Fig. 1.

Identifying small-molecule allosteric modulators of β-arrestin recruitment to the β₂AR. (A) Schematic of screening methodologies for cAMP production and β-arrestin binding. Cells were treated with ISO ± test compounds. The primary high throughput screen utilized a luciferase-based cAMP biosensor, GloSensor (Promega), and an enzyme complementation-based assay, PathHunter (DiscoveRx). Independent secondary screening utilized a cAMP ELISA and a BRET assay for β-arrestin recruitment. (B) Structure of DFPQ. (C) Secondary screen for cAMP production by ELISA. HEK 293 cells stably expressing β₂AR were preincubated with 0.1% DMSO (negative control) or 10 μM DFPQ for 30 min and then stimulated with or without 1 μM ISO for 10 min. Cells were lysed and cAMP production was measured. Data are normalized to 1 μM ISO and are the mean % ± SEM, n = 3. (D) Dose–response curve for DFPQ as measured by BRET. HEK 293 cells cotransfected with β-arrestin2-GFP10 and β₂AR-RlucII were preincubated with 0.1% DMSO (negative control) or the indicated concentrations of DFPQ for 30 min. Cells were incubated with Coelenterazine 400a for 2 min and then stimulated with 1 μM ISO. Data for the dose–response curve was taken 12 min post-ISO addition. Data are the mean ± SEM, n = 3. (E) HEK 293 cells cotransfected with β-arrestin2-GFP10 and β₂AR-RLucII were preincubated with the indicated concentrations of DFPQ for 30 min. Cells were then incubated with Coelenterazine 400a for 2 min and then stimulated with the indicated concentrations of ISO. Data for dose–response curves were taken 12 min post-ISO addition and are the mean ± SEM, n = 3. (F) HEK 293 cells stably expressing β₂AR were preincubated with 0.1% DMSO (negative control), 1 μM DFPQ, or 10 μM DFPQ and stimulated with the indicated concentrations of ISO for 10 min. Cells were lysed and cAMP production was measured by ELISA. Data are normalized to 1 μM ISO and are the mean % ± SEM, n = 3.