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Cited by 154Protein Kinase Regulation and GTPase SignalingProtein Tyrosine PhosphatasesProtein Degradation and Inhibitors

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Chemical acylation of an acquired serine suppresses oncogenic signaling of K-Ras(G12S)

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Zhang, Ziyang, Guiley, Keelan Z., Shokat, Kevan M.·2022·Nature Chemical Biology
DOI 10.1038/s41589-022-01065-9PMC PMC9596369
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Abstract

Drugs that directly impede the function of driver oncogenes offer exceptional efficacy and a therapeutic window. The recently approved mutant selective small-molecule cysteine-reactive covalent inhibitor of the G12C mutant of K-Ras, sotorasib, provides a case in point. KRAS is the most frequently mutated proto-oncogene in human cancer, yet despite success targeting the G12C allele, targeted therapy for other hotspot mutants of KRAS has not been described. Here we report the discovery of small molecules that covalently target a G12S somatic mutation in K-Ras and suppress its oncogenic signaling. We show that these molecules are active in cells expressing K-Ras(G12S) but spare the wild-type protein. Our results provide a path to targeting a second somatic mutation in the oncogene KRAS by overcoming the weak nucleophilicity of an acquired serine residue. The chemistry we describe may serve as a basis for the selective targeting of other unactivated serines.

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Figures

Fig. 1 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. 1 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 dataCC BY · PubMed Central
Fig. 2 A β-lactone ligand acylates the mutant serine of K-Ras(G12S). a, Structures of β-lactones G12Si-1 and G12Si-2. b, Covalent modification of 4 µM recombinant K-Ras(G12S)•GDP or wild-type K-Ras•GDP proteins treated with 10 µM G12Si-1 or G12Si-2 assessed by whole-protein MS (n = 3). Error bars represent s.d. c, Differential scanning fluorimetry of unmodified K-Ras(G12S)•GDP and the K-Ras(G12S)•GDP•G12Si-1 complex (n = 3). d, Illustration of a biochemical assay that monitors nucleotide exchange using a fluorescent GDP analog. e, Nucleotide exchange of wild-type K-Ras, K-Ras(G12S) or K-Ras(G12S)•G12Si-1 adduct in the presence of magnesium (intrinsic), Sos or EDTA (n = 3).
Fig. 2 A β-lactone ligand acylates the mutant serine of K-Ras(G12S). a, Structures of β-lactones G12Si-1 and G12Si-2. b, Covalent modification of 4 µM recombinant K-Ras(G12S)•GDP or wild-type K-Ras•GDP proteins treated with 10 µM G12Si-1 or G12Si-2 assessed by whole-protein MS (n = 3). Error bars represent s.d. c, Differential scanning fluorimetry of unmodified K-Ras(G12S)•GDP and the K-Ras(G12S)•GDP•G12Si-1 complex (n = 3). d, Illustration of a biochemical assay that monitors nucleotide exchange using a fluorescent GDP analog. e, Nucleotide exchange of wild-type K-Ras, K-Ras(G12S) or K-Ras(G12S)•G12Si-1 adduct in the presence of magnesium (intrinsic), Sos or EDTA (n = 3).CC BY · PubMed Central
Fig. 3 G12Si-1 binds covalently to Ser12 in the switch II pocket of K-Ras(G12S). a, Scheme of the nucleophilic ring opening of the β-lactone in G12Si-1 by serine 12. b, The X-ray co-crystal structure of the K-Ras(G12S)•GDP•G12Si-1 complex. 2Fo – Fc map for the ligand G12Si-1 and serine 12 is depicted in gray mesh (1.0σ). c, Crystal structure of the K-Ras(G12S)•GDP•G12Si-1 adduct and the K-Ras(G12C)•GDP•adagrasib adduct (PDB: 6USZ). d, Superimposition of the conformations of covalent ligands of the S-IIP: G12Si-1 (this work), adagrasib (PDB: 6USZ), sotorasib (PDB: 6OIM), ARS1620 (PDB: 5V9U). For X-ray crystallography data collection and refinement statistics, see Supplementary Table 1.
Fig. 3 G12Si-1 binds covalently to Ser12 in the switch II pocket of K-Ras(G12S). a, Scheme of the nucleophilic ring opening of the β-lactone in G12Si-1 by serine 12. b, The X-ray co-crystal structure of the K-Ras(G12S)•GDP•G12Si-1 complex. 2Fo – Fc map for the ligand G12Si-1 and serine 12 is depicted in gray mesh (1.0σ). c, Crystal structure of the K-Ras(G12S)•GDP•G12Si-1 adduct and the K-Ras(G12C)•GDP•adagrasib adduct (PDB: 6USZ). d, Superimposition of the conformations of covalent ligands of the S-IIP: G12Si-1 (this work), adagrasib (PDB: 6USZ), sotorasib (PDB: 6OIM), ARS1620 (PDB: 5V9U). For X-ray crystallography data collection and refinement statistics, see Supplementary Table 1.CC BY · PubMed Central
Fig. 4 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 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 dataCC BY · PubMed Central
Extended Data Fig. 1 Stability of G12Si-1, G12Si-2 and G12Si-3 in phosphate-buffered saline (PBS) pH 7.4. Data is presented as mean values ± standard deviations (n = 3).
Extended Data Fig. 1 Stability of G12Si-1, G12Si-2 and G12Si-3 in phosphate-buffered saline (PBS) pH 7.4. Data is presented as mean values ± standard deviations (n = 3).CC BY · PubMed Central
Extended Data Fig. 2 G12Si-1 does not react with GppNHp-loaded K-Ras(G12S). Modification of K-Ras(G12D)•GDP and K-Ras(G12D)•GppNHp by G12Si-1 was monitored by whole protein mass spectrometry.
Extended Data Fig. 2 G12Si-1 does not react with GppNHp-loaded K-Ras(G12S). Modification of K-Ras(G12D)•GDP and K-Ras(G12D)•GppNHp by G12Si-1 was monitored by whole protein mass spectrometry.CC BY · PubMed Central
Extended Data Fig. 3 Comparison of the chemical structures of G12Si-1 (this work), adagrasib, ARS1620, and sotorasib. The electrophile portion of each compound is highlighted in red (serine-targeting) or orange (cysteine-targeting).
Extended Data Fig. 3 Comparison of the chemical structures of G12Si-1 (this work), adagrasib, ARS1620, and sotorasib. The electrophile portion of each compound is highlighted in red (serine-targeting) or orange (cysteine-targeting).CC BY · PubMed Central
Extended Data Fig. 4 Measurement of kinetic constants for G12Si-1 and G12Si-5. Measurement of Ki and kinact for the reactions a) between K-Ras(G12S)•GDP and G12Si-1, b) between K-Ras(G12S)•GDP and G12Si-5. For each kinetic curve, kobs was determined by fitting the data to a first-order kinetic model. Replicates are plotted as individual data points in the right panel. See Methods section for additional details on data analysis.
Extended Data Fig. 4 Measurement of kinetic constants for G12Si-1 and G12Si-5. Measurement of Ki and kinact for the reactions a) between K-Ras(G12S)•GDP and G12Si-1, b) between K-Ras(G12S)•GDP and G12Si-5. For each kinetic curve, kobs was determined by fitting the data to a first-order kinetic model. Replicates are plotted as individual data points in the right panel. See Methods section for additional details on data analysis.CC BY · PubMed Central
Extended Data Fig. 5 Co-crystal structure of the K-Ras(G12S)•GDP•G12Si-5 adduct. a) Co-crystal structure of the K-Ras(G12S)•GDP•G12Si-5 adduct. b) Superimposed conformations of G12Si-1 and G12Si-5 in their respective co-crystal structures with K-Ras(G12S)•GDP. For X-ray crystallography data collection and refinement statistics, see Supplementary Table 1.
Extended Data Fig. 5 Co-crystal structure of the K-Ras(G12S)•GDP•G12Si-5 adduct. a) Co-crystal structure of the K-Ras(G12S)•GDP•G12Si-5 adduct. b) Superimposed conformations of G12Si-1 and G12Si-5 in their respective co-crystal structures with K-Ras(G12S)•GDP. For X-ray crystallography data collection and refinement statistics, see Supplementary Table 1.CC BY · PubMed Central
Extended Data Fig. 6 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
Extended Data Fig. 6 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 dataCC BY · PubMed Central
Extended Data Fig. 7 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).
Extended Data Fig. 7 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).CC BY · PubMed Central
Extended Data Fig. 8 Growth inhibition of KRAS p. G12S mutant cell lines by G12Si-5. Relative growth of (a) A549, (b) LS123 and (c) KMS20 cells after treatment with G12Si-5 for 72 h. Data is presented as mean values ± standard deviations (n = 3).
Extended Data Fig. 8 Growth inhibition of KRAS p. G12S mutant cell lines by G12Si-5. Relative growth of (a) A549, (b) LS123 and (c) KMS20 cells after treatment with G12Si-5 for 72 h. Data is presented as mean values ± standard deviations (n = 3).CC BY · PubMed Central

