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

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.

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Figure 1: Tethering compounds selectively bind to oncogenic K-Ras(G12C).
Figure 2: Electrophilic compounds bind to S-IIP of K-Ras(G12C) and disrupt switch-I and switch-II.
Figure 3: Compound binding to S-IIP changes nucleotide preference of K-Ras from GTP to GDP.
Figure 4: Compounds block K-Ras(G12C) interactions, decrease viability and increase apoptosis of G12C-containing lung cancer cell lines.

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Protein Data Bank

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Gibbs, J. B., Sigal, I. S., Poe, M. & Scolnick, E. M. Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules. Proc. Natl Acad. Sci. USA 81, 5704–5708 (1984)

    Article  CAS  ADS  Google Scholar 

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

    Article  CAS  ADS  Google Scholar 

  7. Scherer, A. 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)

    Article  CAS  Google Scholar 

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

    Article  CAS  ADS  Google Scholar 

  9. Burlingame, M. A., Tom, C. T. M. B. & Renslo, A. R. Simple one-pot synthesis of disulfide fragments for use in disulfide-exchange screening. ACS Comb. Sci. 13, 205–208 (2011)

    Article  CAS  Google Scholar 

  10. Sadowsky, J. D. 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)

    Article  CAS  ADS  Google Scholar 

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

    Google Scholar 

  12. Bar-Sagi, D. A Ras by any other name. Mol. Cell. Biol. 21, 1441–1443 (2001)

    Article  CAS  Google Scholar 

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

    Article  CAS  ADS  Google Scholar 

  14. Taveras, A. G. 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)

    Article  CAS  Google Scholar 

  15. Naven, R. T., Kantesaria, S., Nadanaciva, S., Schroeter, T. & Leach, K. L. High throughput glutathione and Nrf2 assays to assess chemical and biological reactivity of cysteine-reactive compounds. Toxicol. Rev. 2, 235–244 (2013)

    CAS  Google Scholar 

  16. John, J. 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)

    CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  19. Hall, B. E., Yang, S. S., Boriack-Sjodin, P. A., Kuriyan, J. & Bar-Sagi, D. 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)

    Article  CAS  Google Scholar 

  20. Pai, E. F. 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)

    Article  CAS  ADS  Google Scholar 

  21. Sung, Y.-J., Carter, M., Zhong, J.-M. & Hwang, Y.-W. Mutagenesis of the H-ras p21 at glycine-60 residue disrupts GTP-induced conformational change. Biochemistry 34, 3470–3477 (1995)

    Article  CAS  Google Scholar 

  22. Hwang, M.-C. C., Sung, Y.-J. & Hwang, Y.-W. 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)

    Article  CAS  Google Scholar 

  23. Sunaga, N. 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)

    Article  CAS  Google Scholar 

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

    Article  CAS  ADS  Google Scholar 

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

    Article  CAS  Google Scholar 

  26. Nishimura, A. 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)

    Article  CAS  ADS  Google Scholar 

  27. Maurer, T. 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)

    Article  CAS  ADS  Google Scholar 

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

    Article  Google Scholar 

  29. Shima, F. 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)

    Article  CAS  ADS  Google Scholar 

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

    Article  CAS  ADS  Google Scholar 

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).

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Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Kevan M. Shokat.

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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.

Extended data figures and tables

Extended Data Figure 1 Comparison of co-crystal structure of 6 with K-Ras(G12C) to known structures of Ras.

a, Compound 6 (cyan) bound in the S-IIP of K-Ras(G12C). b, Compound 6 (aligned and overlayed) with GDP-bound wild-type H-Ras showing groove near S-IIP (PDB accession 4Q21)13. c, Clash of compound 6 (aligned and overlayed) with GTPγS-bound K-Ras(G12D), which shows glycerol molecule adjacent to S-IIP (PDB accession 4DSO)27.

