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Background/Aims: The MT₂ melatonin receptor is a potential target for treating circadian rhythm sleep disorders. This study aims to characterize the recently identified MT₂ melatonin receptor agonist. Methods: The pharmacological properties of the MT₂ melatonin receptor-selective agonist as exemplified by compound 1 [N-(2-[7-benzyl-1,6-dihydro-2H-indeno(5,4-b)furan-8-yl]ethyl)acetamide] were evaluated by use of cell-free binding and cell-based functional assays. Results: Competition binding assays using 2-[¹²⁵I]iodomelatonin revealed rapid, reversible, and high-affinity binding of compound 1 to human, mouse, and rat MT₂ melatonin receptors. cAMP, ERK1/2, and PathHunter β-arrestin recruitment assays revealed partial agonist activities. However, compound 1 induced a more intense internalization of human MT₂ melatonin receptor than melatonin. Based on studies using structurally related analogs of compound 1, we further demonstrated that the extent of internalization is independent of the intrinsic efficacy of agonists. Conclusion: These findings provide novel insights into the relationship between intrinsic agonist efficacy and agonist-induced internalization and demonstrate that compound 1 could serve as a pharmacological tool for future studies to elucidate the detailed molecular mechanism of MT₂ receptor internalization.

BACKGROUND AND PURPOSEMelanin-concentrating hormone receptor 1 (MCH1 receptor) antagonists are being considered as anti-obesity agents. The present study reports a new class of MCH1 receptor antagonists with an 8-methylquinoline scaffold. The molecular mechanism of MCH1 receptor blockade by these antagonists was examined. EXPERIMENTAL APPROACHThe pharmacological properties of the 8-methylquinolines as exemplified by MQ1 were evaluated by use of multiple biophysical and cell-based functional assays. KEY RESULTSMultiple signalling pathways for Gαi and Gαq, and β-arrestin were inhibited by MQ1. Furthermore, MQ1 produced an insurmountable antagonism, causing a rightward shift of the curve for concentration-dependent binding of MCH along with a progressive reduction of the maximal response. The dissociation kinetics for MQ1 were determined from washout experiments as well as by affinity selection-MS. In short, MQ1 was shown to be a slowly dissociating reversible MCH1 receptor blocker with a low Koff value. CONCLUSION AND IMPLICATIONSThis is the first time that a slowly dissociating negative allosteric modulator of the MCH1 receptor has been demonstrated to inhibit the numerous signalling pathways of this receptor. The characteristics of MQ1 are superior and distinct from previously reported MCH1 receptor antagonists, making members of this chemotype attractive as drug candidates. Abbreviations