Figure 2.

Compound 11 disrupts KRAS–Raf interaction. (A) Fluorescence polarization of ^BGTP-γ-SKRAS (0.5 μM) as a function of varying concentration of GST–Raf^RBD in the absence (red) and presence (blue) of 1 μM compound 11. Shown above the curves is the K_D for KRAS–Raf^RBD binding obtained by fitting the data to , where P1 is the polarization of free KRAS, P2 is the polarization of Raf-bound KRAS, c is the total concentration of KRAS, and x is the total concentration of Raf^RBD. (B) Amount of GFP–KRAS^G12D pulled down by GST–Raf^RBD after treatment of cell lysates with compound at the indicated concentrations (representative westerns shown at the top). An equal volume of lysates was used, and the data were normalized to GST–RBD and DMSO control, which also serves as the loading control. The RBD sequence length was 1–149 and 51–131 in the fluorescence polarization and pull-down assays, respectively. Whereas the shorter RBD is sufficient for biochemical assays, the extra amino acids in the longer RBD increases the size and thereby enhances the signal-to-noise ratio in the fluorescence polarization assay. (C) GFP fluorescence lifetime from FLIM–FRET using cells expressing GFP–KRAS^G12D alone or with RFP–Raf (full-length cRaf: residues 1–648), with or without treatment by 1 μM compound 11. (D) GFP fluorescence lifetime from FLIM–FRET using cells expressing GFP–KRAS^G12D or HRAS^G12V with an empty vector pC1 or mCherry-Raf^RBD (RBD: residues 51–131), with or without treatment with 1 μM compound 11. In (B–D), data are shown as mean ± standard error (SE) from three separate experiments; significance was estimated by one-way analysis of variance (ANOVA) relative to the control for each bar in B, second bar in C, and second and fourth bars in D, or relative to the bar immediately to the left of bar 3 in C and bars 3 and 6 in D.