.H.), Neuroendocrinology Unit, GIGA-Neurosciences (D.F., A.-S.P.), Laboratory of Protein Signaling and Interactions, GIGA-Molecular Biology of Diseases (J.-C.T.), University of Liège, Liège, Belgium; and Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, Luxembourg (M.S., A.C.).
Andy
A
Chevigné
Laboratory of Molecular Pharmacology, GIGA-Molecular Biology of Diseases (N.D., C.L., J.G., P.G., A.S., J.H.), Laboratory of Medicinal Chemistry, Center for Interdisciplinary Research on Medicines (N.D., B.P., J.H.), Neuroendocrinology Unit, GIGA-Neurosciences (D.F., A.-S.P.), Laboratory of Protein Signaling and Interactions, GIGA-Molecular Biology of Diseases (J.-C.T.), University of Liège, Liège, Belgium; and Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, Luxembourg (M.S., A.C.).
Jean-Claude
JC
Twizere
Laboratory of Molecular Pharmacology, GIGA-Molecular Biology of Diseases (N.D., C.L., J.G., P.G., A.S., J.H.), Laboratory of Medicinal Chemistry, Center for Interdisciplinary Research on Medicines (N.D., B.P., J.H.), Neuroendocrinology Unit, GIGA-Neurosciences (D.F., A.-S.P.), Laboratory of Protein Signaling and Interactions, GIGA-Molecular Biology of Diseases (J.-C.T.), University of Liège, Liège, Belgium; and Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, Luxembourg (M.S., A.C.) j.hanson@ulg.ac.be.
Julien
J
Hanson
D000595:Amino Acid Sequence; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D049409:Luciferases, Firefly; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000235:genetics / Q000378:metabolism*; D000071560:beta-Arrestin 2 / Q000819:agonists* / Q000235:genetics / Q000378:metabolism*
C111343:Gpr27 protein, mouse; D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D000071560:beta-Arrestin 2; D049409:Luciferases, Firefly