Dupuis Nadine, Laschet Céline, Franssen Delphine, Szpakowska Martyna, Gilissen Julie, Geubelle Pierre, Soni Arvind, Parent Anne-Simone, Pirotte Bernard, Chevigné Andy, Twizere Jean-Claude, Hanson Julien
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.).
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.)
Mol Pharmacol. 2017 Jun;91(6):595-608. doi: 10.1124/mol.116.107714. Epub 2017 Mar 17.
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 -arrestin 2 recruitment, no ligand-independent activity could be measured. However, we observed a recruitment of -arrestin 2 to a GPR27V 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 [-[4-(anilinocarbonyl)phenyl]-2,4-dichlorobenzamide (ChemBridge, San Diego, CA; ID5128535) and 2,4-dichloro--{4-[(1,3-thiazol-2-ylamino)sulfonyl]phenyl}benzamide (ChemBridge ID5217941)] sharing a -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 -arrestin 2 assay, imaging of green fluorescent protein-tagged -arrestin 2, and PathHunter -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.
G蛋白偶联受体是治疗人类疾病最重要的药物靶点。其中相当一部分仍未找到经证实的配体。GPR27就是这些孤儿受体之一，其特点是在脊椎动物中具有高度保守性，且在中枢神经系统中占主导性表达。此外，它最近还与胰岛素分泌有关。然而，缺乏GPR27的内源性或替代配体使得对其生物学功能的研究变得复杂。我们的目的是验证GPR27信号通路，因此我们试图筛选一个多样化导向合成文库，以鉴定GPR27特异性替代激动剂。为了选择最佳筛选试验，我们研究了GPR27的配体非依赖性活性。在G蛋白介导的信号通路和β - 抑制蛋白2募集过程中，均未检测到配体非依赖性活性。然而，我们观察到在膜锚定的G蛋白偶联受体激酶-2存在的情况下，β - 抑制蛋白2被募集到GPR27V嵌合体上。因此，我们优化了一种萤火虫荧光素酶互补试验，以针对这种嵌合受体进行筛选。我们鉴定出两种化合物[-[4-(苯胺羰基)苯基]-2,4-二氯苯甲酰胺（ChemBridge公司，加利福尼亚州圣地亚哥；ID5128535）和2,4-二氯-β-{4-[(1,3-噻唑-2-基氨基)磺酰基]苯基}苯甲酰胺（ChemBridge公司ID5217941）]，它们共享一个β - 苯基-2,4-二氯苯甲酰胺支架，对GPR27的选择性高于其密切相关的家族成员GPR85和GPR173。通过NanoLuc二元技术β - 抑制蛋白2试验、绿色荧光蛋白标记的β - 抑制蛋白2成像和PathHunterβ - 抑制蛋白2试验证实了活性的特异性。有趣的是，这些化合物激活GPR27后未检测到G蛋白激活。我们的研究为孤儿GPR27提供了首批选择性替代激动剂。
