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

The proteasome regulates cellular processes as diverse as cell cycle progre ssion and NF-KB activation. In this study, we show that the potent antitumo r natural product epoxomicin specifically targets the proteasome. Utilizing biotinylated-epoxomicin as a molecular probe, we demonstrate that epoxomic in 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 ac tivity. The trypsin-like and peptidyl-glutamyl peptide hydrolyzing catalyti c activities also are inhibited at 100- and 1,000-fold slower rates, respec tively. In contrast to peptide aldehyde proteasome inhibitors, epoxomicin d oes not inhibit nonproteasomal proteases such trypsin, chymotrypsin, papain , calpain, and cathepsin B at concentrations of up to 50 mu M. In addition, epoxomicin is a more potent inhibitor of the chymotrypsin-like activity th an lactacystin and the peptide vinyl sulfone NLVS. Epoxomicin also effectiv ely inhibits NF-KB activation in vitro and potently blocks in vivo inflamma tion in the murine ear edema assay. These results thus define epoxomicin as a novel proteasome inhibitor that likely will prove useful in exploring th e role of the proteasome in various in vivo and in vitro systems.