Photoaffinity labeling (PAL) in combination with a chemical probe to covalently bind its target upon UV irradiation has demonstrated considerable promise in drug discovery for identifying new drug targets and binding sites. In particular, carbene-mediated photoaffinity labeling (cmPAL) has been widely used in drug target identification owing to its excellent photolabeling efficiency, minimal steric interference and longer excitation wavelength.Specifically, diazirines, which are among the precursors of carbenes and have higher carbene yields and greater chemical stability than diazo compounds, have proved to be valuable photolabile reagents in a diverse range of biological systems. This review highlights current advances of cmPAL in medicinal chemistry, with a focus on structures and applications for identifying small molecule-protein and macromolecule-protein interactions and ligand-gated ion channels, coupled with advances in the discovery of targets and inhibitors using carbene precursor-based biological probes developed in recent decades.

Target validation of current drugs remains the major challenge for target-based drug discovery, especially for agrochemical discovery. The bactericide 0 represents a novel lead structure and has shown potent efficacy against those diseases that are extremely difficult to control, such as rice bacterial leaf blight. However, no detailed target analysis of this bactericide has been reported. Here, we developed a panel of 0-derived probes 1–6, in which a conservative modification (alkyne tag) was introduced to keep the antibacterial activity of 0 and provide functionality for target identification via click chemistry. With these cell-permeable probes, we were able to discover dihydrolipoamide S-succinyltransferase (DLST) as an unprecedented target in living cells. The probes showed good preference for DLST, especially probe 1, which demonstrated distinct selectivity and reactivity. Also, we reported 0 as the first covalent DLST inhibitor, which has been used to confirm the involvement of DLST in the regulation of energy production.

This review focuses on the identification of serine hydrolases and their inhibitors in mammals and microbes with activity-based protein profiling (ABPP).