The antitumor drug aclacinomycin A, which inhibits the degradation of ubiquitinated proteins, shows selectivity for the chymotrypsin-like activity of the bovine pituitary 20 S proteasome

The antitumor drug aclacinomycin A was previously shown to inhibit the degradation of ubiquitinated proteins in rabbit reticulocyte lysates with an IC50 of 52 mu M (Isoe, T., Naito, M., Shirai, A., Hirai, R., and Tsuruo, T. (1992) Biochim. Biophys. Acta 1117, 131-135). We report here that from all the catalytic activities of the 20 S proteasome tested, the chymotrypsin-like activity was the only one affected by the antitumor drug, An important requirement for inhibition of the chymotrypsin-like activity seemed to be the presence of hydrophobic nonpolar residues in positions P-1 to P-3. Degradation of Z-E(OtBu)AL-pNA and Z-GGL-AMC at pH 7.5 was dramatically (87-98%) inhibited by 50 mu M of the drug, while that of Z-GGL-pNA (containing uncharged polar residues in positions P-2 and P-3) and succinyl-LLVY-AMC (containing an uncharged polar residue in the P-1 position) was inhibited only 11 and 24%, respectively, Aclacinomycin A had no effect on cathepsin B, stimulated trypsin, and inhibited chymotrypsin and, to a lesser extent, calpain, The aglycone and sugar moieties of the cytotoxic drug are essential for inhibition. The results presented here support a major role for the chymotrypsin-like activity in the degradation of ubiquitinated proteins, Aclacino mycin A is the first described non-peptidic inhibitor showing discrete selectivity for the chymotrypsin-like activity of the 20 S proteasome.