Oncogenic KRAS G12C: Kinetic and redox characterization of covalent inhibition

The recent development of mutant-selective inhibitors for the oncogenic KRAS(G12C) allele has generated considerable excitement. These inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRAS(G12C) protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain. Here, we show by NMR and an independent biochemical assay that the pK(a) of the C12 thiol is depressed (pK(a) similar to 7.6), consistent with susceptibility to chemical ligation. Using a validated fluorescent KRAS(Y137W) variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRAS(G12C) fluorescence changes upon addition of ARS-853 or AMG 510, noting that at low temperatures, ARS-853 addition elicited both a rapid first phase of fluorescence change (attributed to binding, K-d = 36.0 +/- 0.7 mu M) and a second, slower pH-dependent phase, taken to represent covalent ligation. Consistent with the lower pK(a) of the C12 thiol, we found that reversible and irreversible oxidation of KRAS(G12C) occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRAS(G12C) Cys12 to a sulfinate altered RAS conformation and dynamics to be more similar to KRAS(G12D) in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRAS(G12C) drug discovery efforts, and identify the occurrence of G12C oxidation with currently unknown biological ramifications.

Automated summary

The recent development of mutant-selective inhibitors for the oncogenic KRAS(G12C) allele has generated excitement. These inhibitors covalently engage the mutant C12 thiol, locking the KRAS(G12C) protein in an inactive state. However, there are still mechanistic questions regarding the reactivity of this thiol.