Kinetic and Redox Characterization of KRAS G12C Inhibition

Abstract

The development of mutant-selective inhibitors for the KRAS G12C allele has generated considerable excitement. These KRAS G12C 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, motivating the present studies. Measurement of the C12 thiol pKa by NMR and an independent biochemical assay found a depressed pKa (relative to free cysteine) of 7.6 consistent with its susceptibility to chemical ligation. Using a novel and 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 ARS-853 addition at 5°C elicited both a rapid first phase (attributed to binding, yielding a Kd of 36.0 ± 0.7 μM), and a second, slower pH-dependent phase taken to represent covalent ligation. Consistent with the lower pKa 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 thiol to sulfinic acid alters 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 as well as identifying the occurrence of G12C oxidation with currently unknown biological ramifications.

Genetic differences between smokers and never-smokers with lung cancer

“…Oxidation of this cysteine residue blocks covalent binding of KRAS Cys12 inhibitors. Therefore, assessment of KRAS Cys12 oxidation status in tumors prior to treatment may facilitate finding the optimal therapy for patients with this mutation (123). New computational methods enable the determination of receptor molecular structure and flexibility in order to design direct inhibitors of KRAS Cys12 (124).…”