Research Article
Oncogenic KRAS G12C: Kinetic and redox characterization of covalent inhibition

https://doi.org/10.1016/j.jbc.2022.102186Get rights and content
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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.

Keywords

KRASG12C
oxidation
covalent inhibitors
lung cancer
thiol alkylators
RAS GTPase

Abbreviations

ABC
ammonium bicarbonate
ABD-F
4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole
ACN
acetonitrile
CD
circular dichroism
d5-NEM
N-ethyl-d5-maleimide
DTT
1,4-dithiothreitol
ESI-TOF
electrospray ionization time-of-flight
GEF
guanine nucleotide exchange factor
MD
molecular dynamics
NEM
N-ethylmaleimide
SOS1
Son-of-Sevenless

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Present address for G. Aaron Hobbs: Medical University of South Carolina, Charleston, SC 29425, USA.