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Metadata

Authors
Zhang, Z., Guiley, K. Z., & Shokat, K. M.
Affiliations
Howard Hughes Medical Institute (US)
Corresponding author
Shokat, Kevan M.
ORCID
Venue
Nature Chemical Biology
Publisher
Springer Science and Business Media LLC
Published
2022-07-21
Volume
18
Issue
11
Pages
1177-1183
ISSN
  • 1552-4450 (ISSN-L)
  • 1552-4469
DOI
10.1038/s41589-022-01065-9
PMC
PMC9596369
OpenAlex
W4286203125
Cited by
153
References
57
Citations by year
2026: 23 · 2025: 36 · 2024: 51 · 2023: 34 · 2022: 9
FWCI
10.70
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Chemical Biology (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by)
Funders
  • Samuel Waxman Cancer Research Foundation
  • Howard Hughes Medical Institute
  • U.S. Department of Health & Human Services | National Institutes of Health (R01CA244550)
  • The Emerald Foundation The Mark Foundation
  • Damon Runyon Cancer Research Foundation (DRG-2281-17, DRG-2399-20)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Tyrosine Phosphatases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
Keywords
KRAS oncogenic signaling, covalent inhibitors, small molecule inhibitors, sotorasib
MeSH terms
Acylation, Cysteine, Humans, Mutation, Oncogenes, Serine, Proto-Oncogene Proteins p21(ras)

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