Extended Data Figure 2 Additional insights into Ras-compound binding and its biochemical effects.

a, Compound 6 (cyan) is attached to Cys 12 of K-Ras(G12C) and extends into an allosteric binding pocket beneath switch-II (blue), the S-IIP. The binding pocket in K-Ras (surface representation of the protein shown) fits 6 tightly and includes hydrophobic sub-pockets (dashed lines). An extension of the pocket is occupied by water molecules (red spheres) and might provide space for modified compound analogues. bd, X-ray crystallographic studies of K-Ras(G12C) bound to several additional electrophilic analogues (14, 15 and 16, respectively) reveal a similar overall binding mode. All compounds follow a similar trajectory from Cys 12 into S-IIP but show some variability in the region of the piperidine/piperazine. The respective switch-I regions of the protein can be disordered. e, Overlay of the two different crystal forms of K-Ras(G12C) bound to 9 (space group C2 (grey) and P212121 (cyan)) is shown. The ligand orientation and conformation shows minimal changes, whereas switch-II of the protein appears disordered in the C2 form and atypical in the P212121 form. f, An overlay for several compounds including the disulphide 6 is shown (16-green, 6-yellow, 7-orange, 9-cyan). Key hydrophobic residues are labelled and hydrophobic interaction between the compounds and the (p-) or (o-) sub-pockets are indicated by dashed lines.

Extended Data Figure 3 Analysis of compound labelling rate and in vitro specificity.

a, Percentage modification of K-Ras(G12C) by compounds 9 and 12 over time (n = 3, error bars denote s.d.). b, Selective single labelling of K-Ras(G12C) by compound 12 in the presence of BSA. c, Quantitative single labelling of BSA and multiple labelling of K-Ras(G12C) by DTNB. d, Comparison of modification of K-Ras(G12C) and wild-type by 12 (n = 3, error bars denote s.d.).

Extended Data Figure 4 Comparison of active conformation and compound bound form of Ras.

a, X-ray crystal structure of the active conformation of H-Ras(G12C) with GMPPNP shows interactions of the γ-phosphate with key residues (Tyr 32, Thr 35 and Gly 60) that hold switch-I (red) and switch-II (blue) in place. The inactive GDP-bound structure of H-Ras(G12C) reveals the absence of these key interactions and increased distances between these residues and the position of the γ-phosphate (positions from GMPPNP structure indicated by spheres) coinciding with large conformational changes in both switch regions. In the P212121 crystal form of 9 bound to K-Ras(G12C) GDP switch-I is ordered (often disordered by compounds, see Extended Data Table 4), but the structure shows displacement of the γ-phosphate-binding residues beyond their positions in the inactive state. b, As indicated by the X-ray structures, removal of the γ-phosphate leads to relaxation of the ‘spring-loaded’ Ras-GTP back to the GDP state, with opening of switch-II. Compound binding moves switch-II even further away and interferes with GTP binding itself.

Extended Data Figure 5 Inhibitor sensitivity, K-Ras GTP levels and K-Ras dependency of lung cancer cell lines.

a, Percentage viability after treatment for 72 h with 12 relative to DMSO (n = 3 biological replicates, error bars denote s.e.m.). b, K-Ras GTP levels determined by incubating lysates with glutathione S-transferase (GST)-tagged RBD (Ras-binding domain of C-Raf) immobilized on glutathione beads (n = 3 biological replicates). c, Viability of cell lines evaluated 72 h after transfection with KRAS siRNA (n = 3 biological replicates). d, K-Ras immunoblot showing knockdown after KRAS siRNA (n = 3 biological replicates).

Extended Data Table 1 Hit fragments and percentage modification from the primary tethering screen
Extended Data Table 2 Overview of obtained and previously published co-crystal structures and their respective compound–protein binding interfaces
Extended Data Table 3 Extent of labelling after 24 h at 10 μM inhibitor
Extended Data Table 4 Increased distance (Å) between position-12 Cα and Gly 60 Cα correlates with disordering of switch-I

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Ostrem, J., Peters, U., Sos, M. et al. K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions. Nature 503, 548–551 (2013). https://doi.org/10.1038/nature12796

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