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 μM (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 P1 to P3. Degradation of Z-E(OtBu)AL-pNA and Z-LLL-AMC at pH 7.5 was dramatically (87-98%) inhibited by 50 μm of the drug, while that of Z-GGL-pNA (containing uncharged polar residues in positions P2 and P3) and succinyl-LLVY-AMC (containing an uncharged polar residue in the P1 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. Aclacinomycin A is the first described non-peptidic inhibitor showing discrete selectivity for the chymotrypsin-like activity of the 20 S proteasome.

The eukaryotic 20 S proteasome or multicatalytic proteinase complex (MPC) 1 is a multimeric 700-kDa enzyme with subunits of similar size but different charges, arranged in four stacked rings with heptameric symmetry (reviewed in Ref. 2). The subunits of the eukaryotic 20 S proteasome of known amino acid sequence have been divided into two superfamilies with similarities to either the ␣or the ␤-type subunits of the archaebacterial MPC (3). Subunit arrangement may vary among different populations of the eukaryotic 20 S proteasome (3), but they are organized as symmetrical dimers with the ␣-type subunits forming the outer rings (4).
Recent x-ray crystallographic and site-directed mutagenesis studies showed that the active site of the archaebacterial 20 S proteasome resides in the ␤-type subunit and that its N-terminal threonine contributes to the nucleophilic attack in the active site (5,6). Lactacystin, an irreversible inhibitor of the mammalian 20 S proteasome, was shown to covalently modify the N-terminal threonine of a ␤-type subunit, strongly suggesting that the residue may play a catalytic role in the eukaryotic molecule as well (7). Three of the cloned ␤-type subunits in the eukaryotic 20 S proteasome lack the conserved N-terminal threonine residue and were proposed to be catalytically inactive (5,6).
The eukaryotic MPC has numerous distinct catalytic centers, an advantage for an enzyme that plays a major role in the degradation of nuclear and cytosolic proteins and polypeptides. The catalytic activities include those that hydrolyze peptide bonds on the carboxyl side of basic (trypsin-like), hydrophobic (chymotrypsin-like), acidic (peptidylglutamyl peptide or PGP), branched chain, and small neutral amino acids (8 -11). The initial breakdown of ␤-casein was shown to be accomplished by a catalytic center (caseinolytic activity) different from the first three peptidase activities described above (12)(13)(14)(15). Studies on dissociation and reassociation of the mammalian MPC indicate that structural integrity is required for expression of proteolytic and peptidase activities (16).
Attempts have been made to relate different catalytic activities with specific subunits of the eukaryotic 20 S proteasome. Of the 14 cloned yeast subunits, mutations in the pre1 and pre2 genes produced strains defective in chymotrypsin-like activity (17,18), mutations in pre3 and pre4 led to a deficiency in PGP activity (19,20), and PUP3 mutants were impaired in trypsinlike activity (21,22). Subunit-binding studies with a specific inhibitor of the chymotrypsin-like activity showed preferential incorporation of diisopropyl fluorophosphate into the smallest subunit of the chicken liver (23) and bovine lens MPC (24). Investigations with leupeptin, an inhibitor of the trypsin-like activity, identified only one (25) or two (26) mammalian MPC subunits that specifically bound the arginine peptide aldehyde.
In eukaryotic cells, non-ubiquitinated and ubiquitinated protein substrates are respectively degraded by MPC or by a larger complex, the 26 S proteasome (ubiquitin/ATP-dependent proteinase), of which MPC is the catalytic core (reviewed in Ref. 27). The enzymatic centers involved in the initial degradation of different protein substrates have not been identified. Previous work from another laboratory showed that the degradation of ubiquitinated proteins, but not ubiquitination itself, was inhibited by the DNA-intercalative agent aclacinomycin A, also known as aclarubicin (1). We present evidence that the inhibitory potency of the antibiotic is most effective against the MPC activity measured with short synthetic substrates containing nonpolar hydrophobic residues in positions P 1 to P 3 . Furthermore, we show that aclacinomycin A is the first non-peptidic drug with apparent selectivity for the chymotrypsin-like activity of MPC. Both aglycone and sugar moieties of the cytotoxic drug are essential for its inhibitory properties. These studies suggest that a rate-limiting step in the degradation of ubiquitinated proteins involves cleavage after a series of nonpolar hydrophobic residues.

