Inhibition of lysosomal cysteine proteases by chrysotherapeutic compounds: a possible mechanism for the antiarthritic activity of Au(I)

Although Au(I) complexes have been used to treat rheumatoid arthritis for over 75 years, their mechanism of action is still poorly understood. A family of enzymes responsible for joint destruction in rheumatoid arthritis, the cathepsins, has been discussed as a possible biological target of Au(I). In this study, inhibition of the cathepsins by known Au(I) drugs and related compounds was investigated. The compounds tested inhibited cathepsin activity with IC50 values as low as 600 nM. More typical IC50 values were in the 50-200 microM range. Although the gold complexes are not extremely potent cathepsin inhibitors, it is likely that this inhibition is biologically relevant given the high concentrations of Au(I) in the serum and joints of patients undergoing chrysotherapy. While it is likely that there are multiple targets of Au(I) in vivo, inhibition of the cathepsins would provide protection against the joint destruction that is a hallmark of rheumatoid arthritis and is one possible mechanism for Au(I) antiarthritic activity.     

3-Acylamino-azetidin-2-one as a novel class of cysteine proteases inhibitors

A new class of inhibitors for cysteine proteases cathepsin B, L, K and S is described. These inhibitors are based on the beta-lactam ring designed to interact with the nucleophilic thiol of the cysteine in the active site of cysteine proteases. Some 3-acylamino-azetidin-2-one derivatives showed very potent inhibition activities for cathepsins L, K and S at the nanomolar or subnanomolar IC(50) values.     

Cathepsin V, a novel and potent elastolytic activity expressed in activated macrophages

Atherosclerosis is characterized by a thickening and loss of elasticity of the arterial wall. Loss of elasticity has been attributed to the degradation of the arterial elastin matrix. Cathepsins K and S are papain-like cysteine proteases with known elastolytic activities, and both enzymes have been identified in macrophages present in plaque areas of diseased blood vessels. Here we demonstrate that macrophages express a third elastolytic cysteine protease, cathepsin V, which exhibits the most potent elastase activity yet described among human proteases and that cathepsin V is present in atherosclerotic plaque specimens. Approximately 60% of the total elastolytic activity of macrophages can be attributed to cysteine proteases with cathepsins V, K, and S contributing equally. From this 60%, two-thirds occur extracellularly and one-third intracellularly with the latter credited to cathepsin V. Ubiquitously expressed glycosaminoglycans (GAGs) such as chondroitin sulfate specifically inhibit the elastolytic activities of cathepsins V and K via the formation of specific cathepsin-GAG complexes. In contrast, cathepsin S, which does not form complexes with chondroitin sulfate is not inhibited; thus suggesting a specific regulation of elastolytic activities of cathepsins by GAGs. Because the GAG content is reduced in atherosclerotic plaques, an increase of cathepsins V and K activities may accelerate the destruction of the elastin matrix in diseased arteries.     

Cathepsin expression during skeletal development.

Cysteine proteinases, cathepsins B, H, K, L and S, have been implicated in several proteolytic processes during development, growth, remodeling and aging, as well as in a variety of pathological processes. For systematic analysis of cathepsin gene expression we have produced cDNA clones for mouse and human cysteine cathepsins. Northern analysis of a panel of total RNAs isolated from 16-19 different human and mouse tissues revealed the presence of mRNAs for cathepsin B, H, K, L and S in most tissues, but each with a distinct profile. Of the different cathepsin mRNAs, those for cathepsin K were clearly the highest in bone and cartilage. However, relatively high mRNA levels for the other cathepsins were also present in these tissues. To better understand the roles of different cathepsins during endochondral ossification in mouse long bones, cathepsin mRNAs were localized by in situ hybridization. Cathepsin K mRNAs were predominantly seen in multinucleated chondroclastic and osteoclastic cells at the osteochondral junction and on the surface of bone spicules. The other cathepsin mRNAs were also seen in osteoclasts, and in hypertrophic and proliferating chondrocytes. These observations were confirmed by immunohistochemistry and suggest that all cysteine cathepsins are involved in matrix degradation during endochondral ossification.     

