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.     

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.     

A cathepsin L-specific inhibitor preferentially inhibits degradation of autophagosomal LC3 and GABARAP in HeLa and Huh-7 cells

CAA0225 ((2S,3S)-oxirane-2,3-dicarboxylic acid 2-[((S)-1-benzylcarbamoyl-2-phenyl-ethyl)-amide] 3-{[2-(4-hydroxy-phenyl)-ethyl]-amide}) is a cathepsin L-specific inhibitor recently selected out by extensive screening of a series of new epoxysuccinyl peptides. CAA0225 inhibited rat liver cathepsin L with IC50 values of 1.9 nM, but not rat liver cathepsin B (IC50, >1000-5000 nM). We compared effects of CAA0225 on autophagy with those of CA-074 that was previously developed as a cathepsin B-specific inhibitor. In HeLa and Huh-7 cells cultured under nutrient-deprived conditions both CAA0225 and CA-074 significantly and comparably inhibited degradation of long-lived proteins. Meanwhile, CAA0225 effectively inhibited degradation of LC3-II and GABARAP, whereas CA-074-OMe had only a marginal effect on their levels. Therefore, cathepsin L does not seem to play a general role in the degradation of proteins in the lumen of autophagosomes, but is involved more specifically in the degradation of autophagosomal membrane markers.     

Host cell cathepsins potentiate Moloney murine leukemia virus infection

The roles of cellular proteases in Moloney murine leukemia virus (MLV) infection were investigated using MLV particles pseudotyped with vesicular stomatitis virus (VSV) G glycoprotein as a control for effects on core MLV particles versus effects specific to Moloney MLV envelope protein (Env). The broad-spectrum inhibitors cathepsin inhibitor III and E-64d gave comparable dose-dependent inhibition of Moloney MLV Env and VSV G pseudotypes, suggesting that the decrease did not involve the envelope protein. Whereas, CA-074 Me gave a biphasic response that differentiated between Moloney MLV Env and VSV G at low concentrations, at which the drug is highly selective for cathepsin B, but was similar for both glycoproteins at higher concentrations, at which CA-074 Me inhibits other cathepsins. Moloney MLV infection was lower on cathepsin B knockout fibroblasts than wild-type cells, whereas VSV G infection was not reduced on the B-/- cells. Taken together, these results support the notion that cathepsin B acts at an envelope-dependent step while another cathepsin acts at an envelope-independent step, such as uncoating or viral-DNA synthesis. Virus binding was not affected by CA-074 Me, whereas syncytium induction was inhibited in a dose-dependent manner, consistent with cathepsin B involvement in membrane fusion. Western blot analysis revealed specific cathepsin B cleavage of SU in vitro, while TM and CA remained intact. Infection could be enhanced by preincubation of Moloney MLV with cathepsin B, consistent with SU cleavage potentiating infection. These data suggested that during infection of NIH 3T3 cells, endocytosis brings Moloney MLV to early lysosomes, where the virus encounters cellular proteases, including cathepsin B, that cleave SU.     

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.     

Involvement of cathepsin B- and L-like proteinases in silk gland histolysis during metamorphosis of Bombyx mori

To identify proteinases involved in programmed cell death of the silk glands of Bombyx mori, we measured enzyme activities in silk gland homogenates. Several peptidyl-4-methylcoumaryl-7-amides (MCAs) and bovine hemoglobin were used as substrates in the presence and absence of proteinase inhibitors. The hydrolysis of t-butyloxycarbonyl-Phe-Ser-Arg-MCA (Boc-FSR-MCA), benzyloxy-carbonyl-Phe-Arg-MCA (Z-FR-MCA), and Z-Arg-Arg-MCA (Z-RR-MCA) was optimal at pH 5.5, 5.0, and 5.5, respectively. It was stimulated by the sulfhydryl compounds or EDTA and inhibited by both cysteine proteinase inhibitors and a cathepsin B-specific inhibitor, l-3-trans-(propyl-carbamoyl)oxirane-2-carbonyl)-L-isoleucyl-L-prolin (CA-074). The hemoglobin hydrolysis at the optimum pH 3.5 was inactivated by cysteine proteinase inhibitors, but stimulated slightly by pepstatin. The cleavage of Arg-MCA (R-MCA) and Leu-MCA (L-MCA) at optimum pH of 7.0 was strongly inhibited by an aminopeptidase inhibitor, puromycin, and by sulfhydryl compounds. The Boc-FSR-MCA, Z-FR-MCA, Z-RR-MCA, and hemoglobin hydrolyzing activities increased in the silk glands dramatically after cocoon formation, while the R-MCA and L-MCA cleaving activities declined. The results strongly suggest the involvement of cathepsin B- and cathepsin L-like proteinases in the histolysis of the silk gland during metamorphosis.     

