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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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).
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.
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. b–d, 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.
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.).
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).
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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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