Epoxomicin, a potent and selective proteasome inhibitor, exhibits in vivo antiinflammatory activity

The Proteasome regulates cellular processes as diverse as cell cycle progression and NF-kappaB 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 microM. 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-kappaB activation in vitro and potently blocks in vivo inflammation 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 various in vivo and in vitro systems.