Cleavage site specificity of cathepsin K toward cartilage proteoglycans and protease complex formation

Cathepsin K is a potent extracellular matrix-degrading protease that requires interactions with soluble glycosaminolycans for its collagenolytic activity in bone and cartilage. The major sources of glycosaminoglycans in cartilage are aggrecan aggregates. Therefore, we investigated whether cathepsin K activity is capable to hydrolyze aggrecan into fragments allowing the formation of glycosaminoglycan-cathepsin K complexes and determined the cleavage site specificity of cathepsin K toward the cartilage-resident link protein and aggrecan. The cleavage site specificity was compared with those of cathepsins S and L. All three cathepsins released glycosaminoglycans from native bovine cartilage at lysosomal pH and to a lesser degree at neutral extracellular pH. Cathepsin-predigested aggrecan complexes and cartilage provided suitable glycosaminoglycan fragments that allowed the formation of collagenolytically active cathepsin K complexes. A detailed analysis of the degradation of aggrecan aggregates revealed two cathepsin K cleavage sites in the link protein and several sites in aggrecan, including one site within the interglobular domain E1. In summary, these results demonstrate that cathepsin K is capable to degrade aggrecan complexes at specific cleavage sites and that cathepsin K activity alone is sufficient to self-provide the glycosaminoglycan fragments required for the formation of its collagenolytically active complex.     

Serum cathepsin K levels of patients with longstanding rheumatoid arthritis: correlation with radiological destruction

Cathepsin K is a cysteine protease that plays an essential role in osteoclast function and in the degradation of protein components of the bone matrix by cleaving proteins such as collagen type I, collagen type II and osteonectin. Cathepsin K therefore plays a role in bone remodelling and resorption in diseases such as osteoporosis, osteolytic bone metastasis and rheumatoid arthritis. We examined cathepsin K in the serum of 100 patients with active longstanding rheumatoid arthritis. We found increased levels of cathepsin K compared with a healthy control group and found a significant correlation with radiological destruction, measured by the Larsen score. Inhibition of cathepsin K may therefore be a new target for preventing bone erosion and joint destruction in rheumatoid arthritis. However, further studies have to be performed to prove that cathepsin K is a valuable parameter for bone metabolism in patients with early rheumatoid arthritis.     

Serum cathepsin K levels of patients with longstanding rheumatoid arthritis: correlation with radiological destruction

Cathepsin K is a cysteine protease that plays an essential role in osteoclast function and in the degradation of protein components of the bone matrix by cleaving proteins such as collagen type I, collagen type II and osteonectin. Cathepsin K therefore plays a role in bone remodelling and resorption in diseases such as osteoporosis, osteolytic bone metastasis and rheumatoid arthritis. We examined cathepsin K in the serum of 100 patients with active longstanding rheumatoid arthritis. We found increased levels of cathepsin K compared with a healthy control group and found a significant correlation with radiological destruction, measured by the Larsen score. Inhibition of cathepsin K may therefore be a new target for preventing bone erosion and joint destruction in rheumatoid arthritis. However, further studies have to be performed to prove that cathepsin K is a valuable parameter for bone metabolism in patients with early rheumatoid arthritis.     

Ovarian cysteine proteinases in the teleost Fundulus heteroclitus: molecular cloning and gene expression during vitellogenesis and oocyte maturation

The cysteine proteinases cathepsins B and L are members of the multigene family of lysosomal proteases that have been implicated in the processing of yolk proteins (YPs) in teleost oocytes. However, the full identification of the type of cathepsins expressed in fish ovarian follicles and embryos, as well as their regulatory mechanisms and specific function(s), are not yet elucidated. In this study, cDNAs encoding cathepsins B, L, F, K, S, Z, C, and H have been isolated from the teleost Fundulus heteroclitus, and the analysis of their deduced amino acid sequences revealed highly similar structural features to vertebrate orthologs, and confirmed in this species the existence of cathepsin L-like, cathepsin B-like, and cathepsin F-like subfamilies of cysteine proteinases. While all identified cathepsins were expressed in ovarian follicles, the corresponding mRNAs showed different temporal expression patterns. Thus, similar mRNA levels of cathepsins L, F, S, B, C, and Z were found throughout the oocyte growth or vitellogenesis period, whereas those for cathepsin H and K appeared to decrease as vitellogenesis advanced. During oocyte maturation, a transient accumulation of cathepsins L, S, H, and F mRNAs, approximately a 3-, 1.5-, 1.6-, and 6-fold increase, respectively, was detected in ovarian follicles within the 20-25 hr after hormone stimulation, coincident with the maximum proteolysis of the oocyte major YPs. The specific temporal pattern of expression of these genes may indicate a potential role of cathepsin L-like and cathepsin F proteases in the YP processing events occurring during fish oocyte maturation and/or early embryogenesis.     

