Comparison of Different Competitive Proteome Profiling Approaches in Target Identification of Covalent Inhibitors

Abstract

Competitive proteome profiling is a powerful approach for the identification of targets of small molecules. This approach usually employs an inhibitor‐derived probe or a cysteine‐reactive probe such as an IA‐alkyne in a comparison between inhibitor‐treated and untreated samples, thus enabling distinction between genuine targets and nonspecific labeling. We have developed an active probe derived from an EGFR inhibitor, afatinib, and a cysteine reactive probe, an alkyne‐containing α,β‐unsaturated amide, to compare their characterization of cellular targets. In both approaches, myosin heavy chain 9 (MYH9) was identified as an off‐target. Subsequent functional validation experiments suggested that MYH9 might be involved in the function of afatinib.