Abstract
A resurging interest in targeted covalent inhibitors (TCIs) focus on compounds capable of irreversibly reacting with nucleophilic amino acids in a druggable target. p97 is an emerging protein target for cancer therapy, viral infections and neurodegenerative diseases. Extensive efforts were devoted to the development of p97 inhibitors. The most promising inhibitor of p97 was in phase 1 clinical trials, but failed due to the off-target-induced toxicity, suggesting the selective inhibitors of p97 are highly needed. We report herein a new type of TCIs (i.e., FL-18) that showed proteome-wide selectivity towards p97. Equipped with a Michael acceptor and a basic imidazole, FL-18 showed potent inhibition towards U87MG tumor cells, and in proteome-wide profiling, selectively modified endogenous p97 as confirmed by in situ fluorescence scanning, label-free quantitative proteomics and functional validations. FL-18 selectively modified cysteine residues located within the D2 ATP site of p97. This covalent labeling of cysteine residue in p97 was verified by LC‒MS/MS-based site-mapping and site-directed mutagenesis. Further structure-activity relationship (SAR) studies with FL-18 analogs were established. Collectively, FL-18 is the first known small-molecule TCI capable of covalent engagement of p97 with proteome-wide selectivity, thus providing a promising scaffold for cancer therapy.