Fig 1.
Chemical structures of BIMs.
Fig 2.
Effect of PKC and other signaling pathways inhibition on OCT1 activity.
HEK-OCT1 cells were exposed, or not, for 1 h to (A) 2 μM Ro 31–8220, 20 μM KN62, 20 μM PP2, 200 nM wortmannin or 5 μM U0126, (B) 2 μM Ro 31–8220, 5 μM Gö 6983, 1 μM staurosporine or 50 μM chelerythrine or (C) 100 nM PMA. Cells were next incubated with 40 μM [14C]-TEA for 5 min at 37°C in the (A-C) absence or (C) presence of 50 μM verapamil, used here as a reference OCT1 inhibitor. After washing with ice-cold PBS, intracellular accumulation of TEA was determined by scintillation counting. Data are expressed as % of accumulation of TEA found in untreated control cells, set at 100%, and are the means ± SEM of at least three independent experiments. *, p < 0.05 when compared with untreated cells (ANOVA followed by Dunnett's post-hoc test).
Fig 3.
Direct cis-inhibition of OCT1 activity by Ro 31–8220.
(A) HEK-OCT1 and HepaRG cells were incubated with 40 μM [14C]-TEA for 5 min at 37°C, in the presence or absence of 50 μM verapamil or 2 μM Ro 31–8220. After washing with ice-cold PBS, intracellular accumulation of TEA was determined by scintillation counting. Data are expressed as % of accumulation of TEA found in control cells, set at 100%, and are the means ± SEM of at least three independent experiments. *, p < 0.05 when compared with untreated cells (ANOVA followed by Dunnett's post-hoc test). (B) HEK-OCT1 cells were incubated with 40 μM [14C]-TEA for 5 min at 37°C, in the presence of various concentrations of Ro 31–8220 (from 0 to 10 μM). After washing with ice-cold PBS, intracellular accumulation of TEA was determined by scintillation counting. Data are expressed as % of accumulation of TEA found in control cells, set at 100%, and are the means ± SEM of four independent experiments. Ro 31–8220 IC50 value is indicated at the top of the graph. (C) HEK-OCT1 cells were incubated with 1 μM DAPI for 5 min at 37°C, in the presence or absence of 50 μM verapamil or 2 μM Ro 31–8220. After washing with ice-cold PBS, accumulation of DAPI was determined by spectrofluorimetry as described in Materials and Methods. Data are expressed as percentage of DAPI accumulation in control cells and are the means ± SEM of four independent experiments. *, p < 0.05 when compared with untreated cells (ANOVA followed by Dunnett's post-hoc test). (D) HEK-OCT1 cells were either untreated or exposed to 50 μM verapamil or 10 μM Ro 31–8220 for 5 min. After washing, cells were re-incubated in drug-free culture medium for 1 h. Cells were next incubated with 40 μM [14C]TEA for 5 min at 37°C. Intracellular accumulation of labelled TEA was then determined by scintillation counting. Data are expressed as % of accumulation of TEA found in untreated control cells, set at 100%, and are the means ± SEM of three independent experiments. *, p < 0.05 when compared with untreated cells (ANOVA followed by Dunnett's post-hoc test).
Fig 4.
Lineweaver-Burk plot of the inhibitory effect of Ro 31–8220 on TEA uptake in HEK-OCT1 cells.
HEK-OCT1 cells were incubated with various concentrations of TEA (from 10 to 1040 μM) for 5 min at 37°C, in the absence or presence of 0.5 μM Ro 31–8220. After washing with ice- cold PBS, intracellular accumulation of TEA was determined by scintillation counting and normalized to total protein content. Data shown are the means ± SEM of four independent experiments.
Fig 5.
Trans-inhibition of OCT1 activity by Ro 31–8220.
HEK-OCT1 cells were either untreated or exposed to 2 mM unlabelled TEA or 10 μM Ro 31–8220 for 1 h. After washing, cells were next incubated with 40 μM [14C]TEA for 5 min at 37°C. Intracellular accumulation of labelled TEA was then determined by scintillation counting. Data are expressed as % of accumulation of TEA found in untreated control cells, set at 100%, and are the means ± SEM of four independent experiments. *, p < 0.05 when compared with untreated cells (ANOVA followed by Dunnett's post-hoc test).
Fig 6.
Inhibition of OCT1 activity by various BIM-related molecules.
HEK-OCT1 cells were incubated with 40 μM [14C]-TEA for 5 min at 37°C in the presence or absence of 50 μM verapamil or of various BIMs, used each at 5 μM. After washing with ice-cold PBS, intracellular accumulation of [14C]-TEA was determined by scintillation counting. Data are expressed as % of accumulation of TEA found in control cells, set at 100%, and are the means ± SEM of three independent experiments. *, p < 0.05 when compared with untreated cells (ANOVA followed by Dunnett's post-hoc test).
Table 1.
Repartition of molecular descriptors correlated with OCT1 activity inhibition for BIMs (|r|>0.80).
Table 2.
Molecular descriptors highly correlated with OCT1 activity inhibition for BIMs (r>0.90).
Fig 7.
Effect of Ro 31–8220 on OCT2, MATE1 and MATE2-K activities.
(A) HEK-OCT2 cells were incubated with 40 μM [14C]-TEA for 5 min at 37°C and pH = 7.4, in the presence or absence of (Left) 500 μM amitriptyline or 5 μM Ro 31–8220, or of (Right) various concentrations (from 0.01 to 10 μM) of Ro 31–8220. After washing by ice-cold PBS, intracellular accumulation of TEA was determined by scintillation counting. Data are expressed as % of accumulation of TEA found in control cells, set at 100%, and are the means ± SEM of three independent experiments. (Left) *, p < 0.05 when compared with untreated cells (ANOVA followed by Dunnett's post-hoc test); (Right) Ro 31–8220 EC50 value towards OCT2 activity is indicated at the top of the graph. (B) HEK-MATE1 or (C) HEK-MATE2-K cells were incubated with 40 μM [14C]-TEA for 5 min at 37°C and pH = 8.4, in the presence or absence of (Left) 200 μM verapamil or 5 μM Ro 31–8220, or of (Right) various concentrations (from 0.003 to 10 μM) of Ro 31–8220. After washing by ice-cold PBS, intracellular accumulation of TEA was determined by scintillation counting. Data are expressed as % of accumulation of TEA found in control cells, set at 100%, and are the means ± SEM of three independent experiments. (Left) *, p < 0.05 when compared with untreated cells (ANOVA followed by Dunnett's post-hoc test); (Right) Ro 31–8220 IC50 values towards (B) MATE1 and (C) MATE2-K activities are indicated at the top of the graphs.