Letter | Published:

K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions

Nature volume 503, pages 548551 (28 November 2013) | Download Citation

Abstract

Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies1,2,3. Efforts to target this oncogene directly have faced difficulties owing to its picomolar affinity for GTP/GDP4 and the absence of known allosteric regulatory sites. Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis5,6. With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent on relative nucleotide affinity and concentration. This gives GTP an advantage over GDP7 and increases the proportion of active GTP-bound Ras. Here we report the development of small molecules that irreversibly bind to a common oncogenic mutant, K-Ras(G12C). These compounds rely on the mutant cysteine for binding and therefore do not affect the wild-type protein. Crystallographic studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath the effector binding switch-II region. Binding of these inhibitors to K-Ras(G12C) disrupts both switch-I and switch-II, subverting the native nucleotide preference to favour GDP over GTP and impairing binding to Raf. Our data provide structure-based validation of a new allosteric regulatory site on Ras that is targetable in a mutant-specific manner.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Data deposits

Atomic coordinates and structure factors for the reported crystal structures have been deposited with the Protein Data Bank (PDB), and accession numbers can be found in Extended Data Table 2.

References

  1. 1.

    et al. K-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung. N. Engl. J. Med. 323, 561–565 (1990)

  2. 2.

    et al. KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib. PLoS Med. 2, e17 (2005)

  3. 3.

    et al. KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer Res. 66, 3992–3995 (2006)

  4. 4.

    et al. Kinetics of interaction of nucleotides with nucleotide-free H-ras p21. Biochemistry 29, 6058–6065 (1990)

  5. 5.

    , , & Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules. Proc. Natl Acad. Sci. USA 81, 5704–5708 (1984)

  6. 6.

    & A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science 238, 542–545 (1987)

  7. 7.

    et al. Crystallization and preliminary X-ray analysis of the human c-H-ras-oncogene product p21 complexed with GTP analogues. J. Mol. Biol. 206, 257–259 (1989)

  8. 8.

    et al. Site-directed ligand discovery. Proc. Natl Acad. Sci. USA 97, 9367–9372 (2000)

  9. 9.

    , & Simple one-pot synthesis of disulfide fragments for use in disulfide-exchange screening. ACS Comb. Sci. 13, 205–208 (2011)

  10. 10.

    et al. Turning a protein kinase on or off from a single allosteric site via disulfide trapping. Proc. Natl Acad. Sci. USA 108, 6056–6061 (2011)

  11. 11.

    et al. The catalogue of somatic mutations in cancer (COSMIC). Curr. Protoc. Hum. Genet. 57, 10.11.1–10.11.26 (2008)

  12. 12.

    A Ras by any other name. Mol. Cell. Biol. 21, 1441–1443 (2001)

  13. 13.

    et al. Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins. Science 247, 939–945 (1990)

  14. 14.

    et al. Ras oncoprotein inhibitors: the discovery of potent, ras nucleotide exchange inhibitors and the structural determination of a drug–protein complex. Bioorg. Med. Chem. 5, 125–133 (1997)

  15. 15.

    , , , & High throughput glutathione and Nrf2 assays to assess chemical and biological reactivity of cysteine-reactive compounds. Toxicol. Rev. 2, 235–244 (2013)

  16. 16.

    et al. Kinetic and structural analysis of the Mg2+-binding site of the guanine nucleotide-binding protein p21H-ras. J. Biol. Chem. 268, 923–929 (1993)

  17. 17.

    & Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol. Cell. Biol. 8, 3235–3243 (1988)

  18. 18.

    & Dominant inhibitory mutations in the Mg2+-binding site of RasH prevent its activation by GTP. Mol. Cell. Biol. 11, 4822–4829 (1981)

  19. 19.

    , , , & Structure-based mutagenesis reveals distinct functions for Ras switch 1 and switch 2 in Sos-catalyzed guanine nucleotide exchange. J. Biol. Chem. 276, 27629–27637 (2001)

  20. 20.

    et al. Structure of the guanine-nucleotide-binding domain of the Ha-ras oncogene product p21 in the triphosphate conformation. Nature 341, 209–214 (1989)

  21. 21.

