Identification of Covalent Ligands – from Single Targets to Whole Proteome

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There has been a surge of interest and efforts in the discovery of covalent ligands for diverse proteins as tool compounds or therapeutic candidates in recent years. We present two studies that involve applications of a target‐centric approach and a ligand‐centric approach toward covalent ligand discovery. By targeting a rare cysteine residue in a receptor tyrosine kinase EphB3, we were able to rapidly identify potent inhibitors of EphB3 with extraordinary proteomic selectivity supported by activity‐based probe profiling. While characterizing an activity‐based probe intended for EphB3 using ABPP, we made a surprising discovery that its primary cellular target was a catalytic subunit of V‐ATPase through its covalent engagement with a cryptic pocket on V‐ATPase. These two approaches will be increasingly used in combination to develop covalent ligands with high potency and yield comprehensive target profiles to accelerate the rate of therapeutic discovery in the future.