CA-074, but not its methyl ester CA-074Me,is a selective inhibitor of cathepsin B within living cells

Studies using inhibitors that reportedly discriminate between cathepsin B and related lysosomal cysteine proteinases have implicated the enzyme in a wide range of physiological and pathological processes. The most popular substance to selectively inhibit cathepsin B in vivo is CA-074Me, the methyl ester of the E-64 derivative CA-074. However, we now have found that CA-074Me inactivates both cathepsin B and cathepsin L within murine fibroblasts. In contrast, exposure of these cells to the parental compound CA-074 leads to the selective inhibition of endogenous cathepsin B, while intracellular cathepsin L remains unaffected. These results indicate that CA-074 rather than CA-074Me should be used to specifically inactivate cathepsin B within living cells.     

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%.     

Selective inhibition of cathepsin B with cell-permeable CA074Me negatively affects L6 rat myoblast differentiation.

Active cathepsin B, in concert with other cellular proteases, has been implicated in the catabolic restructuring associated with myotube formation during skeletal myoblast cell differentiation (i.e., myogenesis). We have examined this role in differentiating myoblasts using the cell-permeable, cathepsin B selective inhibitor CA074Me. Cathepsin B activity levels in differentiating L6 rat myoblasts treated with CA074Me were significantly lower than levels in control myoblasts. Inhibition of cathepsin B activity by CA074Me occurred at each stage of differentiation and was dose related. Myotube size and number and induced levels of fusion-related creatine phosphokinase activity and myosin heavy-chain protein were reduced from 30 to 50% in CA074Me-treated myoblasts. These reductions were also dose related. In contrast, CA074Me did not affect levels of myogenin, an early marker of myogenesis, or levels of cathepsin L type and myokinase activities, two nonspecific enzymes. The negative effects associated with CA074Me were reversed when the drug was removed. Collectively, these data suggest that active cathepsin B plays a role in myoblast-myoblast fusion and consequently may be necessary for the complete expression of those genes associated with the fusion process.     

Substrate specificity of bovine cathepsin B and its inhibition by CA074, based on crystal structure refinement of the complex

The crystal structure of the bovine spleen cathepsin B (BSCB)-CA074 complex was refined to R = 0.152 using X-ray diffraction data up to 2.18 A resolution. BSCB is characterized by an extra Cys148-Cys252 disulfide bridge, as compared with rat and human CBs. Although the crystal structures of these enzymes showed similar overall folding, a difference was observed in the occluding loop, a structural element specific only to CB. Comparison of the torsion angles indicated the different flexibilities of their loop structures. The oxirane C6 atom of CA074 was covalently bonded to the Cys29 S(gamma) atom (C3-S(gamma)=1.81 A), where the S-configuration was transformed to the R-form. Concerning the oxirane carbon atom that participates in the covalent bonding with the Cys residue, an acceptable rule has been proposed. The substrate specificities at the Sn (n = 1-3) and Sn' (n=1 and 2) subsites of CB, together with the interaction features as to CA074, have been discussed in comparison with the crystal structure of the papain-CA028 (a CA074-related inhibitor) complex.     

Nitroarginine methyl ester and canavanine lower intracellular reduced glutathione

In rat glial cells, arginine analogs N(G)-nitroarginine methyl ester (both D- and L-stereoisomer) and L-canavanine lower the intracellular levels of reduced glutathione, stimulate the pentose phosphate pathway, increase the level of malonyldialdehyde, and increase the leakage of lactate dehydrogenase. These effects are not related to the inhibition of nitric oxide synthase and depend on the oxidation of intracellular thiols; indeed, there are no signs of lipoperoxidation and cytotoxicity in cells previously loaded with glutathione. Furthermore, these arginine analogs elicit an oxidative burst in N11 cells and decrease the detectable level of both glutathione and dithiothreitol in cell-free experiments. These effects were not observed with the arginine analog N(G)-monomethyl-L-arginine, suggesting that the substituting moiety in (or near) the guanidine group could modify the reactivity of the arginine analogs with thiol compounds.     

Fluorimetric assays for cathepsin B and cathepsin H with methylcoumarylamide substrates.

Benzyloxycarbonyl-phenylalanyl-arginine 4-methyl-7-coumarylamide was found to be an excellent substrate for the fluorimetric assay of cathepsin B, and arginine 4-methyl-7-coumarylamide for cathepsin H. Procedures were developed that are very convenient, and avoid the hazards associated with the use of naphthylamides.     

