Kazunori Hanada

1. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) at a concentration of 0.5 mM had no effect on the serine proteinases plasma kallikrein and leucocyte elastase or the metalloproteinases thermolysin and clostridial collagenase. In contrast, 10 muM-E-64 rapidly inactivated the cysteine proteinases cathepsins B, H and L and papain (t0.5 = 0.1-17.3s). The streptococcal cysteine proteinase reacted much more slowly, and there was no irreversible inactivation of clostripain. The cysteine-dependent exopeptidase dipeptidyl peptidase I was very slowly inactivated by E-64. 2. the active-site-directed nature of the interaction of cathepsin B and papain with E-64 was established by protection of the enzyme in the presence of the reversible competitive inhibitor leupeptin and by the stereospecificity for inhibition by the L as opposed to the D compound. 3. It was shown that the rapid stoichiometric reaction of the cysteine proteinases related to papain can be used to determine the operational molarity of solutions of the enzymes and thus to calibrate rate assays. 4. The apparent second-order rate constants for the inactivation of human cathepsins B and H and rat cathepsin L by a series of structural analogues of E-64 are reported, and compared with those for some other active-site-directed inhibitors of cysteine proteinases. 5. L-trans-Epoxysuccinyl-leucylamido(3-methyl)butane (Ep-475) was found to inhibit cathepsins B and L more rapidly than E-64. 6. Fumaryl-leucylamido(3-methyl)butane (Dc-11) was 100-fold less reactive than the corresponding epoxide, but was nevertheless about as effective as iodoacetate.

A new thiol protease inhibitor, named E-64, was isolated from the extract of a solid culture of Aspergillus japonicus TPR-64 freshly isolated from soil. E-64 was obtained as white needles and the empirical formula was estimated to be C15N5H27O5 (M. W. 357). This was almost neutral in its electrophoretic behavior and proved to be a specific and strong inhibitor toward thiol protease such as papain and cathepsin B. They combine equimolecularly and irreversibly.

A series of new epoxysuccinyl peptides were designed and synthesized to develop a specific inhibitor of cathepsin B. Of these compounds, N ‐(L‐3‐ trans ‐ethoxycarbonyloxirane‐2‐carbonyl)‐L‐isoleucyl‐L‐proline (compound CA‐030) and N ‐(L‐3‐ trans ‐propylcarbamoyloxirane‐2‐carbonyl)‐L‐isoleucyl‐L‐proline (compound CA‐074) were the most potent and specific inhibitors of cathepsin B in vitro. The carboxyl group of proline and the ethyl ester group or n ‐propylamide group in the oxirane ring were necessary, the ethyl ester group or the n ‐propylamide group being particularly effective for distinguishing cathepsin B from other cysteine proteinases such as cathepsins L and H, and calpains.

Epiregulin, a novel epidermal growth factor (EGF)-related growth regulating peptide, was purified from conditioned medium of the mouse fibroblast-derived tumor cell line NIH3T3/clone T7. It was a 46-amino-acid single chain polypeptide, and its amino acid sequence exhibited 24-50% amino acid sequence identity with sequences of other EGF-related growth factors. Epiregulin exhibited bifunctional regulatory properties: it inhibited the growth of several epithelial tumor cells and stimulated the growth of fibroblasts and various other types of cells. Epiregulin bound to the EGF receptors of epidermoid carcinoma A431 cells much more weakly than did EGF, but was nevertheless much more potent than EGF as a mitogen for rat primary hepatocytes and Balb/c 3T3 A31 fibroblasts. These findings suggest that epiregulin plays important roles in regulating the growth of epithelial cells and fibroblasts by binding to receptors for EGF-related ligands.

New derivatives of E-64 (compound CA-030 and CA-074) were tested in vitro and in vivo for selective inhibition of cathepsin B. They exhibited 10,000-30,000 times greater inhibitory effects on purified rat cathepsin B than on cathepsin H and L: their initial Ki values for cathepsin B were about 2-5 nM, like that of E-64-c, whereas their initial Ki values for cathepsins H and L were about 40 200 microM. In in vivo conditions, such as intraperitoneal injection of compound CA-030 or CA-074 into rats, compound CA-074 is an especially potent selective inhibitor of cathepsin B, whereas compound CA-030 does not show selectivity for cathepsin B, although both compounds CA-030 and CA-074 show complete selectivity for cathepsin B in vitro.