Structure-based development of pyridoxal propionate derivatives as specific inhibitors of cathepsin K in vitro and in vivo

We found that pyridoxal phosphate shows considerable inhibition of cathepsins. CLIK-071, in which the phosphate ester of position 3 of pyridoxal phosphate was replaced by propionate, strongly inhibited cathepsin B. Three new types of synthetic pyridoxal propionate derivatives showing specific inhibition of cathepsin K were developed. New synthetic pyridoxal propionate derivatives, -162, -163, and -164, in which the methyl arm of position 6 of CLIK-071 was additionally modified, strongly inhibited cathepsin K and cathepsin S weakly, but other cathepsins were not inhibited. CLIK-166, in which the position 4 aldehyde of CLIK-071 is replaced by a vinyl radical and position 5 is additionally modified, showed cathepsin K-specific inhibition at 10(-5) M. Pit formation due to bone collagen degradation by cathepsin K of rat osteoclasts was specifically suppressed by administration of CLIK-164, but not by inhibitors of cathepsin L or B.     

Manipulating substrate and pH in zymography protocols selectively distinguishes cathepsins K, L, S, and V activity in cells and tissues

Cathepsins K, L, S, and V are cysteine proteases that have been implicated in tissue-destructive diseases such as atherosclerosis, tumor metastasis, and osteoporosis. Among these four cathepsins are the most powerful human collagenases and elastases, and they share 60% sequence homology. Proper quantification of mature, active cathepsins has been confounded by inhibitor and reporter substrate cross-reactivity, but is necessary to develop properly dosed therapeutic applications. Here, we detail a method of multiplex cathepsin zymography to detect and distinguish the activity of mature cathepsins K, L, S, and V by exploiting differences in individual cathepsin substrate preferences, pH effects, and electrophoretic mobility under non-reducing conditions. Specific identification of cathepsins K, L, S, and V in one cell/tissue extract was obtained with cathepsin K (37 kDa), V (35 kDa), S (25 kDa), and L (20 kDa) under non-reducing conditions. Cathepsin K activity disappeared and V remained when incubated at pH 4 instead of 6. Application of this antibody free, species independent, and medium-throughput method was demonstrated with primary human monocyte-derived macrophages and osteoclasts, endothelial cells stimulated with inflammatory cytokines, and normal and cancer lung tissues, which identified elevated cathepsin V in lung cancer.     

Synthesis and evaluation of arylaminoethyl amides as noncovalent inhibitors of cathepsin S. Part 3: heterocyclic P3

A series of N(alpha)-2-benzoxazolyl-alpha-amino acid-(arylaminoethyl)amides were identified as potent, selective, and noncovalent inhibitors of cathepsin S. Structure-activity relationships including strategies for modulating the selectivities among cathepsins S, K, and L, and in vivo pharmacokinetics are discussed. A X-ray structure of compound 3 bound to the active site of cathepsin S is also reported.     

Regulation of collagenase activities of human cathepsins by glycosaminoglycans

Cathepsin K, a lysosomal papain-like cysteine protease, forms collagenolytically highly active complexes with chondroitin sulfate and represents the most potent mammalian collagenase. Here we demonstrate that complex formation with glycosaminoglycans (GAGs) is unique for cathepsin K among human papain-like cysteine proteases and that different GAGs compete for the binding to cathepsin K. GAGs predominantly expressed in bone and cartilage, such as chondroitin and keratan sulfates, enhance the collagenolytic activity of cathepsin K, whereas dermatan, heparan sulfate, and heparin selectively inhibit this activity. Moreover, GAGs potently inhibit the collagenase activity of other cysteine proteases such as cathepsins L and S at 37 degrees C. Along this line MMP1-generated collagen fragments in the presence of GAGs are stable against further degradation at 28 degrees C by all cathepsins but cathepsin K, whereas thermal destabilization at 37 degrees C renders the fragments accessible to all cathepsins. These results suggest a novel mechanism for the regulation of matrix protein degradation by GAGs. It further implies that cathepsin K represents the only lysosomal collagenolytic activity under physiologically relevant conditions.     

Potency and selectivity of inhibition of cathepsin K, L and S by their respective propeptides.

