In vitro activity: ARS-853 is a novel, potent, selective, covalent inhibitor of KRAS(G12C). It inhibits mutant KRAS-driven signaling by binding to the GDP-bound oncoprotein and preventing activation. KRAS gain-of-function mutations occur in approximately 30% of all human cancers. To date, no targeted therapy has been discovered for cancers with KRAS mutations. Based on the rates of engagement and inhibition observed for ARS-853, along with a mutant-specific mass spectrometry-based assay for assessing KRAS activation status, the nucleotide state of KRAS(G12C) is in a state of dynamic flux that can be modulated by upstream signaling factors. These studies provide convincing evidence that the KRAS(G12C) mutation generates a 'hyperexcitable' rather than a 'statically active' state and that targeting the inactive, GDP-bound form is a promising approach for generating novel anti-RAS therapeutics.

Kinase Assay: GDP-loaded, hexahistidine-tagged, truncated (1-169) KRAS proteins (G12C, WT, G13D, as indicated) at 2 μmol/L final concentration were incubated with the test compounds at the doses and time points indicated in a buffer containing 20 mmol/L HEPES pH 7.5, 150 mmol/L NaCl, 1 mmol/L MgCl2, and 1 mmol/L DTT. Reactions were quenched by adding formic acid to 0.2%. The extent of covalent modification was determined by liquid chromatography, electrospray mass spectrometry analysis of the intact proteins on either a time of flight (TOF; Agilent 6530) or Q-Exactive (Thermo) mass spectrometer.

Cell Assay: Cells (35 × 103) adhered overnight were treated with compound at the indicated concentration and incubation time. After treatment, cells were washed twice with PBS buffer, and proteins were extracted using a buffer containing 9 mol/L urea, 10 mmol/L DTT, and 50 mmol/L ammonium bicarbonate, pH 8. Following iodoacetamide alkylation and trypsin digestion, the samples were analyzed by targeted LC/MS-MS analysis on a Dionex RSLCnano LC (Thermo Scientific) coupled with a Q-Exactive quadrupole orbitrap mass spectrometer (Thermo Scientific). Detailed descriptions of the sample processing and LC/MS-MS methods can be found in Supplementary Methods and Supplementary Table S6. KRASG12C mutant cells (H358) were treated with ARS853 for 5 hours. The effect on the level of active, or GTP-bound, KRAS was determined by a RAS-binding domain pull-down (RBD:PD) assay and immunoblotting with a KRAS-specific antibody.