Inhibition of HIV-1 replication by amphotericin B methyl ester: selection for resistant variants

Membrane cholesterol plays an important role in human immunodeficiency virus type 1 (HIV-1) particle production and infectivity. Here, we have investigated the target and mechanism of action of a cholesterol-binding compound, the polyene antifungal antibiotic amphotericin B methyl ester (AME). We found that AME potently inhibited the replication of a highly divergent panel of HIV-1 isolates in various T-cell lines and primary cells irrespective of clade or target cell tropism. The defects in HIV-1 replication caused by AME were due to profoundly impaired viral infectivity as well as a defect in viral particle production. To elucidate further the mechanism of action of AME, we selected for and characterized AME-resistant HIV-1 variants. Mutations responsible for AME resistance mapped to a highly conserved and functionally important endocytosis motif in the cytoplasmic tail of the transmembrane glycoprotein gp41. Interestingly, truncation of the gp41 cytoplasmic tail in the context of either HIV-1 or rhesus macaque simian immunodeficiency virus also conferred resistance to AME. The infectivity of HIV-1 virions bearing murine leukemia virus or vesicular stomatitis virus glycoproteins was unaffected by AME. Our data define the target and mechanism of action of AME and provide support for the concept that cholesterol-binding compounds should be pursued as antiretroviral drugs to disrupt HIV-1 replication.     

Specific spectrophotometric assays for cathepsin B1.

Cathepsin B₁ from bovine spleen was partially purified by acetone fractionation and by chromatography on Sephadex G-150 and DEAE Sephadex A-50. The enzyme was shown to catalyze the hydrolysis of p-nitrophenyl benzyloxycarbonylglycinate and p-nitrophenyl α-N-benzyloxycarbonyl-l-lysinate. Under the assay conditions, cathepsin B₁ is the major enzyme present in bovine spleen homogenates hydrolyzing these substrates. The kinetic parameters for the hydrolysis of p-nitrophenyl benzyloxycarbonylglycinate and p-nitrophenyl α-N-benzyloxycarbonyl-l-lysinate were measured and compared with those obtained for other cathepsin B₁ substrates. These results form the basis of an improved spectrophotometric assay for this enzyme in which the liberation of p-nitrophenol from either the N-benzyloxycarbonyl glycine or lysine p-nitrophenyl ester is monitored continuously at 326 nm.     

Activation of cathepsin D by glycine ethyl ester.

Cathepsin D purified from bovine spleen is activated by glycine ethyl ester. A maximum of 70% activation was observed at a glycine ethyl ester concentration of 0.1 M and at pH 4.5. The activation effect appears to be reversible.     

Fluorescent microplate assay for cancer cell-associated cathepsin B.

Cathepsin B and in particular cell-surface and secreted cathepsin B has been implicated in the invasive and metastatic phenotype of numerous types of cancer. We describe here a method to easily survey cancer cell lines for cathepsin B activity using the highly selective substrate Z-Arg-Arg-AMC. Intact human U87 glioma cells hydrolyze Z-Arg-Arg-AMC with a Km of 460 microM at pH 7.0 and 37 degrees C. This is nearly the same as the Km of 430 microM obtained with purified cathepsin B assayed under the same conditions. The pericellular (i.e. both cell-surface and released) cathepsin B activity was inhibited by the cysteine protease inhibitors E-64, leupeptin, Mu-Np2-HphVS-2Np, Mu-Leu-HpHVSPh and the cathepsin B selective inhibitor Mu-Tyr(3,5 I2)-HphVSPh with IC50 values similar to those observed for the inhibition of purified human liver cathepsin B. Other human cancer cell lines with measurable pericellular cathepsin B activity included HT-1080 fibrosarcoma, MiaPaCa pancreatic, PC-3 prostate and HCT-116 colon. Cathepsin B activity correlated with protein levels of cathepsin B as determined by immunoblot analysis. Pericellular cathepsin B activity was also detected in the rat cell lines MatLyLu prostate and Mat B III adenocarcinoma and in the murine lines B16a melanoma and Lewis lung carcinoma. The ability to determine pericellular cathepsin B activity will be useful in selecting appropriate cell lines for use in vivo when analyzing the effects of inhibiting cathepsin B activity on tumor growth and metastasis.     

Reduced toxicity of amphotericin B methyl ester (AME) vs. amphotericin B and fungizone in tissue culture.

