In vitro activity: SR17018 is a μ-opioid-receptor (MOR) agonist, binding with GTPγS, with an EC50 of 97 nM. SR17018 shows no obvious effect on inducing βarrestin2 recruitment to the MOR at below 10 μM. SR17018 promotes signaling through G proteins or βarrestin2.

Kinase Assay: To determine βarrestin2 recruitment to the human MOR a commercial enzyme fragment complementation assay (β-galactosidase) was used. U2OS-βarrestin-hMOR PathHunter cells were plated at a density of 5,000 cells per well of a 384-well, white-walled assay microplate (Greiner Bio-One) in Assay Complete Cell Plating 5 Reagent (DiscoveRx) 16-20 hr prior to measuring the signal. Cells were treated for 90 min with increasing concentrations of test compounds at 37°C and βarrestin2 recruitment was determined using the PathHunter Detection Kit with the β-galactosidase substrate to detect functional β-galactosidas. The resulting increase in luminescence was measured using a SpectraMax M5e Microplate Reader (Molecular Devices). The average vehicle for the PathHunter U2OS OPRM1 βarrestin cells was 446 ± 25 RLU and the average fold over vehicle for DAMGO was 36 ± 1.

Cell Assay: CHO-hMOR, -hDOR and -hKOR cells were plated at a density of 4,000 cells per well of a 384-well, white-walled, 30 μl-volume microplate (Greiner Bio-One) in Opti-MEM containing 1% FBS 4 hr prior to assaying. Cells were treated with 20 μM forskolin, 25 μM 4-(3-Butoxy-4-methoxybenzyl)imidazolidin-2-one (Ro-20-1724) and increasing concentrations of test compounds for 30 min at 25°C. Inhibition of cAMP was then determined using the Homogeneous Time-Resolved Fluorescence resonance energy transfer (FRET) cAMP HiRange assay by Cisbio (Cisbio-62AM6PEC). Fluorescence was measured at 620 and 665 nm using an Envision Multilabel Reader (PerkinElmer). FRET was calculated by the ratio of 665 nm / 620 nm. The average vehicle ratio for CHO-hMOR cells was 3134 ± 99 and the average fold over vehicle for DAMGO was 2.2 ± 0.04. The average vehicle ratio for CHO-hDOR cells was 2962 ± 181 and the average fold over vehicle for SNC80 was 1.6 ± 0.04. The average vehicle ratio for CHO-hKOR cells was 2965 ± 153 and the average fold over vehicle for U69,593 was 1.9 ± 0.12.