Mitsuo Murata

Novel epoxysuccinyl peptides Selective inhibitors of cathepsin B, in vitro

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.

Novel epoxysuccinyl peptides A selective inhibitor of cathepsin B, in vivo

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 10000–30000 times greater inhibitory effects on purified rat cathepsin B than on cathepsin H and L; their initial K 1 values for cathepsin B were about 2–5 nM, like that of E‐64‐c, whereas their initial K 1 values for cathepsins H and L were about 40–200 μM. In in vivo conditions, such us 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.

CA074 methyl ester: A proinhibitor for intracellular cathepsin B

The specificity of compound CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-pro line] for the inactivation of cathepsin B was quantified in in vitro measurements with cysteine endopeptidases from cattle, it being found that the compound is a very rapid inactivator of cathepsin B (rate constant 112,000 M-1.s-1), with barely detectable action on cathepsins H, L, and S or m-calpain. Conversion of the proline carboxyl group of the inhibitor to the methyl ester virtually abolished the effect on cathepsin B, and a possible explanation for the importance of the carboxyl is presented on the basis of the tertiary structure of cathepsin B. It was found that CA074 methyl ester (1 microM, 3 h) caused selective inactivation of the intracellular cathepsin B of human gingival fibroblasts in culture, in contrast to other available agents, and we suggest that CA074 methyl ester will be of value in the elucidation of the biological functions of cathepsin B.

Stimulation of glucose uptake in muscle cells by prolonged treatment with scriptide, a histone deacetylase inhibitor.

Glucose incorporation is regulated mainly by GLUT4 in skeletal muscles. Here we report that treatment of L6 myotubes with scriptide, a hydroxamic acid-based histone deacetylase (HDAC) inhibitor, stimulated 2-deoxyglucose uptake. The effect appeared only after 24 hr, resulting in 2.4-fold glucose uptake at treatment day 6. Scriptide acted synergistically with insulin, indicating it stimulated a distinct pathway from the insulin signaling pathway. It was not observed in undifferentiated myoblasts or 3T3-L1 adipocytes, suggesting a muscle-specific effect of scriptide. A five-carbon chain and hydroxamic acid, essential for histone deacetylase inhibition, were indispensable for this effect, and trichostatin A stimulated glucose uptake as well. Scriptide increased the cellular content of GLUT4, and induced GLUT4 translocation, but GLUT4 mRNA level did not change, indicating scriptide functions posttranslationally. Our results indicated a novel function for HDAC inhibitors of increasing GLUT4 content and its translocation in muscle cells, resulting in stimulation of glucose uptake.

Crystallization and preliminary X-ray study of the cathepsin B complexed with CA074, a selective inhibitor

Cathepsin B from bovine spleen has been purified and crystallized as a complex with a specific inhibitor CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L- isoleucyl-L-proline], using the hanging-drop method. The complex crystals obtained from 50 mM-citrate buffer (pH 3.5) belong to the tetragonal space group P4(1) (or P4(3)) with a = 73.06 A and c = 141.59 A, and diffract beyond 2.2 A resolution. There are two complex molecules per asymmetric unit giving a packing density of 3.37 A3/Da and indicating a high solvent content of 63.5%.

Maturation and degradation of β-galactosidase in the post-Golgi compartment are regulated by cathepsin B and a non-cysteine protease

Lysosomal β‐galactosidase precursor is processed to a mature form and associated with protective protein in lysosomes. In this study we used two cysteine protease proinhibitors, E64‐d for cathepsins B, S, H, and L, and CA074Me for cathepsin B. They are converted intracellularly to active forms, E‐64c and CA074, respectively. Both active compounds inhibited maturation of the exogenous β‐galactosidase precursor, but E‐64c did not inhibit further degradation to an inactive 50‐kDa product. We concluded that cathepsin B participated exclusively in maturation of β‐galactosidase, and a non‐cysteine protease was involved in further degradation and inactivation of the enzyme molecule.

Clarification of substrate specificity of papain by crystal analyses of complexes with covalent-type inhibitors

In order to investigate the stereo specificity of papain Sn subsites (n = 1-4) at the atomic level, two kinds of covalent-type inhibitors were designed based on the previous results on papain-E-64 and papain-E-64-c interactions, and their complex crystals with papain were analyzed by X-ray diffraction. The results show that the hydrophobic regions consisting of Val-133, Val-157 and Asp-158 and of Tyr-61, Gly-66 and Tyr-67 residues interact with the hydrophobic P2 and P3 side chains of inhibitors, thus indicating the function of the former and latter binding pockets as S2 and S3 subsites, respectively. Furthermore, the X-ray analysis suggests that the papain has no definite Sn subsite of n > or = 4, and the S3-P3 hydrophobic interaction is significantly affected by the Pn side chain (n > or = 4) of both the substrate and the inhibitor.