Metalloprotein-Catalyzed Click Reaction for In Situ Generation of a Potent Inhibitor

In situ click chemistry has great potential for identifying enzyme inhibitors. However, conventional in situ click chemistry provides extremely low yields of the products, making it incompatible with direct activity-based assays. Here, to address this issue, we focused on the catalysis of azide-alkyne cycloaddition (AAC) by the metal ion in metalloproteins. We chose 2-ethynyl N-heterocompounds as alkyne fragments which are activated by coordination to the metal ion. For proof of concept, we applied metal ion-catalyzed in situ AAC to identify inhibitors of Fe(II)-dependent lysine demethylase 5C (KDM5C). The triazole product was obtained in dramatically high yield, dependently on Fe(II) in KDM5C, and the metalloprotein-catalyzed click reaction was compatible with activity-based high-throughput screening, enabling us to discover a potent KDM5C inhibitor. Thus, metal-catalyzed in situ AAC should be generally applicable to other metalloproteins.