Intracellular and extracellular cathepsin B facilitate invasion of MCF-10A neoT cells through reconstituted extracellular matrix in vitro

Lysosomal cysteine proteinase cathepsin B is implicated in remodeling the extracellular matrix, a crucial step in the process of tumor cell invasion. In this study the contributions of intracellular and extracellular cathepsin B activities in the invasion of ras-transformed human breast epithelial cells, MCF-10A neoT, were assessed using specific cathepsin B neutralizing monoclonal antibody (Mab) 2A2, together with other general and specific cysteine proteinase inhibitors. We showed that the degradation of extracellular matrix by living MCF-10A neoT cells was predominantly intracellular, as imaged by confocal assays using quenched fluorescent substrate DQ-collagen IV. CA-074, a membrane-impermeable cathepsin B-selective inhibitor and its membrane-permeable analogue CA-074Me showed similar inhibition of invasion at 10 microM, i.e., 24.9 and 27.0%, respectively. Neutralizing monoclonal antibody exhibited a significantly higher inhibitory effect, decreasing invasion at 0.5 microM by 42.7%. Tumor cells may internalize monoclonal antibody; therefore, 2A2 Mab could impair both the intracellular and the extracellular fractions of cathepsin B activity. However, both 2A2 Mab and cathepsin B-selective inhibitors were less potent than the general cysteine proteinase inhibitors chicken cystatin and E-64, indicating that other cysteine proteinases, presumably cathepsin L, are involved in invasion. Our results show that intracellular and extracellular cathepsin B activity contribute to in vitro invasion of MCF-10A neoT cells and suggest that inhibitors capable of impairing both fractions have a potential as new anticancer drugs.     

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 K₁ values for cathepsin B were about 2–5 nM, like that of E-64-c, whereas their initial K₁ 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.     

Effects of selective inhibition of cathepsin B and general inhibition of cysteine proteinases on lysosomal proteolysis in rat liver in vivo and in vitro.

Intraperitoneal administration of N-(L-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-prolin e (CA-074) to rats at a dose of 4 mg/100 g greatly inhibited cathepsin-B activity in both liver and kidney for at least 4 h. Its inhibitory effect was selective for cathepsin-B activity in the liver but not in the kidney. The effects of selective inhibition of cathepsin-B activity by CA-074 treatment, and general inhibition of cysteine proteinases by N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl-3-methylbutylamid e (E-64-c) on the degradation of fluorescein isothiocyanate (FITC)-labeled asialofetuin in liver lysosomes, were examined in vivo. Undegraded or partially degraded FITC-labeled asialofetuin and its FITC-labeled degradation products were both found in the lysosomes and were easily separated by Sephadex G-25' column chromatography. The FITC-labeled degradation products were mainly lysine with an FITC-labeled epsilon-amino group. Accumulation of undegraded or partially degraded FITC-labeled asialofetuin in the lysosomes was marked after E-64-c treatment, but slight after CA-074 treatment. Under the marked inhibition of general lysosomal cysteine-proteinase activity by E-64-c or marked selective inhibition of cathepsin-B activity by CA-074 in vitro, degradation of FITC-labeled asialofetuin by disrupted lysosomes was analyzed on the basis of measurement of FITC-labeled degradation products by Sephadex G-25 column chromatography. It was suppressed markedly but incompletely by E-64-c as well as by CA-074, but more weakly than by E-64-c. These results shows that E-64-sensitive cysteine proteinases are important in lysosomal protein degradation, but cathepsin B has only a role in part and that an E-64-resistant proteinase(s) may also be important.     

