Emerging Roles of Cysteine Cathepsins in Disease ...

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Emerging Roles of Cysteine Cathepsins in Disease and their Potential as Drug Targets Olga Vasiljeva1,2, Thomas Reinheckel1, Christoph Peters1, Duan Turk2, Vito Turk2, Boris Turk2,* 1 2

Institut für Molekulare Medizin und Zellforschung, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany and Department of Biochemistry and Molecular Biology, J. Stefan Institute, Ljubljana, Slovenia Abstract: The general view on cysteine cathepsins, which were long believed to be primarily involved in intracellular protein turnover, has dramatically changed in last 10 to 15 years. The discovery of new cathepsins, such as cathepsins K, V, X, F and O, and their tissue distribution suggested that at least some of them are involved in very specific cellular processes. Moreover, gene ablation experiments revealed that cathepsins play a vital role in numerous physiological processes, such as antigen processing and presentation, bone remodelling, prohormone processing and wound healing. Their involvement in several pathologies, including osteoporosis, rheumatoid arthritis, osteoarthritis, bronchial asthma and cancer have also been confirmed and today several of them have been validated as relevant targets for therapies. Compounds targeting cathepsins S and K are already in clinical evaluation, whereas others are in experimental phases. The cathepsin K inhibitor AAE-581 (balicatib) as the most advanced of them passed Phase II clinical trials in 2005. In this review, we discuss the current view on cathepsins as an emerging group of targets for several diseases and the development of cathepsin K and S inhibitors for treatment of osteoporosis and various immune disorders.

INTRODUCTION Cysteine proteases from the C1 family (papain family) of CA clan comprise one of the largest and best characterized families of cysteine peptidases. Although not that abundant in humans with eleven members existing at the gene level (cathepsins B, C (J, dipeptidyl peptidase I), F, H, K (O, O2), L, O, S, V (L2), W (lymphopain), X (P, Y, Z); [1]) human clan CA proteases or the cysteine cathepsins have a long history dating back to the 1940’s, when cathepsin C was discovered [2]. Although they were relatively well characterized biochemically, until 1990’s is their physiological and pathophysiological roles were poorly understood. Traditionally cysteine cathepsins were believed to execute non-specific bulk proteolysis within the lysosome [3]. However, there is growing evidence for specific intra- and extracellular functions for these papain-like enzymes [2]. Collectively, cysteine cathepsins participate in numerous important physiological and pathological processes. Many of these specific functions of cysteine cathepsins have been confirmed and/or revealed by gene-knockout analyses (Table 1). Cysteine cathepsins are involved in precursor protein activation (including proenzymes and prohormones), MHC-II-mediated antigen presentation, bone remodelling, keratinocyte differentiation, hair cycle, reproduction, and apoptosis. They have also been implied to participate in tumour progression and metastasis, inflammatory diseases, such as inflammatory rheumatoid arthritis, atherosclerosis, and periodontitis. In addition, mutations in cathepsin genes result in human hereditary disorders, *Address correspondence to this author at the Department of Biochemistry and Molecular Biology, J. Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia; Tel: +386 1 477 3772; Fax: +386 1 477 3984; E-mail: [email protected] 1381-6128/07 $50.00+.00

as shown for cathepsin K gene mutations in pycnodysostosis, and for the Papillon–Lefevre and Haim–Munk syndromes caused by defects in the cathepsin C gene. The following review comprises several issues concerning structure, function, activity regulation and involvement of cysteine cathepsins in several physiological and pathological processes, especially with respect to their potential application as diagnostic and/or prognostic markers and drug targets in order to prevent inappropriate proteolysis, which could be harmful or lethal. The impact of cysteine cathepsins in cancer biology, immune system, pathophysiology of bone and joints, as well as the development of cathepsin S and K inhibitors will be discussed. STRUCTURE AND CATHEPSINS

FUNCTION

OF

CYSTEINE

Cysteine cathepsins are all relatively small monomeric proteins with Mw around 30 kDa with the exception of the tetrameric cathepsin C with Mw around 200 kDa. They are all based on the common fold of a papain-like structure, which consists of two domains (L(eft)-domain and R(ight)domain referring to the standard view shown in Fig. 1a), reminiscent of a closed book with the spine at the front. The domains separate at the top in a V-shaped active-site cleft, in the middle of which the catalytic residues, Cys25 from the L-domain and His159 from the R-domain, reside (papain numbering). The most prominent feature of the L- domain is the central ~30 residues long - helix on top of which Cys25 is located. The R-domain forms a kind of -helical barrel, which includes a shorter - motif. In the active form, Cys25 and His159 form a thiolate-imidazolium ion pair, which is essential for the activity of proteases. The ion pair is characterized by an unusually low pKa value of the reactive thiolate group (pKa ~2.5-3.5; [4]). © 2007 Bentham Science Publishers Ltd.

