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. 2013 Nov 28;503(7477):548-51.
doi: 10.1038/nature12796. Epub 2013 Nov 20.

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

Jonathan M Ostrem  1 , Ulf Peters, Martin L Sos, James A Wells, Kevan M Shokat
Affiliations

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

Jonathan M Ostrem et al. Nature. .

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 therapies. Efforts to _target this oncogene directly have faced difficulties owing to its picomolar affinity for GTP/GDP and the absence of known allosteric regulatory sites. Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis. 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 GDP 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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Conflict of interest statement

The authors declare competing financial interests: details are available in the online version of the paper.

Figures

Extended Data Figure 1
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). 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).
Extended Data Figure 2
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. 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.
Extended Data Figure 3
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
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
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).
Figure 1
Figure 1. Tethering compounds selectively bind to oncogenic K-Ras(G12C)
a, Crystal structure of K-Ras(G12C) GDP shows Cys 12 (yellow), switch-I (red) and switch-II (blue). Switch-II is partially disordered. b, Percentage modification by compounds 6H05 and 2E07 (n = 3, error bars denote s.d.). c, 6H05 analogue structure–activity relationship. Relative potency = (fragment DR50)/(6H05 DR50), in which DR50 denotes the dose ratio resulting in 50% modification; see Methods. d, Co-crystal structure of 6 (cyan) and K-Ras(G12C) with GDP (grey) and Ca2+ (green). e, Fo − Fc omit map (grey mesh, 2.5σ) of 6 and Cys 12 from d. f, Surface representation of S-IIP around 6 showing hydrogen bonds (yellow lines). Indicated residues make hydrophobic contacts with 6.
Figure 2
Figure 2. Electrophilic compounds bind to S-IIP of K-Ras(G12C) and disrupt switch-I and switch-II
a, Subset of vinyl sulphonamide analogues. b, Subset of acrylamide analogues. Percentages in a and b represent adduct formation after 24 h with 10 μM compound. c, Overlay of co-crystal structures of 8, 9 and 11 with GDP-bound K-Ras(G12C). d–f, Binding of tethering compound 6 or electrophilic compound 8 to Cys 12 (yellow) of K-Ras GDP leads to displacement (arrows) of switch-II (blue) as compared to active Ras (H-Ras(G12C) GMPPNP; d). In the case of tethering compound 6 (e), switch-I (red) resembles the inactive GDP-bound conformation, however electrophile 8 (f) causes partial disordering of switch-I.
Figure 3
Figure 3. Compound binding to S-IIP changes nucleotide preference of K-Ras from GTP to GDP
a, EDTA-mediated competition between mant-dGDP loaded on K-Ras(G12C) and free unlabelled GDP. The experiment was carried out with full-length K-Ras(G12C) alone (squares), or modified by 8 (upwards triangles) or 12 (downwards triangles) (n = 3). Data from a representative experiment is shown fitted to a sigmoidal curve for each protein. b, EDTA-mediated competition between bound mant-dGDP and free unlabelled GTP. c, Quantification of the GDP and GTP titrations in a and b (n = 3; error bars denote s.d.; IC50 obtained from sigmoidal fits). d, e, Schematic representation of experiments shown in a (d) and b (e).
Figure 4
Figure 4. Compounds block K-Ras(G12C) interactions, decrease viability and increase apoptosis of G12C-containing lung cancer cell lines
a–c, SOS-catalysed nucleotide exchange for full-length K-Ras(G12C) alone (a), or K-Ras(G12C) labelled with 8 (b) or 12 (c). d, Schematic representation of a–c. e, Half-life of exchange for a–c (n = 3 biological replicates, error bars denote s.d.). f, Co-immunoprecipitation (IP) of B-Raf and C-Raf with Ras from K-Ras(G12C) cell lines after treatment with compound 12 (n = 3 biological replicates). WCL, whole cell lysate. g, Viability of K-Ras(G12C)-mutant cell lines (H1792, H358, Calu-1 and H23) and cell lines lacking this mutation (A549, H1299 and H1437) after treatment with 12 (n = 3 biological replicates, error bars denote s.e.m.). h, Induction of apoptosis after 48 h with 10 μM 12. i, H1792 cell viability assays carried out as in g, with range of concentrations of 10, 12 and 17 (n = 3 biological replicates, error bars denote s.e.m.).
See this image and copyright information in PMC

Comment in

  • Drug discovery: Pocket of opportunity.
    Bollag G, Zhang C. Bollag G, et al. Nature. 2013 Nov 28;503(7477):475-6. doi: 10.1038/nature12835. Epub 2013 Nov 20. Nature. 2013. PMID: 24256732 No abstract available.
  • Oncogenes: direct hit on mutant RAS.
    Seton-Rogers S. Seton-Rogers S. Nat Rev Cancer. 2014 Jan;14(1):8-9. doi: 10.1038/nrc3650. Epub 2013 Dec 5. Nat Rev Cancer. 2014. PMID: 24304874 No abstract available.

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