Fig. 4
Abd-7 disrupting RAS-effector interactions. HEK293T cells were transfected with different BRET-based RAS biosensor expression vectors to evaluate the inhibition of RAS PPI in cells by compound Abd-7. Transfection vector encoded full-length RAS was fused to the donor molecule RLuc8 and the effectors fused to the acceptor molecule GFP2. a Effect of Abd-2 and Abd-7 on KRASG12D interaction with PI3Kα, PI3Kγ, CRAF or RALGDS. The BRET signal is plotted as a % of control cells treated with DMSO only and dose response to 5, 10 and 20 μM of each compound. b Effect of Abd-2 and Abd-7 on the BRET signal from interaction of KRASG12 mutants (Rluc8-KRASG12) and full-length CRAF (GFP2-CRAF FL). c, d Effect of Abd-2 and Abd-7 on the interaction of NRASQ61H (c) and HRASG12V (d) with various RAS effectors domain and with full-length CRAF. The BRET ratio corresponds to the light emitted by the GFP2 acceptor constructs (515 nm ± 30) upon addition of Coelenterazine 400a divided by the light emitted by the RLuc8 donor constructs (410 nm ± 80). The normalized BRET ratio is the BRET ratio normalized to the DMSO negative and calculated as follows: (BRETcompound/BRETDMSO) x 100, where BRETcompound corresponds to the BRET ratio for the compound-treated cells, BRETDMSO to the DMSO-treated cells. Each experiment was repeated at least three times. Statistical analyses were performed using a one-way ANOVA followed by Dunnett’s post-tests (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Where error bars are presented, they correspond to mean values ± SD of biological repeats (a–c)
