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Somaticmutations in thesmallGTPase K-Ras are themost common activating lesions found in human cancer, and are generally associated with poor response to standard therapies1-3. 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 activeGTP-boundRas.Herewe 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 thereforedo 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 bindingswitch-II region. Binding of these inhibitors toK-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.
To target K-Ras(G12C) we took advantage of the unique nucleophilicity of cysteine thiols by exploring cysteine-reactive small molecules. This strategy has the added advantage of allowing selectivity for the mutant over wild-type K-Ras. Notably, the mutant Cys 12 sits in close proximity to both the nucleotide pocket and the switch regions involved in effector interactions (Fig. 1a).To identify a chemical starting point, we used a disulphide-fragment-based screening approach called tethering8. Wescreened a library of 480 tethering compounds againstK-Ras(G12C) in theGDPstate using intact proteinmass spectrometry9,10 (seeMethods and Extended Data Table 1). Fragments 6H05 (9461% (mean6s.d.)) and2E07 (84.660.3%) gave the greatest degree of modification (Fig. 1b, c). Reaction with wild-type K-Ras, which contains three native cysteine residues, was not detected. Conversely, both compounds modify the oncogenic G12C mutant of the highly homologous protein H-Ras11,12 (Fig. 1b). Binding was not diminished by 1mMGDPin the presence of EDTA, suggesting that the compounds bind in an allosteric site not overlapping with GDP. Pre-loading of K-Ras with GTP significantly impairs modification by both compounds, indicating incompatibility between compound binding and the active conformation of Ras.
We chose to...