Roles of cathepsins in reperfusion-induced apoptosis in cultured astrocytes

Astrocytic apoptosis may play a role in the central nervous system injury. We previously showed that reperfusion of cultured astrocytes with normal medium after exposure to hydrogen peroxide (H(2)O(2))-containing medium causes apoptosis. This study examines the involvement of the lysosomal enzymes cathepsins B and D in the astrocytic apoptosis. Reperfusion after exposure to H(2)O(2) caused a marked increase in caspase-3 and cathepsin D activities and a marked decrease in cathepsin B activity. Pepstatin A, an inhibitor of cathepsin D, and acetyl-L-aspartyl-L-methionyl-L-glutaminyl-L-aspart-1-aldehyde (Ac-DMQD-CHO), a specific inhibitor of caspase-3, blocked the H(2)O(2)-induced decrease in cell viability and DNA ladder formation in cultured rat astrocytes. The (L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl)-L-isoleucyl-L-proline methyl ester (CA074 Me), a specific inhibitor of cathepsin B, did not affect the H(2)O(2)-induced cell injury. On the other hand, CA074 Me decreased cell viability with DNA ladder formation when cultured in the presence of Ac-DMQD-CHO. This caspase-independent apoptosis was attenuated by the addition of the cathepsin D inhibitor pepstatin A. Caspase-3 like activity was markedly inhibited by Ac-DMQD-CHO and partially by pepstatin A. Pepstatin A and CA074 Me inhibited cathepsin B and cathepsin D activities, respectively, in the presence and absence of Ac-DMQD-CHO. These results suggest that cathepsins B and D are involved in astrocytic apoptosis: cathepsin D acts as a death-inducing factor upstream of caspase-3 and the caspase-independent apoptosis is regulated antagonistically by cathepsins B and D.     

Intracellular proteolytic activity of cathepsin B is associated with capillary-like tube formation by endothelial cells in vitro

The lysosomal cysteine protease cathepsin B is implicated in degradation of extracellular matrix (ECM), a crucial step in a variety of physiological and pathological processes, including tumor dissemination and angiogenesis. In this study, we analyzed the contribution of extracellular and intracellular cathepsin B activity on the formation of capillary-like tubular structures by human umbilical vein endothelial cells (HUVECs) grown on Matrigel matrix, using general and specific cysteine protease inhibitors. We demonstrated, by confocal assay using quenched fluorescent protein substrate DQ-collagen IV, that endothelial cells degrade ECM both intracellularly and pericellularly. Intracellular cathepsin B activity detected by degradation of Z-Arg-Arg cresyl violet substrate was co-localized with the products of DQ-collagen IV degradation in the perinuclear region and in the capillary-like tubular structures. Treatment of cells with membrane-permeable CA-074 Me effectively abolished intracellular cathepsin B activity, and resulted in reduced tube length (32.3+/-9.4% at 10 microM), total tubule area (49.6+/-12.4% at 10 microM), and the number of branch points of tubules (47.5+/-7.7% at 10 microM) in a dose-dependent manner. In contrast, CA-074 (0.1-10 microM), a membrane-impermeable cathepsin B specific inhibitor, general cysteine protease inhibitors chicken cystatin (5 microM) and E-64 (10 microM), and the metalloprotease inhibitor Minocycline (10 microM) showed no significant inhibitory effect in our angiogenesis model. These results show that, besides multiple regulatory molecules, intracellular cathepsin B also contributes to the neovascularization process and should be considered as a potential therapeutic target.     

A cathepsin B-like protease is required for host protein degradation in Trypanosoma brucei

Identification and analysis of Clan CA (papain) cysteine proteases in primitive protozoa and metazoa have suggested that this enzyme family is more diverse and biologically important than originally thought. The protozoan parasite Trypanosoma brucei is the etiological agent of African sleeping sickness. The cysteine protease activity of this organism is a validated drug target as first recognized by the killing of the parasite with the diazomethane inhibitor Z-Phe-Ala-CHN(2) (where Z is benzyloxycarbonyl). Whereas the presumed target of this inhibitor was rhodesain (also brucipain, trypanopain), the major cathepsin L-like cysteine protease of T. brucei, genomic analysis has now identified tbcatB, a cathepsin B-like cysteine protease as a possible inhibitor target. The mRNA of tbcatB is more abundantly expressed in the bloodstream versus the procyclic form of the parasite. Induction of RNA interference against rhodesain did not result in an abnormal phenotype in cultured T. brucei. However, induction of RNA interference against tbcatB led to enlargement of the endosome, accumulation of fluorescein isothiocyanate-transferrin, defective cytokinesis after completion of mitosis, and ultimately the death of cultured parasites. Therefore, tbcatB, but not rhodesain, is essential for T. brucei survival in culture and is the most likely target of the diazomethane protease inhibitor Z-Phe-Ala-CHN(2) in T. brucei.     

