Global profiling of AMG510 modified proteins identified tumor suppressor KEAP1 as an off-target.

KRAS inhibitor AMG510 covalently modifies the G12C residue and inactivates the KRAS/G12C function. Because there are many reactive cysteines in the proteome, it is important to characterize AMG510 on-target modification and off-targets. Here, we presented a streamlined workflow to measure abundant AMG510 modified peptides including that of KRAS/G12C by direct profiling, and a pan-AMG510 antibody peptide IP workflow to profile less abundant AMG510 off-targets. We identified over 300 off-target sites with three distinct kinetic patterns, expanding the AMG510 modified proteome involved in the nucleocytoplasmic transport, response to oxidative stress, adaptive immune system, and glycolysis. We found that AMG510 covalently modified cys339 of ALDOA and inhibited its enzyme activity. Moreover, AMG510 modified KEAP1 cys288 and induced NRF2 accumulation in the nuclear of NSCLC cells independent of KRAS/G12C mutation. Our study provides a comprehensive resource of protein off-targets of AMG510 and elucidates potential toxicological sideeffects for this covalent KRASG12C inhibitor.