    , , & Mutagenesis of the H-ras p21 at glycine-60 residue disrupts GTP-induced conformational change. Biochemistry 34, 3470–3477 (1995)

  22. 22.

    , & The differential effects of the Gly-60 to Ala mutation on the interaction of H-Ras p21 with different downstream targets. J. Biol. Chem. 271, 8196–8202 (1996)

  23. 23.

    et al. Knockdown of oncogenic KRAS in non-small cell lung cancers suppresses tumor growth and sensitizes tumor cells to targeted therapy. Mol. Cancer Ther. 10, 336–346 (2011)

  24. 24.

    et al. Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1. Nature 462, 108–112 (2009)

  25. 25.

    et al. Brefeldin A acts to stabilize an abortive ARF–GDP–Sec7 domain protein complex. Mol. Cell 3, 275–285 (1999)

  26. 26.

    et al. Structural basis for the specific inhibition of heterotrimeric Gq protein by a small molecule. Proc. Natl Acad. Sci. USA 107, 13666–13671 (2010)

  27. 27.

    et al. Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity. Proc. Natl Acad. Sci. USA 109, 5299–5304 (2012)

  28. 28.

    et al. Discovery of small molecules that bind to K-Ras and inhibit Sos-mediated activation. Angew. Chem. 124, 6244–6247 (2012)

  29. 29.

    et al. In silico discovery of small-molecule Ras inhibitors that display antitumor activity by blocking the Ras-effector interaction. Proc. Natl Acad. Sci. USA 110, 8182–8187 (2013)

  30. 30.

    et al. Guanosine triphosphatase stimulation of oncogenic Ras mutants. Proc. Natl Acad. Sci. USA 96, 7065–7070 (1999)

Download references

Acknowledgements

We are grateful to M. Burlingame and J. Sadowsky for assistance with the tethering screen; P. Ren and Y. Liu for assistance in chemical design and discussions; N. Younger for preparing several compounds; J. Kuriyan for sharing SOS and H-Ras constructs; F. McCormick and T. Yuan for discussion and sharing K-Ras reagents; R. Goody, K. Shannon and F. Wittinghofer for discussion. U.P. was supported by a postdoctoral fellowship of the Tobacco-related Disease Research Program (19FT-0069). The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under Contract No. DE-AC02-05CH11231. M.L.S. is a fellow of the International Association for the Study of Lung Cancer (IASLC) and receives a Young Investigator Award of the Prostate Cancer Foundation (PCF).

Author information

Author notes

    • Jonathan M. Ostrem
    •  & Ulf Peters

    These authors contributed equally to this work.

Affiliations

  1. Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, California 94158, USA

    • Jonathan M. Ostrem
    • , Ulf Peters
    • , Martin L. Sos
    •  & Kevan M. Shokat
  2. Departments of Pharmaceutical Chemistry and Cellular and Molecular Pharmacology, University of California, San Francisco, California 94158, USA

    • James A. Wells

Authors

  1. Search for Jonathan M. Ostrem in:

  2. Search for Ulf Peters in:

  3. Search for Martin L. Sos in:

  4. Search for James A. Wells in:

  5. Search for Kevan M. Shokat in:

Contributions

J.M.O., U.P., J.A.W. and K.M.S. designed the study. J.M.O., U.P. and K.M.S. designed the molecules and wrote the manuscript. J.M.O. and U.P. performed the initial screen, synthesized the molecules and performed biochemical assays. U.P. expressed and purified the proteins and performed structural studies. J.M.O. and M.L.S. performed the cellular assays. J.M.O., U.P., M.L.S. and K.M.S performed analysis. All authors edited and approved the manuscript.

Competing interests

J.M.O., U.P. and K.M.S. are joint inventors on a UC Regents-owned patent application covering these molecules, which has been licensed to Araxes Pharma LLC. J.M.O., U.P. and K.M.S. hold stock in and are consultants to Araxes Pharma LLC.

Corresponding author

Correspondence to Kevan M. Shokat.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Text and Data, additional references and Supplementary Table 1.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature12796

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.