Cathepsin L and cathepsin B mediate reovirus disassembly in murine fibroblast cells

After attachment to receptors, reovirus virions are internalized by endocytosis and exposed to acid-dependent proteases that catalyze viral disassembly. Previous studies using the cysteine protease inhibitor E64 and a mutant cell line that does not support reovirus disassembly suggest a requirement for specific endocytic proteases in reovirus entry. This study identifies the endocytic proteases that mediate reovirus disassembly in murine fibroblast cells. Infection of both L929 cells treated with the cathepsin L inhibitor Z-Phe-Tyr(t-Bu)-diazomethyl ketone and cathepsin L-deficient mouse embryo fibroblasts resulted in inefficient proteolytic disassembly of viral outer-capsid proteins and decreased viral yields. In contrast, both L929 cells treated with the cathepsin B inhibitor CA-074Me and cathepsin B-deficient mouse embryo fibroblasts support reovirus disassembly and growth. However, removal of both cathepsin B and cathepsin L activity completely abrogates disassembly and growth of reovirus. Concordantly, cathepsin L mediates reovirus disassembly more efficiently than cathepsin B in vitro. These results demonstrate that either cathepsin L or cathepsin B is required for reovirus entry into murine fibroblasts and indicate that cathepsin L is the primary mediator of reovirus disassembly. Moreover, these findings suggest that specific endocytic proteases can determine host cell susceptibility to infection by intracellular pathogens.     

The selective role of cathepsins B and D in the lysosomal degradation of endogenous and exogenous proteins.

A selective inhibitor of cathepsin B, a derivative of E-64 (compound CA-074), and pepstatin-asialofetuin, a potent inhibitor of cathepsin D, were used for an in vivo study of the selective role of these proteinases in lysosomal proteolysis. Administration of compound CA-074 or pepstatinasialofetuin to rats caused only a slight shift of the lysosomal density and no increase in sequestered enzymes in the autolysosomal fraction, although cathepsin B or D activity in the liver was markedly inhibited. These treatments also had little effect on the inhibition of the degradation of endocytosed FITC-labeled asialofetuin. In contrast, leupeptin treatment caused marked inhibition of lysosomal degradation of endogenous and exogenous proteins. These results suggest a small contribution of cathepsins B and D to the initiation of lysosomal proteolysis.     

Distinct cathepsins control necrotic cell death mediated by pyroptosis inducers and lysosome-destabilizing agents

Necrotic cell death triggers a range of biological responses including a strong adaptive immune response, yet we know little about the cellular pathways that control necrotic cell death. Inhibitor studies suggest that proteases, and in particular cathepsins, drive necrotic cell death. The cathepsin B-selective inhibitor CA-074-Me blocks all forms of programmed necrosis by an unknown mechanism. We found that cathepsin B deficiency does not prevent induction of pyroptosis and lysosome-mediated necrosis suggesting that CA-074-Me blocks necrotic cell death by targeting cathepsins other than cathepsin B. A single cathepsin, cathepsin C, drives necrotic cell death mediated by the lysosome-destabilizing agent Leu-Leu-OMe (LLOMe). Here we present evidence that cathepsin C-deficiency and CA-074-Me block LLOMe killing in a distinct and cell type-specific fashion. Cathepsin C-deficiency and CA-074-Me block LLOMe killing of all myeloid cells, except for neutrophils. Cathepsin C-deficiency, but not CA-074-Me, blocks LLOMe killing of neutrophils suggesting that CA-074-Me does not target cathepsin C directly, consistent with inhibitor studies using recombinant cathepsin C. Unlike other cathepsins, cathepsin C lacks endoproteolytic activity, and requires activation by other lysosomal proteases, such as cathepsin D. Consistent with this theory, we found that lysosomotropic agents and cathepsin D downregulation by siRNA block LLOMe-mediated necrosis. Our findings indicate that a proteolytic cascade, involving cathepsins C and D, controls LLOMe-mediated necrosis. In contrast, cathepsins C and D were not required for pyroptotic cell death suggesting that distinct cathepsins control pyroptosis and lysosome-mediated necrosis.     

