Orphan GPCRs: from ligand discovery to physiological role. Application to apelin. Nadia de Mota1, Xavier Iturrioz1, Rodrigo Alvear1, Annabelle Reaux1, Said El Messari1, Nicolas Chartrel2, Dominique Bonnet3, Hubert Dabire4, Alain Berdeaux4, Françoise Moos5, B. Maigret6, JeanLuc Galzi3, Marcel Hibert3, Hubert Vaudry2 and Catherine Llorens-Cortes1*. 1

INSERM U691, Collège de France, 75005 Paris, INSERM U413, Université de Rouen, IRFMP 23, 76821 Mont-Saint-Aignan, 3 CNRS, UMR 7175, Faculté de Pharmacie et Ecole de Biotechnologie, Illkirch Graffenstaden, 4 INSERM, U841, Faculté de Médecine, 94010, Créteil, 5 CNRS, UMR5226, UFR de Pharmacie, 33077 Bordeaux, 6 CNRS UMR 7503, Orpailleur group- LORIA, Université Henri Poincaré, 54003 Nancy. 2

Our efforts to clone an angiotensin III receptor led to the cloning of a gene encoding a rat seven transmembrane domain receptor exhibiting 95% sequence identity with that of the human orphan APJ receptor. This orphan has now been deorphanized and corresponds to apelin receptor. We characterized this receptor pharmacologically and demonstrated that apelin and its receptor were expressed together with vasopressin (AVP) in the magnocellular hypothalamic neurons. We showed that the central injection of apelin in lactating rats decreased the electrical activity of these neurons and the secretion of AVP into the bloodstream, resulting in aqueous diuresis. Apelin is thus a natural inhibitor of the antidiuretic effects of AVP. Finally, in rats and humans, apelin and AVP were found to be regulated in opposite manners by osmotic stimuli, thereby participating to the maintenance of body fluid homeostasis. Apelin and its receptor are also present in the heart, kidney and blood vessels and the systemic injection of apelin decreases arterial blood pressure, improves cardiac contractility and reduces cardiac loading. Apelin may therefore be involved in controlling cardiovascular function. To explore further upstream the role played by this peptide, we used an original FRET-based assay to screen a library of fluorescent (lissamine) compounds on the human EGFP-tagged apelin receptor to identify non peptidic agonists or antagonists of the apelin receptor which do not exist to date,. This screening led to isolate several hits whose the most potent, E339-3D6, displayed an affinity of 53nM, behaved as a partial agonist towards adenylate cyclase and induced the internalization of the apelin receptor. Using molecular modeling, we provided evidence that the binding moieties for E339-3D6 and apelin overlapped within the receptor binding cavity. In addition, E339-3D6 induces a concentration-dependent vasorelaxation of rat aorta precontracted with noradrenaline via NO production and given by central route in mice potently inhibits AVP release in the blood circulation, both effects being comparable to those produced by apelin. This novel ligand represents the first non peptidic agonist of the apelin receptor. Its optimization towards a compound meeting the clinical candidate status requirements will allow to develop new potent agonists of the apelin receptor which could represent a new class of therapeutic agents for the treatment cardiovascular diseases,water retention and/or hyponatremic disorders. *Correspondance : [email protected]