FIGURE 3.

cmp4 affects HRas binding to GEF (RasGRF1) and effector (Raf1-RBD) in a dose-dependent manner. (A) Inhibition of nucleotide dissociation rate on both HRas and HRas^G13D (1 μM) in the presence of 100 μM cmp4 and increasing concentrations of RasGRF1 (range 0.01–0.25 μM). (B) Best fitting pose of cmp4 on HRas-GDP (pink) was superimposed to the structure of nucleotide-free Ras (HRas_nf, in grey) from the crystal structure of the hSos1 catalytic domain associated with HRas (PDB ID: 1bkd). Switch I and II regions are stained darker. GDP is in pink, cmp4 in yellow; (C) Biacore-based direct measurement of 0.5 μM GEF (GST-RasGRF1) binding to His-HRas-GTP in the presence of increasing concentrations of cmp4 (25–1000 μM). In the insert kinetics analysis of RasGRF1 binding to HRas-GDP in the presence of different concentrations of cmp4, relative to SPR curves. All points for initial association rate (v_on, closed symbols, v_off, open symbols) were fitted respectively to a nonlinear ‘growth-sigmoidal Hill’ curve (n = 1), which is reported in the graph as a thin line; (D) Levels of HRas-GTP bound to a Ras binding domain (RBD) of Raf1 in the presence of increasing concentrations of cmp4 (range 0–500 μM), detected with the G-LISA^® kit (Cytoskeleton, Inc. BK131). Data were normalized to Ras-GTP levels measured in the absence of cmp4 (control). All data are significant at 99%, as calculated by Student’s t-test in comparison to control. In the inset, the percentage of inhibition of Ras-GTP bound to RBD as a function of cmp4 concentration, relative to the G-LISA experiment. All points were fitted respectively to a nonlinear ‘growth-sigmoidal Hill’ curve (n = 1).