The proteasome regulates cellular processes as diverse as cell cycle progression and NF-κB activation. In this study, we show that the potent antitumor natural product epoxomicin specifically targets the proteasome. Utilizing biotinylated-epoxomicin as a molecular probe, we demonstrate that epoxomicin covalently binds to the LMP7, X, MECL1, and Z catalytic subunits of the proteasome. Enzymatic analyses with purified bovine erythrocyte proteasome reveal that epoxomicin potently inhibits primarily the chymotrypsin-like activity. The trypsin-like and peptidyl-glutamyl peptide hydrolyzing catalytic activities also are inhibited at 100- and 1,000-fold slower rates, respectively. In contrast to peptide aldehyde proteasome inhibitors, epoxomicin does not inhibit nonproteasomal proteases such trypsin, chymotrypsin, papain, calpain, and cathepsin B at concentrations of up to 50 μM. In addition, epoxomicin is a more potent inhibitor of the chymotrypsin-like activity than lactacystin and the peptide vinyl sulfone NLVS. Epoxomicin also effectively inhibits NF-κB activation in vitro and potently blocksin vivoinflammation in the murine ear edema assay. These results thus define epoxomicin as a novel proteasome inhibitor that likely will prove useful in exploring the role of the proteasome in variousin vivoandin vitrosystems.

Meng, L., Mohan, R., Kwok, B. H. B., Elofsson, M., Sin, N., & Crews, C. M. (1999). Epoxomicin, a potent and selective proteasome inhibitor, exhibitsin vivoantiinflammatory activity. Proceedings of the National Academy of Sciences, 96(18), 10403–10408.