Oncogenic KRAS G12C: Kinetic and redox characterization of covalent inhibition

by Selleckchem | Jul 21 2023

The recent development of mutant-selective inhibitors for the oncogenic KRASG12C allele has generated considerable excitement. These inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRASG12C 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 pKa of the C12 thiol is depressed (pKa ∼7.6), consistent with susceptibility to chemical ligation. Using a validated fluorescent KRASY137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRASG12C 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, Kd = 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 KRASG12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRASG12C Cys12 to a sulfinate altered RAS conformation and dynamics to be more similar to KRASG12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRASG12C drug discovery efforts, and identify the occurrence of G12C oxidation with currently unknown biological ramifications.

Comments:

The recent development of mutant-selective inhibitors for the oncogenic KRASG12C allele has generated significant interest in the field of cancer research. These inhibitors work by covalently targeting and inactivating the mutant KRASG12C protein, which plays a critical role in various cancers. However, there are still unanswered questions regarding the reactivity of the C12 thiol located within the phosphoryl binding loop of RAS, which is targeted by these inhibitors.

A study using nuclear magnetic resonance (NMR) and biochemical assays has shed light on the reactivity of the C12 thiol. The study found that the pKa of the C12 thiol is depressed, with a value of approximately 7.6. This suggests that the thiol is more susceptible to chemical ligation, a process in which a covalent bond is formed between the thiol and another molecule. The researchers used a fluorescent variant of KRAS (KRASY137W) and stopped-flow spectroscopy to investigate the kinetics of fluorescence changes upon the addition of two inhibitors, ARS-853 and AMG 510. At low temperatures, the addition of ARS-853 induced a rapid initial fluorescence change, which was attributed to binding (with a dissociation constant, Kd, of 36.0 ± 0.7 μM). A slower pH-dependent phase was observed, representing covalent ligation of the inhibitor to KRASG12C.

Interestingly, the study also discovered that the C12 thiol of KRASG12C is prone to reversible and irreversible oxidation both in vitro and within cells. This oxidation prevents the covalent binding of ARS-853 and potentially has biological implications. The researchers further investigated the consequences of C12 thiol oxidation by performing molecular dynamics simulations. They found that oxidation of the Cys12 residue to a sulfinate altered the conformation and dynamics of the RAS protein, making it more similar to KRASG12D, another common mutant form of KRAS, as assessed by the simulations.

These findings provide valuable insights for future drug discovery efforts targeting KRASG12C. The knowledge of the depressed pKa of the C12 thiol and its susceptibility to oxidation can guide the development of more effective inhibitors. Additionally, the identification of oxidation-induced conformational and dynamic changes in KRASG12C highlights the complexity of RAS signaling and suggests potential biological implications that warrant further investigation.