The prodomains of several cysteine proteases of the papain family have been shown to be potent inhibitors of their parent enzymes. An increased interest in cysteine proteases inhibitors has been generated with potential therapeutic targets such as cathepsin K for osteoporosis and cathepsin S for immune modulation. The propeptides of cathepsin S, L and K were expressed as glutathione S-transferase-fusion proteins in Escherichia coli. The proteins were purified on glutathione affinity columns and the glutathione S-transferase was removed by thrombin cleavage. All three propeptides were tested for inhibitor potency and found to be selective within the cathepsin L subfamily (cathepsins K, L and S) compared with cathepsin B or papain. Inhibition of cathepsin K by either procathepsin K, L or S was time-dependent and occurred by an apparent one-step mechanism. The cathepsin K propeptide had a Ki of 3.6-6.3 nM for each of the three cathepsins K, L and S. The cathepsin L propeptide was at least a 240-fold selective inhibitor of cathepsin K (Ki = 0.27 nM) and cathepsin L (Ki = 0.12 nM) compared with cathepsin S (Ki = 65 nM). Interestingly, the cathepsin S propeptide was more selective for inhibition of cathepsin L (Ki = 0.46 nM) than cathepsin S (Ki = 7.6 nM) itself or cathepsin K (Ki = 7.0 nM). This is in sharp contrast to previously published data demonstrating that the cathepsin S propeptide is equipotent for inhibition of human cathepsin S and rat and paramecium cathepsin L [Maubach, G., Schilling, K., Rommerskirch, W., Wenz, I., Schultz, J. E., Weber, E. & Wiederanders, B. (1997), Eur J. Biochem. 250, 745-750]. These results demonstrate that limited selectivity of inhibition can be measured for the procathepsins K, L and S vs. the parent enzymes, but selective inhibition vs. cathepsin B and papain was obtained.     

Detecting cathepsin activity in human osteoarthritis via activity-based probes

IntroductionLysosomal cathepsins have been reported to contribute to Osteoarthritis (OA) pathophysiology due to their increase in pro-inflammatory conditions. Given the causal role of cathepsins in OA, monitoring their specific activity could provide means for assessing OA severity. To this end, we herein sought to assess a cathepsin activity-based probe (ABP), GB123, in vitro and in vivo.MethodsProtein levels and activity of cathepsins B and S were monitored by immunoblot analysis and GB123 labeling in cultured primary chondrocytes and conditioned media, following stimuli with tumor necrosis factor alpha (TNFα) and/or Interleukin 1 beta (IL-1β). Similarly, cathepsin activity was examined in sections of intact cartilage (IC) and degraded cartilage (DC) regions of OA. Finally, synovial fluid (SF) and serum from donors with no signs of diseases, early OA, late OA and rheumatoid arthritis (RA) patients were analyzed with GB123 to detect distinct activity levels of cathepsin B and S.ResultsCathepsin activity in cell lysates, conditioned media explants and DC sections showed enhanced enzymatic activity of cathepsins B and S. Further histological analysis revealed that cathepsin activity was found higher in superficial zones of DC than in IC. Examining serum and SF revealed that cathepsin B is significantly elevated with OA severity in serum and SF, yet levels of cathepsin S are more correlated with synovitis and RA.ConclusionsBased on our data, cathepsin activity monitored by ABPs correlated well with OA severity and joint inflammation, directing towards a novel etiological target for OA, which possesses significant translational potential in developing means for non-invasive detection of early signs of OA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0586-5) contains supplementary material, which is available to authorized users.     

Collagenolytic activity of cathepsin K is specifically modulated by cartilage-resident chondroitin sulfates

Cathepsin K is the predominant cysteine protease in osteoclast-mediated bone remodeling, and the protease is thought to be involved in the pathogenesis of diseases with excessive bone and cartilage resorption. Osteoclastic matrix degradation occurs in the extracellular resorption lacuna and upon phagocytosis within the cell's lysosomal-endosomal compartment. Since glycosaminoglycans (GAGs) are abundant in extracellular matrixes of cartilage and growing bone, we have analyzed the effect of GAGs on the activity of bone and cartilage-resident cathepsins K and L and MMP-1. GAGs, in particular chondroitin sulfates, specifically and selectively increased the stability of cathepsin K but had no effect on cathepsin L and MMP-1. GAGs strongly enhanced the stability and, to a lesser extent, the catalytic activity of cathepsin K. To combine the activity and stability parameters, we defined a novel kinetic term, named cumulative activity (CA), which reflects the total substrate turnover during the life span of the enzyme. In the presence of chondroitin-4-sulfate (C-4S), the CA value increased 200-fold for cathepsin K but only 25-fold with chondroitin-6-sulfate (C-6S). C-4S dramatically increased the hydrolysis of soluble as well insoluble type I and II collagens, whereas the effects of C-6S and hyaluronic acid were less pronounced. C-4S acts in a concentration-dependent manner but reaches saturation at approximately 0.1%, a concentration similar to that found in the synovial fluid of arthritis patients. C-4S increased the cathepsin K-mediated release of hydroxyproline from insoluble type I collagen 10-fold but had only a less than 2-fold enhancing effect on the hydrolysis of intact cartilage. The relatively small increase in the hydrolysis of cartilage by C-4S was attributed to the endogenous chondroitin sulfate content present in the cartilage. Although C-4S increased the pH stability at neutral pH, a significant increase in the collagenolytic activity of cathepsin K at this pH was not observed, thus suggesting that the unique collagenolytic activity of cathepsin K at acidic pH is mechanistically determined and not by the enzyme's instability at neutral pH. The selective and significant stabilization and activation of cathepsin K activity by C-4S may provide a rationale for a novel mechanism to regulate the enzyme's activity during bone growth and aging, two processes known for significant changes in the GAG content.     

