Abstract
RA-0003022 (3) was identified as a high-quality covalent chemical probe for Chikungunya nsP2 cysteine protease (nsP2pro). Isoxazole 3 covalently captured the active site C478 and inactivated the enzyme with a k inact/K i ratio of 6000 M-1s-1. A negative control analog RA-0025453 (4) retained the covalent warhead but demonstrated >100-fold decrease in enzyme inhibition. Isoxazoles 3 and 4 were stable across a wide range of pH in solution and upon prolonged storage as solids. The covalent chemical probe 3 was inactive across a panel of 23 human and viral cysteine proteases and demonstrated remarkable proteome-wide selectivity by two chemoproteomic methods. Isoxazole 3 reduced viral titer against infectious isolates of Chikungunya, Mayaro, and Venezuelan Equine Encephalitis viruses demonstrating its activity on both New and Old World alphaviruses. Isoxazole 3 and its negative control 4 will find utility as covalent chemical probes to study the role of the nsP2pro in alphaviral replication and virulence.