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.     

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.     

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.     

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.     

Trypanosoma brucei: chemical evidence that cathepsin L is essential for survival and a relevant drug target

The protozoan parasite causing human African trypanosomiasis, Trypanosoma brucei, displays cysteine peptidase activity, the chemical inhibition of which is lethal to the parasite. This activity comprises a cathepsin B (TbCATB) and a cathepsin L (TbCATL). Previous RNA interference (RNAi) data suggest that TbCATB rather than TbCATL is essential to survival even though silencing of the latter was incomplete. Also, chemical evidence supporting the essentiality of either enzyme which would facilitate a target-based drug development programme is lacking. Using specific peptidyl inhibitors and substrates, we quantified the contributions of TbCATB and TbCATL to the survival of T. brucei. At 100 μM, the minimal inhibitory concentration that kills all parasites in culture, the non-specific cathepsin inhibitors, benzyloxycarbonyl-phenylalanyl-arginyl-diazomethyl ketone (Z-FA-diazomethyl ketone) and (l-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l-proline methyl ester (CA-074Me) inhibited TbCATL and TbCATB by >99%. The cathepsin L (CATL)-specific inhibitor, ((2S,3S)-oxirane-2,3-dicarboxylic acid 2-[((S)-1-benzylcarbamoyl-2-phenyl-ethyl)-amide] 3-{[2-(4-hydroxy-phenyl)-ethyl]-amide}) (CAA0225), killed parasites with >99% inhibition of TbCATL but only 70% inhibition of TbCATB. Conversely, the cathepsin B (CATB)-specific inhibitor, (l-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l-proline (CA-074), did not affect survival even though TbCATB inhibition at >95% was statistically indistinguishable from the complete inhibition by Z-FA-diazomethyl ketone and CA-074Me. The observed inhibition of TbCATL by CA-074 and CA-074Me was shown to be facilitated by the reducing intracellular environment. All inhibitors, except the CATB-specific inhibitor, CA-074, blockaded lysosomal hydrolysis prior to death. The results suggest that TbCATL, rather than TbCATB, is essential to the survival of T. brucei and an appropriate drug target.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Inhibition of cathepsin B reduces beta-amyloid production in regulated secretory vesicles of neuronal chromaffin cells: evidence for cathepsin B as a candidate beta-secretase of Alzheimer's disease

The regulated secretory pathway of neurons is the major source of extracellular A beta that accumulates in Alzheimer's disease (AD). Extracellular A beta secreted from that pathway is generated by beta-secretase processing of amyloid precursor protein (APP). Previously, cysteine protease activity was demonstrated as the major beta-secretase activity in regulated secretory vesicles of neuronal chromaffin cells. In this study, the representative cysteine protease activity in these secretory vesicles was purified and identified as cathepsin B by peptide sequencing. Immunoelectron microscopy demonstrated colocalization of cathepsin B with A beta in these vesicles. The selective cathepsin B inhibitor, CA074, blocked the conversion of endogenous APP to A beta in isolated regulated secretory vesicles. In chromaffin cells, CA074Me (a cell permeable form of CA074) reduced by about 50% the extracellular A beta released by the regulated secretory pathway, but CA074Me had no effect on A beta released by the constitutive pathway. Furthermore, CA074Me inhibited processing of APP into the COOH-terminal beta-secretase-like cleavage product. These results provide evidence for cathepsin B as a candidate beta-secretase in regulated secretory vesicles of neuronal chromaffin cells. These findings implicate cathepsin B as beta-secretase in the regulated secretory pathway of brain neurons, suggesting that inhibitors of cathepsin B may be considered as therapeutic agents to reduce A beta in AD.     

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.     