Significance of cathepsin B accumulation in synovial fluid of rheumatoid arthritis

We measured and compared the activities of various kinds of proteinases, such as cysteine, serine, aspartic, and metalloproteinases, in synovial fluids of 16 patients with rheumatoid arthritis (RA) and 18 patients with osteoarthritis (OA). More than 19-fold higher activity of cathepsin B and about 6-fold higher activity of prolylendopeptidase, compared to those of OA, were accumulated in RA fluid. Moreover, levels of cathepsins B and S using the corresponding sandwich enzyme immunoassays were statistically higher in RA fluid than those in OA. Significant amounts of 41-kDa and 35-kDa procathepsin L were detected in RA fluid using gelatin zymography, while 41-kDa enzyme alone was detected in OA. Cathepsin B in RA fluid could degrade collagen, and this degradation was suppressed by the addition of CA-074, a specific inhibitor of cathepsin B. Therefore, cathepsin B may participate in joint destruction of RA, and its inhibitor may be effective for RA care.     

Trifluoroethylamines as amide isosteres in inhibitors of cathepsin K

The P2-P3 amide of dipeptide cathepsin K inhibitors can be replaced by the metabolically stable trifluoroethylamine group. The non-basic nature of the nitrogen allows the important hydrogen bond to Gly66 to be made. The resulting compounds are 10- to 20-fold more potent than the corresponding amide derivatives. Compound 8 is a 5 pM inhibitor of human cathepsin K with >10,000-fold selectivity over other cathepsins.     

Unmet needs in rheumatoid arthritis

Until the pathophysiology/etiology of rheumatoid arthritis (RA) is better understood, treatment strategies must focus on disease management. Early diagnosis and treatment with disease-modifying antirheumatic drugs (DMARDs) are necessary to reduce early joint damage, functional loss, and mortality. Several clinical trials have now clearly shown that administering appropriate DMARDs early yields better therapeutic outcomes. However, RA is a heterogeneous disease in which responses to treatment vary considerably for any given patient. Thus, choosing which patients receive combination DMARDs, and which combinations, remains one of our major challenges in treating RA patients. In many well controlled clinical trials methotrexate and other DMARDs, including the tumor necrosis factor-alpha inhibitors, have shown considerable efficacy in controlling the inflammatory process, but many patients continue to have active disease. Optimizing clinical response requires the use of a full spectrum of clinical agents with different therapeutic targets. Newer therapies, such as rituximab, that specifically target B cells have emerged as viable treatment options for patients with RA.     

Lack of cathepsin activities alter or prevent the development of lung granulomas in a mouse model of sarcoidosis

Background

Remodeling of lung tissues during the process of granuloma formation requires significant restructuring of the extra-cellular matrix and cathepsins K, L and S are among the strongest extra-cellular matrix degrading enzymes. Cathepsin K is highly expressed in various pathological granulomatous infiltrates and all three enzymes in their active form are detected in bronchoalveolar lavage fluids from patients with sarcoidosis. Granulomatous inflammation is driven by T-cell response and cathepsins S and L are actively involved in the regulation of antigen presentation and T-cell selection. Here, we show that the disruption of the activities of cathepsins K, L, or S affects the development of lung granulomas in a mouse model of sarcoidosis.

Methods

Apolipoprotein E-deficient mice lacking cathepsin K or L were fed Paigen diet for 16 weeks and lungs were analyzed and compared with their cathepsin-expressing littermates. The role of cathepsin S in the development of granulomas was evaluated using mice treated for 8 weeks with a potent and selective cathepsin S inhibitor.

Results

When compared to wild-type litters, more cathepsin K-deficient mice had lung granulomas, but individually affected mice developed smaller granulomas that were present in lower numbers. The absence of cathepsin K increased the number of multinucleated giant cells and the collagen content in granulomas. Cathepsin L deficiency resulted in decreased size and number of lung granulomas. Apoe-/- mice treated with a selective cathepsin S inhibitor did not develop lung granulomas and only individual epithelioid cells were observed.

Conclusions

Cathepsin K deficiency affected mostly the occurrence and composition of lung granulomas, whereas cathepsin L deficiency significantly reduced their number and cathepsin S inhibition prevented the formation of granulomas.