cAMP assay

Intracellular cAMP accumulation was measured using a competitive immunoassay based on homogeneous time-resolved fluorescence technology (cAMP dynamic-2, cisbio). hMC4R HEK293T stable cell lines expressing either the WT or mutant form R165W 3HA-hMC4R-Venus constructs were dispensed in 96-well plates (15 × 10³ cells/well) and processed as described below.

For antagonist potency assessment, cells were preincubated with increasing concentrations of UM0130866 for 1 hour at 37°C on a Heidolph Titramax 100 shaker at speed 900 rpm in complete buffer: 140 mM NaCl, 2.7 mM KCl, 1 mM CaCl₂, 12 mM NaHCO₃, 5.6 mM d-glucose, 0.49 mM MgCl₂, 0.37 mM NaHPO₄, 25 mM HEPES at pH 7.4, 0.75 mM 3-isobutyl-1-methylxanthine, 0.01% BSA (w/v). Then, 25 nM of NDP-α-MSH (corresponding to EC₅₀) in complete buffer was added for 30 minutes at 37°C as above. For maximal response (100%), cells were only incubated with 25 nM NDP-α-MSH as described above. Cells were kept on ice for 5 minutes before proceeding to cAMP measurement. For PC potency assessment of UM0130866, cells were treated the day before the assay with increasing concentrations of UM0130866 for 15 hours. After wash in 1× D-PBS (pH 7.4), cells were incubated 1 hour at 37°C on Heidolph Titramax 100 shaker at speed 900 rpm in complete buffer containing 1 μM NDP-α-MSH. For agonist potency measurement after PC treatment, cells were treated in the presence or absence of 10 μM UM0130866 for 15 hours before the cAMP assay, washed in 1× D-PBS at pH 7.4, and incubated 1 hour at 37°C on Heidolph Titramax 100 shaker at speed 900 rpm in complete buffer containing increasing concentrations of melanocortin agonists. For all assays, plates were then put at –80°C at least 90 minutes and thawed at RT on Heidolph Titramax 100 shaker at speed 1050 rpm. Cell lysates were then transferred to 384-well plates, lysed, and incubated with cAMP labeled with the dye d2 and anti-cAMP M antibody labeled with Cryptate (cAMP dynamic-2 kit, cisbio) according to the manufacturer’s protocol. Reading of the homogeneous time-resolved fluorescence signal was performed on an Artemis time-resolved fluorescence resonance energy transfer plate reader (Cosmo Bio USA).