Fig 1: Analysis of the cellular targets of G12Si-5 using an alkyne analog.a) Structures of probes 6 (an analog of G12Si-5) and 7 (regioisomer of 6). b) Relative growth of Ba/F3:K-Ras(G12S) cells in the presence (10 ng/mL) or absence of IL-3 after treatment with 6 for 72 h. Data is presented as mean values ± standard deviations (n = 3). c) In-gel fluorescence image of SDS-PAGE-resolved lysates of compound-treated A549 cells after click-conjugation of the tetramethylrhodamine (TAMRA) fluorophore. Data is representative of three independent experiments. d) Volcano plot of proteins captured by neutravidin from A549 cells treated with 6 (5 µM) after click conjugation of biotin. These cells were pre-treated with G12Si-5 (10 µM, “Competition”) or DMSO (“No Competition”) for 1 h prior to the addition of probe 6. Proteins were quantified using tandem mass tags. Fold change is calculated as ratio of mean intensities (three biological replicates per condition). p-values are calculated using unpaired, two-tailed Student’s t-test. See Methods section for mass spectrometry methods and Supplementary Dataset 1 for a full list of identified proteins. Source data
Fig 2: G12Si-5 inhibits phospho-ERK signaling in BaF3/K-Ras(G12S) cells.Phospho-ERK levels of BaF3 parental cells (+10 ng/mL IL-3) and BaF3/K-Ras(G12S) cells (no IL-3) after treatment with (a) adagrasib or (b) G12Si-5 for 1 h. Data is presented as mean values ± standard deviations (n = 3).
Fig 3: Optimized β-lactone ligands suppress K-Ras(G12S) signaling in cells.a, Structures of K-Ras(G12S) ligands G12Si-3, G12Si-4 and G12Si-5. b, Time-dependent covalent modification of recombinant K-Ras(G12S)•GDP protein by 10 µM compound at 23 °C assessed by whole-protein MS (n = 3, replicates are plotted as individual data points). c, Immunoblot of A549 cells treated with 10 µM adagrasib, G12Si-3, G12Si-4 or G12Si-5 for 2 h. Data are representative of two independent experiments. d, Immunoblot of A549 cells treated with various concentrations of G12Si-5 for 2 h. Data are representative of two independent experiments. e, Immunoblot of A549, A375, SW1990 and H358 cells treated with DMSO or 10 µM G12Si-5 for 2 h. Data are representative of two independent experiments. WT, wild-type. f, Relative growth of Ba/F3 parental cells (+10 ng ml−1 IL-3) and Ba/F3:K-Ras(G12S) cells (no IL-3) after treatment with adagrasib or G12Si-5 for 72 h. Data are presented as mean ± s.d. (n = 3) and are representative of four independent experiments.Source data
Fig 4: K-Ras(G12S) is an oncogenic driver with intrinsic GTPase activity.a, Immunoblot of Ba/F3 cells expressing wild-type K-Ras (WT), K-Ras(G12S) or K-Ras(G12C). IL-3 was removed from the culture media 10 min before cells were lysed and analyzed. Data are representative of two experiments using independently generated Ba/F3 transductants. For gel source data, see Supplementary Information. b, Growth of Ba/F3 transductants in the absence of IL-3 (n = 3). Data are representative of two experiments using independently generated Ba/F3 transductants. Error bars represent s.d. of three technical replicates. c, X-Ray crystal structure of K-Ras(G12S)•GDP. Insets depict the 2Fo – Fc map for the mutant serine and GDP (1.0σ, gray mesh) and the superimposed structures K-Ras(G12S)•GDP (this structure, gray) and K-Ras(G12C)•GDP (PDB: 4L8G, yellow). For X-ray crystallography data collection and refinement statistics, see Supplementary Table 1. d, Intrinsic and NF1-mediated single-turnover GTPase activity of wild-type K-Ras and K-Ras(G12S) (n = 3). Data points are plotted as the mean. Error bars represent s.d. and are plotted as dashed lines above and below the data points.Source data
Fig 5: Malolactone (R)-G12Di-7 (13) covalently and mutant-selectively modified recombinant and endogenous K-Ras-G12D oncoprotein in cancer cell lines.a, Chemical structure of (R)-G12Di-7. b, Pseudo-first-order K-Ras-G12D labeling kinetics of (R)-G12Di-7. Conditions: K-Ras-G12D (200 nM), (R)-G12Di-7 (10 µM), room temperature. c, Second-order K-Ras-G12D labeling kinetics of (R)-G12Di-7. d, Covalent labeling selectivity against K-Ras WT and mutants. All data points represent individual biological replicates. Data are presented as mean ± standard deviation (n = 3). e, Immunoblot of Ba/F3:K-Ras-G12D, SW1990 AsPC-1, AGS, HCT116, A549, A375 and H1299 cells treated with DMSO or 10 µM (R)-G12Di-7 for 4 h. Data are representative of two independent experiments. f, Stability of covalent complex G12D•(R)-G12Di-7 in cells. Data are representative of two independent experiments. g, Relative growth of Ba/F3:K-Ras-G12D cells (with or without 10 ng ml−1 IL-3) after treatment with (R)-G12Di-7 for 72 h. Data are presented as mean ± standard deviation (n = 3) and are representative of two independent experiments.Source data
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