EXPERIMENTAL PROCEDURES
Materials-The 20 S proteasome was isolated from bovine pituitaries as described (28). Frozen bovine pituitaries were from Pel-Freez Biologicals, Inc. (Rogers, AR). Z-GGL-pNA, Z-DALR-NA, and Z-LLL-AMC were synthesized as described (8,29,30). The synthesis and some properties of Z-E(OtBu)AL-NA, Z-EAL-pNA, and Z-IEAL-pNA were recently described (31). Z-GPAGG-pAB and Z-GPALG-pAB were a generous gift from Dr. C. Cardozo (Department of Pharmacology, Mount Sinai Medical School, New York, NY). Z-LLE-NA, succinyl-LLVY-AMC, dephosphorylated ␤-casein, rabbit muscle calpain, bovine pancreas ␣-chymotrypsin and trypsin were from Sigma. Cathepsin B was prepared in this laboratory by E. Wilk from rat liver as described (32,33). Aclacinomycin A was a generous gift from the Drug Synthesis & Chemistry Branch, Development Therapeutics Program, Division of Cancer Treatment, National Cancer Institute and from Mercian Corp., Fujisawa, Japan. Aclacinomycin B and all of the aclacinomycin A analogs were a generous gift from Mercian Corp. Other reagents were of highest purity available.
Purification of the Aglycone and Sugar Moieties of Aclacinomycin A-Hydrogenolysis of the anthracycline was as described (34). Separation of the aglycone and sugar moieties was accomplished by chromatography on a Sephadex LH-20 column (34).
Enzyme Assays-Enzyme activities with synthetic substrates were determined as described previously, at 37°C (8,9). Substrates were Z-GGL-pNA, Z-DALR-NA, and Z-LLE-NA for determination of the chymotrypsin-like, trypsin-like, and PGP activities of the 20 S proteasome, respectively. MPC hydrolysis of Z-GPALG-pAB (branched chain amino acid preferring activity) and Z-GPAGG-pAB (small neutral amino acid preferring activity) were measured as described (11). Degradation of dephosphorylated ␤-casein by MPC (caseinolytic activity) and by calpain were determined by a gel electrophoretic method (12,35) and quantified by image analysis (13). Treatment of MPC with 3,4 dichloroisocoumarin was as described (15). The activity of cathepsin B was probed with Z-LLR-NA in a 0.2 M sodium acetate, 2 mM EDTA buffer, pH 4.8 (32,33). The activity of chymotrypsin toward Z-GGL-pNA and Z-E(OtBu)AL-pNA was measured in 0.05 M Tris-HCl, pH 7.5, and trypsin activity toward Z-DALR-NA was analyzed in the presence of 1 mM CaCl 2 in 0.05 M Tris-HCl, pH 8.0.
Specific activities are expressed in terms of units/mg of enzyme, where 1 unit of enzymatic activity is defined as the amount of enzyme liberating 1 mol of aromatic amine/h.
Inhibition Assays-Solutions of aclacinomycin A and its analogs were prepared either in H 2 O or in dimethyl sulfoxide. The solutions were added directly to the assay mixtures containing buffer, enzymes, and their respective substrates in a total volume of 100 l. The final dimethyl sulfoxide concentration was 1.5%. All activities were expressed relative to control (vehicle alone) conditions.

Chymotrypsin-like Activity of MPC Measured with Z-LLL-AMC and Z-E(OtBu)AL-pNA-
In an effort to characterize the MPC activity toward Z-LLL-AMC and Z-E(OtBu)AL-pNA, the relationships between substrate concentration and rate of hydrolysis were determined ( Fig. 1). Both reactions followed normal Michaelis-Menten kinetics with apparent K m values of 2.7 and 2.2 mM, and V max of 10 units/mg and 34 units/mg of enzyme with Z-LLL-AMC and Z-E(OtBu)AL-pNA, respectively, estimated by Lineweaver-Burk plots (Fig. 1). Substrate saturation could not be reached with either of the substrates because of their limited solubility.
Changes in the Chymotrypsin-like Activity of the 20 S Proteasome Produced by Aclacinomycin A-The effect of the cytotoxic drug on MPC hydrolysis of peptide bonds after hydrophobic amino acids was studied with six different substrates ( Fig.  2 and Table I). Among the synthetic chromogenic substrates utilized to measure the chymotrypsin-like activity of MPC, Z-LLL-AMC and Z-E(OtBu)AL-pNA were the most sensitive to inhibition by aclacinomycin A, with IC 50 values of 16 and 18 M, respectively (Fig. 2). Low concentrations of the cytotoxic drug (up to 10 M) slightly stimulated hydrolysis of the L-pNA bond in Z-E(OtBu)AL-pNA (Fig. 2).
Surprisingly, hydrolysis of Z-EAL-pNA, a substrate identical to Z-E(OtBu)AL-pNA except for the unblocked glutamate residue, was not affected by aclacinomycin A concentrations up to 100 M (Table I). Insertion of an additional nonpolar hydrophobic group (Ile), as in Z-IEAL-pNA, significantly increased the inhibitory effect of the drug. MPC hydrolysis of Z-EAL-pNA and Z-IEAL-pNA followed normal Michaelis-Menten kinetics with apparent K m values of 1.0 and 0.86 mM and V max of 6.1 and 6.4 units/mg of enzyme, respectively, estimated by Lineweaver-Burk plots (not shown).
