Fig. 4.
Compound Ch-3 disrupts RAS-effector interactions. Assessment of the inhibition of RAS protein–protein interactions in cells by the chemical series compounds Ch-1, Ch-2, and Ch-3 using different BRET-based RAS biosensor expression vectors. (A–C) Data from BRET assays using RLuc8-KRASG12D with either anti-RAS VHY6-GFP2 (A) with dematured anti-RAS VHY6dm-GFP2 (B) or with full-length CRAFFL-GFP2 (C). (D) Data from BRET assay using a negative control BRET-based biosensor LMO2/VH576dm. The VH576dm is a dematured anti-LMO2 VH. The data are computed relative to cells treated with DMSO vehicle only (open bar) or with Ch-1, Ch-2, or Ch-3 (shaded bars). (E and F) The effect of the Ch-3 compound on mutant KRASG12X (E) and NRASQ61H and HRASG12V (F) interactions with CRAFFL. (G–I) The effect of Ch-3 on the interaction of KRASG12D (G), NRASQ61H (H), and HRASG12V (I) with various RAS effector domains (PI3Kα, PI3Kγ, CRAF, and RALGDS). The range of concentration of the compounds was 5, 10, and 20 μM. Each experiment was repeated at least twice (biological replicates). Statistical analyses were performed using a one-way ANOVA followed by Dunnett’s posttests (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Error bars correspond to mean values ± SD of biological repeats. RLuc8-KRAS, NRAS, and HRAS all comprised full-length RAS components.