Effect of cysteine proteinase inhibitors on murine B16 melanoma cell invasion in vitro

Various types of proteinases are implicated in the malignant progression of human and animal tumors. Proteinase inhibitors may therefore be useful as therapeutic agents in anti-invasive and anti-metastatic treatment. The aims of this study were (1) to estimate the relative importance of proteinases in B16 cell invasion in vitro using synthetic, class-specific proteinase inhibitors and (2) to assess the inhibitory effect of some naturally occurring cysteine proteinase inhibitors. Serine proteinase inhibitor reduced invasiveness by up to 24%, whereas inhibition of aspartic proteinases reduced invasion by 11%. Synthetic inhibitors of cysteine proteinases markedly impaired invasion: cathepsin B inhibitors, particularly Ca-074Me, inhibited invasion from 20-40%, whereas cathepsin L inhibitor Clik 148 reduced invasion by 11%. The potato cysteine proteinase inhibitor PCPI 8.7 inhibited invasion by 21%, whereas another potato inhibitor, PCPI 6.6, and the mushroom cysteine proteinase inhibitor clitocypin had no effects. As the inhibitors that inhibited cathepsin B were in general more efficient at impairing the invasiveness, we conclude that of the two cysteine proteinases, cathepsin B plays a more important role than cathepsin L in murine melanoma cell invasion.     

Exocytosis of active cathepsin B enzyme activity at pH 7.0, inhibition and molecular mass.

Lysosomal cathepsin B has been implicated in parasitic, inflammatory and neoplastic diseases. Most of these pathologies suggest a role for cathepsin B outside the cells, although the origin of extracellular active enzyme is not well defined. The activity of extracellular cathepsin B is difficult to assess because of the presence of inhibitors and inactivation of the enzyme by oxidizing agents. Therefore, we have developed a continuous assay for measurement of cathepsin B activity produced pericellularly by living cells. The kinetic rate of Z-Arg-Arg-NHMec conversion was monitored and the assay optimized for enzyme stability, cell viability and sensitivity. To validate the assay, we determined that human liver cathepsin B was stable and active under the conditions of the assay and its activity could be inhibited by the selective epoxide derivative CA-074. Via this assay, we were able to demonstrate that active cathepsin B was secreted pericellularly by viable cells. Both preneoplastic and malignant cells secreted active cathepsin B. Pretreatment of cells with the membrane-permeant proinhibitor CA-074Me completely abolished pericellular and total cathepsin B activity whereas pretreatment with the active drug CA-074 had no effect. Immunoprecipitation and immunoblotting experiments suggested that the active enzyme species was 31-kDa single-chain cathepsin B. Exocytosis of cathepsin B was not related to secretion of proenzyme or secretion from mature lysosomes. Our results suggest an alternative pathway for exocytosis of active cathepsin B.     

Insights into the roles of cathepsins in antigen processing and presentation revealed by specific inhibitors

Eleven human cathepsins have been identified, however, the in vivo roles of individual cathepsins are still largely unknown. In this brief review we will summarize the functions of individual cathepsins in antigen processing and presentation, which are the initial steps of the immune response. Two general inhibitors of papain-like cysteine proteases, E-64 and pyridoxal phosphate, can completely suppress antigen presentation in vivo. To evaluate the contribution of individual cathepsins, specific inhibitors have been developed based on cathepsin tertiary structures: CA-074 for cathepsin B, CLIK-148 and -195 for cathepsin L, CLIK-60 for cathepsin S. Administration of CA-074, a cathepsin B inhibitor, suppresses the response to exogenous antigens, such as hepatitis B virus antigen, ovalbumin and Leishmania major antigen, and induces switching of the helper T cell responses from Th-2 to Th-1 of CD4+ T cells, thereby downregulating the production of IgE and IgG1. Administration of the cathepsin S inhibitor CLIK-60 impairs presentation of an autoantigen, alpha-fodrin, in Sjogren's syndrome and suppresses the Th-1 response and autoantibody production.