The comparative toxicities of amphotericin B methyl ester (AME), the parent antibiotic amphotericin B (AB), and the deoxycholate solubilized complex of AB, Fungizone (FZ), toward five cell lines has been determined as measured by early membrane damage (51Cr release), 24 hr survival, 72 hr viability, and growth rate. Cells used were of turtle (TH-1), marsupial (PT K2), human MA 160), rabbit (RK-13) and hamster (BHK-21) origin. AME: (a) caused less membrane damage at 1 hr than AB or FZ; (b) was less toxic than AB or FZ as indicated by 24 hr cell survival and 72 hr cell viability; and (c) was required in higher levels than AB or FZ to reduce the growth rate of all five cell lines. Spectrophotometric analysis of residual polyene levels indicated that AME had good stability in tissue culture medium. Previous studies have indicated that AME has the same in vitro antifungal activity as the parent antibiotic AB (1, 2). These findings suggest that AME may prove to be superior to AB and FZ for use as an antifungal agent in tissue culture systems.     

Rat liver thiol proteinases: cathepsin B, cathepsin H and cathepsin L

Data on following points of lysosomal thiol proteinases (cathepsins B, H and L) from rat liver are described in this paper: Partial amino acid sequence of cathepsin B, substrate specificity of cathepsin L, immunological studies of cathepsin B and H and effectiveness of E-64, specific thiol proteinase inhibitor in vivo.     

N-bromoacetyl-p-azido-L-phenylalanine methyl ester: a bifunctional chemically and photo-activated cross-linking reagent

A cross-linking reagent with individually controllable functional groups, N-bromoacetyl-p-azido-l-phenylalanine methyl ester, has been developed. When reacted with endoplasmic reticulum membranes stripped of their ribosomes, the bromoacetyl group is most active at basic pH, reacts only with protein, and has biphasic reaction kinetics. Although the azido function also has biphasic reaction kinetics, the extent of the irradiated reaction is different from that of the dark reaction. In addition, the azido function reacts both with protein and with RNA. In a cross-linking experiment, no cross-linking occurred until the reagent-membrane complex was irradiated, even though the complex underwent limited exposure to light. Upon irradiation, extensive cross-linking occurred.     

Reduced toxicity of amphotericin B methyl ester (AME) vs. amphotericin B and fungizone in tissue culture

Summary The comparative toxicities of amphotericin B methyl ester (AME), the parent antibiotic amphotericin B (AB), and the deoxycholate solubilized complex of AB, Fungizone² (FZ), toward five cell lines has been determined as measured by early membrane damage (⁵¹Cr release), 24 hr survival, 72 hr viability, and growth rate. Cells used were of turtle (TH-1), marsupial (PT K2), human MA 160), rabbit (RK-13) and hamster (BHK-21) origin. AME: (a) caused less membrane damage at 1 hr than AB or FZ; (b) was less toxic than AB or FZ as indicated by 24 hr cell survival and 72 hr cell viability; and (c) was required in higher levels than AB or FZ to reduce the growth rate of all five cell lines. Spectrophotometric analysis of residual polyene levels indicated that AME had good stability in tissue culture medium. Previous studies have indicated that AME has the same in vitro antifungal activity as the parent antibiotic AB (1, 2). These findings suggest that AME may prove to be superior to AB and FZ for use as an antifungal agent in tissue culture systems. Fungizone^R. Trade mark. E. R. Squibb and Sons. This investigation was supported in part by Contract NIH 69-2161, NIH Grant No. AI-02095 and NIH Training Grant No. GM 507 from the National Institute of General Medical Sciences.     

Structural requirements for cathepsin B and cathepsin H inhibition by kininogens

Domain 3 (D3) of human kininogens, the major cysteine proteinase inhibitors in plasma, has been shown to be the tightest binding inhibitory domain for cathepsins B and H. D3 was expressed in three fragments as its exon products as follows: exon 7 (Gly235-Gln292), exon 8 (Gln292-Gly328), and exon 9 (Gln329-Met357). Exon products 7, 8, and 9 alone as well as exon product 7 + 9 each exhibited an IC₅₀ value 5- to 30-fold higher (5–30M) than exon products 7 + 8 and 8 + 9 (0.9–1.3M) for cathepsins B and H, respectively. However, in turn, the exon products 7 + 8 and 8 + 9 seemed to be less potent inhibitors than the intact D3 (10, 200 nM) or HK (200, 500 nM) molecule. These results clearly indicate that an intact molecule of HK or its domain 3 as a whole is required for optimal inhibition of cathepsins B and H.