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Table 1.

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Phenotypes of Cysteine Cathepsine Knock-Out Mice

Targeted Cysteine Cathepsin Cathepsin B

Spontaneous Phenotype

Challenge Phenotype

- Absence of immune phenotype [91]

- Reduced trypsinogen activation in experimental pancreatitis [205]

- Impaired solubilization and processing of thyroglobulin [204]

- Reduced TNF- induced hepatocyte apoptosis [44] - Delayed development of mammary cancer and reduced metastasis [80] - Reduced tumour vascularization and growth with impaired tumour invasion in RIP1-Tag2 transgenic tumour model [34]

Cathepsin C

- Lack of granzyme and chymase activation [102, 206]

- Protection against autoimmune experimental arthritis [157, 207] - Increased survival in sepsis [208]

Cathepsin F

- Neuronal lipofuscinosis and progressive late-onset neurological disease [101]

Cathepsin K

- Osteopetrosis and impaired resorption of bone matrix, phenocoping human pycnodysostosis [144, 145]

- Reduced atherosclerosis progression and increased plaque stability in experimental atherosclerosis [209]

- Impaired processing of thyroglobulin and reduced release of thyroid hormone [204]

- Increased lung fibrosis after administration of bleomycin [210]

- Impaired hair follicle morphogenesis and cycling [37, 211]

- Reduced endothelial progenitor cell dependent neovascularization in a hind limb ischemia model [63]

- Epidermal hyperplasia due to a hyperproliferation of basal keratinocytes [37, 38]

- Reduced type 1 autoimmune diabetes [216]

Cathepsin L

- Dilated cardiomyopathy [212, 213]

- Impaired tumour cell proliferation and growth in RIP1-Tag2 tumour model [34]

- Impaired positive selection of T-helper cells [92, 214] - Reduced enkephalin processing [215] - Impaired solubilization of thyroglobulin and reduced release of thyroid hormone [204] - Decreased trabecular bone volume [140] Cathepsin S

- Immune defect: impaired invariant chain degradation in antigen presenting cells [95]

- Diminished susceptibility to an experimentally elicited autoimmunity, collagen-induced arthritis [95] - Reduced atherosclerosis in atherosclerosis-prone LDL receptor– deficient mice [217] - Reduced wound-healing–associated neovascularization [57] - Decreased plaque size and the incidence of acute plaque rupture in animal model of spontaneous plaque destabilization [218] - Resistant to the development of experimental autoimmune myasthenia gravis [219] - Reduced angiogenesis and growth of solid tumours in RIP1-Tag2 transgenic tumour model [34, 56]

Cathepsin W

- Absence of immune phenotype [220]

Crystal structures of substrate analogue inhibitors revealed that substrates bind along the active site cleft in an extended conformation with the side chain positions alternating against the L- and R- domain. The surface of the substrate binding sites is formed by four chain segments comprising two shorter loops on the L-domain (19-25, 61-69) and two longer loops on the R-domain (136-162, 182-213). A third loop from the L-domain might be named as well if, the disulfide (Cys 22 - Cys 65), which connects the two L-domain loops at the top, is considered an additional loop closure. The substrate binding sites S2, S1 and S1’ are well defined among all of the cathepsins [5, 6]. Superimposition of the nine

crystal structures of cathepsins currently available shows that these sites are equipositioned in all of them. In these sites, main chain atoms of substrate residues interact with the highly conserved residues Gly 66, Gln 29 and Trp 177, using papain nomenclature. The S2 binding site is a deep pocket, whereas the S1 and S1' sites provide a binding surface. The S2 and S1’ sites are the major specificity determinants, whereas the positioning of P3 residue is mediated only by sidechain interactions. For this reason, the binding geometries of the latter are scattered over a broad area and are unique for each substrate (Fig. 1b).

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Fig. (1). (A) Fold of cathepsin L, the typical representative of cysteine cathepsins, is shown in blue in the standard orientation, which keeps the positioning of the right and left domains on their corresponding sides. The side chains of catalytic residues Cys25 and His163 are shown in yellow as a ball and stick model. (B) A view of the active site cleft from the top. A polyalanine substrate model (shown in green) binds in an extended conformation along the active site cleft. The main chain atoms of the substrate form hydrogen bonds (shown as white broken stick) with conserved residues beneath the surface of the enzyme. Cathepsin L surface is shown in grey apart from the area, which belongs to the catalytic cysteine residue, shown in yellow. The substrate binding sites from S3 to S2' are marked.

