MC2 Human Melanocortin GPCR Cell Based Antagonist cAMP Assay. MC2R antagonism measured by cAMP accumulation was performed at Eurofins (catalog #2241) using MC2R stably expressed in HEK293 cells as previously described . Briefly, compounds were screened at 30 uM for single concentration (SC) response and serially, in 3-fold dilutions, from 100 uM to 0.015 nM to obtain concentration response curves (CRC). Reference antagonist ACTH (1-39) was added to all cells except the basal control at a concentration of 100nM. Cells were suspended in HBSS buffer (Invitrogen), 20 mM HEPES (pH 7.4), and 500 uM IBMX and then dispensed to microplates at a concentration of approximately 100 cells/well. Cells were then incubated for 5 minutes in the presence of the reference antagonist, the test compound, or HBSS as a basal control. After incubation for 10 minutes at room temperature, cells are lysed and the fluorescent donor and acceptor compounds were added (D2-labeled cAMP and anti-cAMP antibody labeled with europium cryptate, respectively) and allowed to incubate for 60 minutes at room temperature. Florescence transfer was then recorded (excitation: 337nm; emission: 620 and 665 nm) using a microplate reader (Envision, Perkin Elmer). Compounds were measured in duplicate, and inhibition was calculated by measuring the cAMP accumulation, which was determined by dividing the 655 nm signal by the 620 nm signal and expressed as percent inhibition relative to ACTH agonism. Concentration response curves are provided in Supporting Information. For the selectivity panel we estimated EC50 from single concentration screens with a hyperbolic equation, %inh = ([I]/([I] + IC50), where [I] is the concentration of the inhibitor and %inh is the percent inhibition, assuming 100% inhibition at infinite [I] .