Photoaffinity probes combined with the chemical proteomic platform have emerged as versatile tools for ligand and target discovery. However, photoaffinity probes with retained activity cannot always label the known target, indicating that it is challenging to profile a ligand’s targets based on its photoaffinity probe modified at a single site. Herein, we construct a series of site-diversified probes (P1-P6) of 4-anilinoquinazoline, a scaffold shared by several marketed EGFR-targeted drugs, via attaching a “fully functionalized” diazirine tag to six different sites, respectively. Chemical proteomic analysis revealed that these probes show different proteome-wide profiles and distinct competition patterns by erlotinib. Remarkably, low activity P4 towards EGFR inhibition has better EGFR labelling efficiency than the higher one, P5, which highlights the dominance of labelling accessibility of diazirine over probe affinity. In addition, the integrated analysis of protein targets of site-diversified probes can also help distinguish false positive targets. We anticipate that site-diversification of the probes of a given scaffold is an indispensable strategy to truly harness the power of photoaffinity-based chemoproteomics in drug discovery.
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Evaluation of Site-Diversified, Fully Functionalized Diazirine Probes for Chemical Proteomic Applications