β-arrestin recruitment assay

The PathHunter eXpress OPRM1 CHO-K1 β-Arrestin GPCR assay (Eurofins DiscoverX Corporation) was performed according to the manufacturer’s protocol. Briefly, cells were seeded in 100 µL cell plating reagent in 96 well plates and allowed to incubate at 37 °C (5% CO₂, 95% relative humidity) for 48 h. Each compound was assessed using an 11 point fivefold serial dilution with a starting concentration of 10 µM. Aliquots of compound solution were added to cells (final concentrations were 20 μM forskolin, and 0.4% DMSO) and incubated at 37 °C and 5% CO₂ for 30 min. Detection solution was then added to cells and incubated at room temperature protected from light for 1 h. Luminescence was quantified using a SpectraMax iD5 multi-mode microplate reader with SoftMax Pro software (Molecular Devices, San Jose, CA). Data were normalized to control values and analyzed using nonlinear regression to determine maximal and EC₅₀ values (Prism 8.4 for Mac; Graphpad San Diego, CA).