NMR-based fragment screen identifies Ras-binding compounds. (A) One-dimensional proton spectra showing representative screening results. (i) The 1D reference spectrum of a compound pool containing DCAI. Arrows point to the peaks of DCAI. (ii) The STD spectrum of the DCAI-containing pool. (B) These panels show the ¹H¹⁵N HSQC of at a concentration of 100 μM titrated with 0, 250, 500, 1,000, 1,500, and 4,000 μM DCAI (from black to purple). Residues V8, T74, G75, L56, and D57 make up one contiguous site in the Ras protein structure. Side-chain resonances are labeled “sc.”

Ras-ligand cocrystal structures reveal an expandable small-molecule binding pocket in KRas. (A) Structure of KRas_m bound to GTPγS-Mg²⁺. The switch I/II regions are colored in green and orange, respectively. The spheres indicate residues with chemical shift perturbations upon DCAI binding in the NMR experiments. The bound GTPγS is shown as sticks and Mg²⁺ as a blue dot. The disordered region of KRas_m is drawn as a dotted line. Hypothetic cytoplasmic membrane is shown to illustrate its potential interactions with the KRas_m C-terminal domain. (B) The small-molecule binding pocket on KRas. Surface of KRas_m in complex with BZDN (orange) and BZIM (cyan) is colored by electrostatic potential with red being negative and blue being positive. (C) The DCAI-binding pocket on KRas_m. D54 and R41 side chains are shown in rotamer conformation with (cyan) and without (blue) DCAI bound. Arrows indicate the direction of their rotation upon DCAI binding. Residues with chemical shift perturbations in the NMR spectra are colored in gray. (D) A close-up surface rendering of the DCAI 4-chlorine atom (in green) and its interacting surface on KRas_m (in blue) in comparison with the KRas_m surface with no ligand bound (in brown).

DCAI specifically inhibits the SOS^cat-catalyzed nucleotide release and exchange from KRas and KRas_m. (A) Ras/SOS contact surface was calculated using the HRas–SOS1 cocrystal structure (). Ras residues within 4.5 Å of any SOS1 atom in the HRas–SOS1 complex are colored blue; Ras residues that fall outside the HRas–SOS1 interface are colored in white. DCAI (carbon in yellow color) was modeled into the HRas–SOS1 complex by superimposing the nonswitch regions of the KRas_m-DCAI structure onto the apo-HRas structure in the HRas–SOS1 complex. (B) SOS^cat-catalyzed nucleotide release assays were performed in duplicate with 125 nM SOS^cat in the presence of DCAI (red), BZIM (black), or INDL (gray), and the plot shows the rates normalized to that of the DMSO control. Average IC₅₀ for DCAI over three independent experiments under identical conditions was 155 ± 36 μM. (C) SOS^cat-catalyzed nucleotide release assays were performed, in triplicate, as described in C for wild-type KRas (black) and mutant KRas_m (red), except that a higher SOS^cat concentration, 500 nM, was used here to provide an optimal assay window for both KRas_m and KRas. (D)SOS^cat-catalyzed nucleotide exchange reactions were performed in duplicate with KRas^GDP/SOS^cat (black), RhoA^GDP/Dbs (cyan), or Cdc42^GDP/Dbs (magenta) in the presence of DCAI. The plot shows the rates normalized to that of DMSO. Error bars in B–D are the SEM.

DCAI is a competitive inhibitor that blocks the Ras-SOS^cat interaction. (A) Ras coordinates from the DCAI-KRas_m and BZIM-KRas_m complexes were superimposed onto those from the HRas–SOS complex (1NVV; HRas bound to the substrate site of SOS; ref. ) to highlight the locations of the DCAI and BZIM binding sites. The carbon atoms of Ras R41 and D54 are colored in green in the Ras–SOS complex and in yellow in the Ras–DCAI complex. (B) Nucleotide release assays were performed with wild-type Ras^GDP (black) or Ras-R41S^GDP (red) across a range of SOS^cat concentrations. SOS^cat is less effective in catalyzing nucleotide release from Ras-R41S^mant-GDP. (C, Top) Nucleotide-free biotinylated KRas was generated by incubation with EDTA. Streptavidin beads were incubated with SOS^cat alone (lane 1; negative control) or with SOS^cat plus nucleotide-free biotinylated KRas in the presence of increasing concentrations of DCAI (lanes 3–7). Bead-associated SOS^cat was purified, washed, and detected by Western blotting. (Bottom) Identical reactions as above, with the exception that EDTA was omitted in the reaction. Results shown are representative of three independent experiments. (D) Nucleotide release assays were performed, in duplicates, with a dose titration of SOS^cat in the presence of DMSO or of either 125 or 250 μM DCAI. The relative reaction rates were determined by normalizing to those of DMSO control, and the percentage of inhibition by DCAI was plotted for each concentration of SOS^cat. Error bars in B and D are the SEM.