The potency of the aclacinomycin A-inhibition was greatly reduced against those substrates with uncharged polar (Gly or Tyr) residues in positions P 1 -P 3 (Table I,  fold trypsin hydrolysis of Z-DALR-NA, had little effect on cathepsin B, and inhibited calpain degradation of dephosphorylated ␤-casein by 44% (Fig. 3).
Examination of the Leu-X hydrolase activity of chymotrypsin with the substrates Z-GGL-pNA and Z-E(OtBu)AL-pNA in the presence of increasing concentrations of aclacinomycin A yielded curves similar to the ones obtained with the 20 S proteasome (Fig. 4, A and B). At the concentrations of aclacinomycin A tested (up to 50 M) only a small decrease in the hydrolysis of Z-GGL-pNA by either the 20 S proteasome or by chymotrypsin was detected. However, aclacinomycin A significantly decreased hydrolysis of Z-E(OtBu)AL-pNA by MPC or by chymotrypsin with IC 50 values of 18 and 24 M, respectively.
Kinetics of the Aclacinomycin A Inhibition-Lineweaver-Burk analysis established that aclacinomycin A is a noncompetitive inhibitor of the hydrolysis of Z-LLL-AMC by the 20 S proteasome and of the hydrolysis of Z-E(OtBu)AL-pNA by chy-motrypsin (Fig. 5). In both cases the inhibition was reversible.
Effect of Different Analogs of Aclacinomycin A on the Chymotrypsin-like Activity of MPC and on the Activity of Chymotrypsin-Aclacinomycin A (Fig. 6) is an anthracyclic antibiotic consisting of an aglycone moiety (aklavinone) linked to a trisaccharide moiety with one aminosugar (L-rhodosamine) and two deoxysugars, namely 2-deoxy-L-fucose and L-cinerulose A (34,36). To establish the structural requirements of the inhibitory reaction, different analogs of aclacinomycin A described by Oki et al. (34) were tested for their ability to modify the chymotrypsin-like activity of MPC and the activity of chymotrypsin (Table II).
The effect of the aclacinomycin A-analogs on MPC activity toward Z-LLL-AMC and Z-E(OtBu)AL-pNA were similar. Aklavinone, the aglycone moiety of aclacinomycin A and B, had no inhibitory properties, and neither did tetracycline, another antibiotic with a similar aglycone structure but lacking sugars. No significant change in inhibitory potency was detected with the analogs that differed from aclacinomycin A by substitution of the aminosugar for a deoxysugar, such as in U5; by replacement of any of the deoxysugars, such as in aclacinomycin B and MA144-M1; or by deletion of only one deoxysugar, such as in MA144-S1. However, inhibition by MA144-T1, a compound missing two of the deoxysugars, and by daunomycin, another anthracycline antibiotic with a different aglycone (daunomycinone) linked to only one aminosugar (daunosamine), was less  pronounced. Intermediate inhibitory values were obtained in reactions run in the presence of the sugar moiety alone. Comparable results to those described above were obtained with the different aclacinomycin A-analogs on the activity of chymotrypsin toward Z-E(OtBu)AL-pNA (Table II). Of all the compounds listed in Table II, daunomycin was the most effective inhibitor of MPC hydrolysis toward succinyl-LLVY-AMC, decreasing it by 40%. The MPC activity toward Z-GGL-pNA was only modestly reduced by the aclacinomycin A-analogs. DISCUSSION Aclacinomycin A was found to inhibit the degradation of ubiquitinated proteins in reticulocyte lysates at a step following ubiquitin-protein conjugation (1). The antitumor drug did not interfere with ubiquitination and did not interact directly with ubiquitin (1). We explored the possibility that the targets for aclacinomycin A action were the catalytic centers of the 20 S proteasome.
From all the catalytic activities of MPC measured, only the chymotrypsin-like component tested with two substrates containing hydrophobic nonpolar residues in positions P 1 to P 3 (namely, Z-E(OtBu)AL-pNA and Z-LLL-AMC) was highly sensitive to the antibiotic, with IC 50 values of approximately 18 M. Hydrolysis of two other substrates, Z-GGL-pNA and succinyl-LLVY-AMC, frequently utilized to probe the chymotrypsinlike activity of MPC, were only repressed 11 and 24%, respectively, by 50 M aclacinomycin A.
