Recent advances in identifying protein targets in drug discovery.

Phenotype-based screening has emerged as an alternative route for discovering new chemical entities toward first-in-class therapeutics. However, clarifying their mode of action has been a significant bottleneck for drug discovery. For target protein identification, conventionally bioactive small molecules are conjugated onto solid supports and then applied to isolate target proteins from whole proteome. This approach requires a high binding affinity between bioactive small molecules and their target proteins. Besides, the binding affinity can be significantly hampered after structural modifications of bioactive molecules with linkers. To overcome these limitations, two major strategies have recently been pursued: (1) the covalent conjugation between small molecules and target proteins using photoactivatable moieties or electrophiles, and (2) label-free target identification through monitoring target engagement by tracking the thermal, proteolytic, or chemical stability of target proteins. This review focuses on recent advancements in target identification from covalent capturing to label-free strategies.