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

Yini Wang; Bowen Zhong; Caixia Xu; Dongdong Zhan; Songhao Zhao; Hongxing Wu; Mingwei Liu; Xiaoling Lan; Danni Cai; Qian Ding; Biao Zheng; Jiong Lan; Qiang Lv; Yi Wang; Jun Qin

doi:10.1016/j.isci.2023.106080

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

iScience. 2023 Jan 28;26(2):106080. doi: 10.1016/j.isci.2023.106080. eCollection 2023 Feb 17.

Authors

Yini Wang¹, Bowen Zhong¹, Caixia Xu¹, Dongdong Zhan¹, Songhao Zhao², Hongxing Wu³, Mingwei Liu¹, Xiaoling Lan⁴, Danni Cai⁴, Qian Ding⁴, Biao Zheng⁴, Jiong Lan⁴, Qiang Lv⁴, Yi Wang¹, Jun Qin¹

Affiliations

¹ State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (The PHOENIX Center, Beijing), Beijing Institute of Lifeomics, Beijing 102206, China.
² Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China.
³ Beijing Pineal Diagnostics Co., Ltd., Beijing 100195, China.
⁴ GenFleet Therapeutics Inc., Shanghai 201203, China.

Abstract

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.

Keywords: Biochemical assay; Biochemistry methods; Proteomics.