The catalytic activities of the 20 S proteasome toward other short synthetic substrates representing the trypsin-like (Z-DALR-NA), PGP (Z-LLE-NA), branched chain amino acid preferring (Z-GPALG-pAB), and small neutral amino acid preferring (Z-GPAGG-pAB) activities were not affected by concentrations of aclacinomycin A up to 100 M. The caseinolytic activity of 3,4 dichloroisocoumarin-treated MPC probed with dephosphorylated ␤-casein was moderately inhibited (29%) by 100 M of the cytotoxic drug. The effect of the drug on other proteinases was studied. Aclacinomycin A had no effect on cathepsin B, stimulated trypsin, and moderately inhibited calpain. Chymotrypsin-hydrolysis of Z-E(OtBu)AL-pNA was potently inhibited by the drug (IC 50 ϭ 24 M), but that of Z-GGL-pNA was hardly affected.
The finding that aclacinomycin A inhibits the cleavage of substrates containing a series of nonpolar hydrophobic residues preceding the scissile bond and inhibits the degradation of ubiquitinated proteins indicates that the chymotrypsin-like activity of the 20 S proteasome is a major factor in the degradation of ubiquitinated proteins.
Other studies support these findings. The degradation of ubiquitinated proteins either in whole cells or by the reticulo-cyte lysate fraction II was found to be inhibited by the peptidyl aldehyde Z-IE(OtBu)AL-CHO (37) or by the amyloid ␤-protein (38), respectively. Most likely, the mechanism of inhibition of the breakdown of ubiquitinated proteins by aclacinomycin A, Z-IE(OtBu)AL-CHO, and amyloid ␤-protein is not the same. However, all three compounds inhibited primarily the chymotrypsin-like activity of the 20 S proteasome measured with short synthetic substrates containing a series of nonpolar hydrophobic residues preceding the cleavage bond.
Yeast mutants defective in chymotrypsin-like activity accumulate ubiquitinated proteins when subjected to heat stress or when grown in the presence of canavanine (17,18). No deficiency in ubiquitin-dependent proteolysis was detected in yeast mutants defective in PGP activity (19,20). Together, these results suggest that the chymotrypsin-like component may be responsible for a rate-limiting step in the cleavage of ubiquitinated proteins. However, they do not exclude the possibility that other catalytic components of the 20 S proteasome may also play an important role in the degradation of ubiquitinated proteins.
Interestingly, Davies and co-workers (39) proposed that increased proteolytic susceptibility caused by oxidation coincides with partial unfolding and exposure of previously buried hydrophobic domains of the oxidatively modified proteins. Partial unfolding and exposure of hydrophobic domains in proteins could likewise be a recognition signal for degradation by the ubiquitin/ATP-dependent proteinase.
Lactacystin, another non-peptidic compound isolated, like aclacinomycin A, from Streptomyces, was found to inhibit at least three peptidase activities of the 20 S proteasome, namely chymotrypsin-like, trypsin-like, and PGP (7). The ␤-lactone is an irreversible inhibitor of the first two peptidase activities, but it covalently modifies the N-terminal threonine of only one ␤-type subunit of the eukaryotic 20 S proteasome. We show that aclacinomycin A has a greater selectivity for the different catalytic components of the 20 S proteasome than the ␤-lactone, as it primarily inhibits the chymotrypsin-like activity and the degradation of ubiquitinated proteins. Aclacinomycin A is a reversible noncompetitive inhibitor of the hydrolysis of Z-LLL-AMC and Z-E(OtBu)AL-pNA by the 20 S proteasome and chymotrypsin, respectively. Therefore, aclacinomycin A may bind to an allosteric site, causing the distortion of the catalytic site and obstructing its access to the scissile bond (40).
The powerful antitumor properties of aclacinomycin A were shown to result from the disruption of the structure of DNA as it intercalates into the helices. Based on nuclear magnetic resonance structural studies, it was deduced that the elongated aglycone portion of the molecule inserts between the base pairs of the DNA double helix and the trisaccharide tail lies in the TABLE II Effect of analogs of aclacinomycin A on the chymotrypsin-like activity of the 20 S proteasome (2.5 g) and on chymotrypsin (2 g) Enzymatic activities were measured with synthetic substrates (400 M) as described under "Experimental Procedures." All activities are expressed relative to zero drug treatment. The data represent means of at least three experiments for each condition.

Drug
Sugar moiety 20 S proteasome Chymotrypsin Z-GGL-pNA Suc-LLVY-AMC Z-LLL-AMC Z-E(OtBu)AL-pNA Z-E(OtBu)AL-pNA minor grooves (41). Studies in progress are addressing the mechanism of action of aclacinomycin A toward the 20 S proteasome. We established that both the aglycone and sugar moieties of the antitumor drug are required for inhibition of the 20 S proteasome. For maximal effect, the composition of the sugar moiety may vary, but must contain at least two sugars. The 20 S proteasome may play an important role in different disease states, such as inflammation (reviewed in Ref. 42) or muscle atrophy (43). The discovery of a non-peptidic, cell permeable inhibitor of the 20 S proteasome, such as aclacinomycin A, may lead to the development of other, more potent inhibitors, which may be useful as novel therapeutic drugs.