Epoxysuccinyl peptide-derived cathepsin B inhibitors: modulating membrane permeability by conjugation with the C-terminal heptapeptide segment of penetratin

Besides its physiological role in lysosomal protein breakdown, extralysosomal cathepsin B has recently been implicated in apoptotic cell death. Highly specific irreversible cathepsin B inhibitors that are readily cell-permeant should be useful tools to elucidate the effects of cathepsin B in the cytosol. We have covalently functionalised the poorly cell-permeant epoxysuccinyl-based cathepsin B inhibitor [R-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH; R=OMe] with the C-terminal heptapeptide segment of penetratin (R=epsilonAhx-Arg-Arg-Nle-Lys-Trp-Lys-Lys-NH2). The high inhibitory potency and selectivity for cathepsin B versus cathepsin L of the parent compound was not affected by the conjugation with the penetratin heptapeptide. The conjugate was shown to efficiently penetrate into MCF-7 cells as an active inhibitor, thereby circumventing an intracellular activation step that is required by other inhibitors, such as the prodrug-like epoxysuccinyl peptides E64d and CA074Me.     

IL-6/sIL-6R enhances cathepsin B and L production via caveolin-1-mediated JNK-AP-1 pathway in human gingival fibroblasts

Interleukin (IL)-6 has an important role in inflammatory diseases. Lysosomal enzymes cathepsins are widely expressed as cysteine proteases regulating inflammatory process. Caveolin-1 (Cav-1) is a scaffolding/regulatory membrane protein that interacts with signaling molecules. In this study, we investigated the role of Cav-1 on (1) the productivity, and (2) the enzymatic activity of cathepsin B and L in human gingival fibroblasts (HGFs) treated with IL-6 in the presence of soluble form of IL-6 receptor (sIL-6R). At first, we established the siRNA-mediated Cav-1 down-regulating in vitro systems by transient transfection of Cav-1 siRNA. The siRNA-mediated Cav-1 down-regulated cells were treated with IL-6/sIL-6R for indicated times. Then, cell lysates were collected, and examined the IL-6-induced signaling pathway, cathepsin B and L production, and measurement of cathepsins activity. To investigate the cathepsin L activity, cathepsin-(B + L) activity was measured after pretreatment with CA-074Me, a specific inhibitor for cathepsin B. We found that IL-6/sIL-6R enhanced significantly both production and activity of cathepsin B and L in HGFs. Interestingly, IL-6-mediated phosphorylation of both p44/42 MAPK and JNK was dramatically suppressed in Cav-1 down-regulated HGFs treated with IL-6/sIL-6R. In addition, both production and activity of cathepsin B and L were also significantly suppressed. Importantly, we demonstrated that JNK inhibition, but not p44/42 MAPK inhibition, significantly diminished IL-6/sIL-6R-induced cathepsin B and L production. Taken together, we concluded that IL-6/sIL-6R enhances cathepsin B and L production via IL-6/sIL-6R-mediated Cav-1-JNK-AP-1 pathway in HGFs. Our findings indicate that Cav-1 might be a therapeutic target for IL-6-mediated tissue degradation in periodontitis.     

CD4-independent human immunodeficiency virus infection involves participation of endocytosis and cathepsin B

During a comparison of the infectivity of mNDK, a CD4-independent human immunodeficiency virus type 1 (HIV-1) strain, to various cell lines, we found that HeLa cells were much less susceptible than 293T and TE671 cells. Hybridoma cells between HeLa and 293T cells were as susceptible as 293T cells, suggesting that cellular factors enhance the mNDK infection in 293T cells. By screening a cDNA expression library in HeLa cells, cystatin C was isolated as an enhancer of the mNDK infection. Because cathepsin B protease, a natural ligand of cystatin C, was upregulated in HeLa cells, we speculated that the high levels of cathepsin B activities were inhibitory to the CD4-independent infection and that cystatin C enhanced the infection by impairing the excessive cathepsin B activity. Consistent with this idea, pretreatment of HeLa cells with 125 μM of CA-074Me, a cathepsin B inhibitor, resulted in an 8-fold enhancement of the mNDK infectivity. Because cathepsin B is activated by low pH in acidic endosomes, we further examined the potential roles of endosomes in the CD4-independent infection. Suppression of endosome acidification or endocytosis by inhibitors or by an Eps15 dominant negative mutant reduced the infectivity of mNDK in which CD4-dependent infections were not significantly impaired. Taken together, these results suggest that endocytosis, endosomal acidification, and cathepsin B activity are involved in the CD4-independent entry of HIV-1.