Supplementary Material for: Pharmacological Characterization of a Highly Selective and Potent Partial Agonist of the MT<sub>2</sub> Melatonin Receptor

2014-07-19T00:00:00Z (GMT) by Sakurai T. Koike T. Nakayama M.
<b><i>Background/Aims:</i></b> The MT<sub>2</sub> melatonin receptor is a potential target for treating circadian rhythm sleep disorders. This study aims to characterize the recently identified MT<sub>2</sub> melatonin receptor agonist. <b><i>Methods:</i></b> The pharmacological properties of the MT<sub>2</sub> 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. <b><i>Results:</i></b> Competition binding assays using 2-[<sup>125</sup>I]iodomelatonin revealed rapid, reversible, and high-affinity binding of compound 1 to human, mouse, and rat MT<sub>2</sub> 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 MT<sub>2</sub> 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. <b><i>Conclusion:</i></b> 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 MT<sub>2</sub> receptor internalization.