Kinase experiment:

Purified KRAS (1 μM) is incubated EDTA (10 mM) and GDP (1 mM) or GTPγS (1 mM) at room temperature for 1 h followed by addition of MgCl2 (1 mM) to terminate the reaction. ARS853 (1 μM) is then added and the mixture is incubated for another hour at room temperature. HEK293 cells expressing various KRAS mutants are treated with ARS853. Proteins are extracted using a buffer containing 9M urea, 10 mM DTT and 50 mM ammonium bicarbonate, pH 8, heated to 65℃ for 15 min and alkylated using 50 mM iodoacetamide at 37℃ for 30 min. The samples are desalted by gel filtration in Zeba spin desalting plates followed by addition of sequencing-grade trypsin to a concentration of 10 μg/ml, and incubation for one hour at 37℃. Heavy isotopic standards (25 fmol) of the KRASG12C target peptide and KRAS normalization peptide are added to the samples followed by desalting in Strata-X polymeric reverse phase plates. LC-MS/MS analysis is performed in a Q Exactive quadrupole orbitrap mass spectrometer under standard condition. The amount of KRASG12C bound by the drug is determined by the ratio of the modified G12C peptide to that of the heavy isotopic standards[1].

ARS-853 is a selective, covalent KRASG12C inhibitor with an IC50 of 2.5 uM.

ARS853 is designed to bind KRASG12C with high affinity. Treatment of KRASG12C-mutant lung cancer cells with ARS853 reduces the level of GTP-bound KRAS by more than 95% (10 uM). ARS853 inhibits proliferation with an inhibitory concentration 50% (IC50) of 2.5 uM, which is similar to its IC50 for target inhibition. ARS853 (10 uM) inhibits effector signaling and cell proliferation to varying degrees in six KRASG12C mutant lung cancer cell lines, but not in non-KRASG12C models. Similarly, it completely suppresses the effects of exogenous KRASG12C expression on KRAS-GTP levels, KRAS-BRAF interaction, and ERK signaling. ARS-853 treatment also induces apoptosis in four KRASG12C mutant cell lines. ARS853 selectively reduces KRAS-GTP levels and RAS-effector signaling in KRASG12C-mutant cells, while inhibiting their proliferation and inducing cell death[1]. ARS-853 inhibits mutant KRAS-driven signaling by binding to the GDP-bound oncoprotein and preventing activation[2].

References:
[1]. Lito P, et al. Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism. Science. 2016 Feb 5;351(6273):604-8.
[2]. Patricelli MP, et al. Selective Inhibition of Oncogenic KRAS Output with Small Molecules Targeting the Inactive State. Cancer Discov. 2016 Mar;6(3):316-29.