Figure 6

GRK2/3 inhibition and off-target effects by CMPD101. Effect of a range of CMPD101 concentrations on (a) basal (vehicle treated) or (b) 100 µM DAMGO induced μ-OR internalization and (e) basal (vehicle treated) or (f) 37.5 µM DAMGO stimulated β-arrestin2 recruitment in parental HEK293A (WT) cells or HEK293A cell lines with deletion of GRK2 and -3 (ΔGRK2/3). β-arrestin2 recruitment was measured by ebBRET after 6 min stimulation of μ-OR. Data represent the mean ± SEM after buffer (vehicle treated) or vehicle (DAMGO treated) subtraction from 3 independent experiments carried out in duplicate. μ-OR surface expression was determined from donor signals in absence of acceptor for internalization experiments (c) and by ELISA for β-arrestin2 recruitment experiments (g). Data points represent the mean of individual experiments and bars represent the mean ± SEM from three independent experiments with 32 (internalization) or 3 (ELISA) replicates per experiment. In (g) data were normalized to expression in parental cells. (d) Effect of a range of CMPD101 concentrations on donor signals in absence of acceptor. An unaffected donor signal indicates that the compound does not interfere with the assay. Data represent the mean ± SEM from 3 independent experiments carried out in duplicate. Effect of different CMPD101 concentrations on ΔGRK2/3 cells in (a), (b), (d), (e) and (f) was compared to buffer by one-way repeated measures ANOVA with Dunnett’s multiple comparisons test before normalization. Expression in parental and ΔGRK2/3 cells in (c) and (g) was compared by ratio paired t-test before normalization. ***P < 0.001. a.u., arbitrary units.