Chemoproteomics‐Enabled Identification of 4‐Oxo‐β‐Lactams as Inhibitors of Dipeptidyl Peptidases 8 and 9

Dipeptidyl peptidases 8 and 9 (DPP8/9) have gathered interest as drug targets due to their important roles in biological processes like immunity and tumorigenesis. Elucidation of their distinct individual functions remains an ongoing task and could benefit from the availability of novel, chemically diverse and selective chemical tools. Here, we report the activity‐based protein profiling (ABPP)‐mediated discovery of 4‐oxo‐β‐lactams as potent, non‐substrate‐like nanomolar DPP8/9 inhibitors. X‐ray crystallographic structures revealed different ligand binding modes for DPP8 and DPP9, including an unprecedented targeting of an extended S2′ (eS2′) subsite in DPP8. Biological assays confirmed inhibition at both target and cellular levels. Altogether, our integrated chemical proteomics and structure‐guided small molecule design approach led to novel DPP8/9 inhibitors with alternative molecular inhibition mechanisms, delivering the highest selectivity index reported to date. Achieving Dipeptidyl Peptidase 8 and 9 (DPP8/9) selectivity is a current challenge in chemical biology. Supported by activity‐based protein profiling and X‐ray crystallography, we disclose 4‐oxo‐β‐lactams as potent, non‐substrate‐like nanomolar DPP8/9 inhibitors with different binding modes for DPP8/9, including an unprecedented targeting of an extended S2′ subsite in DPP8 and the best selectivity index reported to date.