Whereas in endopeptidases the active site cleft extends along the whole length of the two domain interface, the exopeptidases (cathepsin B, C, H and X) possess additional features that reduce the number of substrate binding sites. The role of these features is dual: they prevent binding of longer peptidyl substrates and they dock with charged N- or Cchain termini of substrates by utilizing selective electrostatic interactions. Carboxypeptidases cathepsins B [7] and X [8] utilize insertion of histidine residues which block the active site cleft on the primed binding side and bind the charged main chain carboxylic group of the C-terminal residue of a substrate. In cathepsin B an about 20 residue insertion, termed the occluding loop, blocks the active site cleft on the primed binding side with two histidine residues, His 110 and His 111, whereas in cathepsin X only a three residue insertion called mini-loop [9] positions the His 23 side chain into the active site cleft. In the carboxydipeptidase cathepsin B binding in sites beyond S2 is blocked, whereas in carboxymonopeptidase cathepsin X binding in sites beyond S1 is blocked. Amino peptidases cathepsins H [10] and C [11, 12] use negatively charged carboxylic groups to anchor the positively charged N-terminal amino group of the substrate, however, they utilize different constructs. In cathepsin H an eight residues long residual of the propeptide, termed mini-chain, that remains attached to the papain-like structure provides its C-terminal Thr 83P carboxylic group, whereas in cathepsin

C the carboxylic group is provided by the side chain of Asp 1 residue, the N-terminal residue of the exclusion domain. The exclusion domain is a unique appendix to the papainlike structure responsible for the substrate exclusion as well as for tetrameric assembly of cathepsin C molecules. ACTIVITY REGULATION As a number of other proteases, cysteine cathepsins are synthesized as inactive zymogens, which require proteolytic removal of the N-terminal propeptide for their activity. Based on the in vitro data, activation can be autocatalytic or facilitated by other proteases such as cathepsin D and pepsin. The crystal structures of procathepsins B, L and K [13-18] show that propeptide runs through the active site cleft in the orientation opposite to that of a substrate, thereby blocking access to the active site, which is already formed in the proenzyme. Based on these data, a bimolecular mechanism of autocatalytic activation was suggested, as the unimolecular intramolecular mechanism initially suggested would require enormous movement of the propeptide to bind into the active site in the proper orientation for autocleavage. This was later confirmed using cathepsins B, S and K as model systems [19-21]. Autocatalytic activation can be facilitated by pH drop and/or by glycosaminoglycans (GAGs), which both facilitate weakening of the interaction between the propeptide and the body of the enzyme [22]. Especially GAGs likely play an important role in autocatalytic activation of the cathepsins also at physiological conditions as they enable the activation at pH values around the neutral [20, 22]. However,


only the endopeptidases can be activated autocatalytically. The true exopeptidases cathepsins X and C require endopeptidases, such as cathepsin L, for their activation [9, 23]. The major regulators of the activity of mature cysteine cathepsins are their endogenous inhibitors. Currently, they can be classified into several completely different families: the cystatins, the thyropins, the serpins, the propeptide-like inhibitors and the -barrel type inhibitors [24, 25]. The best characterized are the cystatins, which can be further subdivided into the intracellular stefins and extracellular cystatins and the kininogens. The stefins probably serve to inhibit cathepsins escaping from lysosomes, as their absence could lead to pathologies, such as neuronal apoptosis, observed in the stefin B-deficient animals [26]. A similar role, although extracellular probably have the cystatins. The other inhibitors likely function as either regulatory or emergency type inhibitors, providing the necessary balance under normal conditions [24]. Glycosaminoglycans (GAGs) are another important regulator of cathepsin activities. In addition to their role in the activation, they were shown to be able to stabilize the cathepsins at neutral pH, thereby delaying their inactivation, as demonstrated for cathepsin B [27]. And last but not least, some GAGs, such as chondroitin and dermatan sulphates, were shown to dramatically increase the collagenolytic activity of cathepsin K, and at the same time reduce the collagenolytic activities of other cathepsins such as L and S, thereby providing a novel mechanism for the regulation of physiological activity of cysteine cathepsins [28, 29]. These are also the mechanisms likely contributing to the pathological role of some of the cathepsins (see below) CYSTEINE CATHEPSINS IN PATHOLOGY Proteases can be very harmful if not strictly controlled. There is a growing body of information that cysteine cathepsins are involved in numerous pathologies, with at least some of them representing suitable targets for therapy. One of the areas, where cysteine cathepsins have gathered a lot of attraction recently is cancer biology, especially after the failure of the MMP inhibitors in clinical trials [30, 31]. The other emerging areas include viral infections, cardiovascular diseases and osteoarthritis, although the major focus is still on osteoporosis, rheumatoid arthritis and other diseases linked with the immune system. CATHEPSINS IN CANCER BIOLOGY There is increasing evidence that cysteine proteases, mostly cathepsins B and L, and to a lesser extend cathepsins H, S, X, and K, contribute to the proteolytic events during tumour progression. Cysteine cathepsins upregulation has been reported for many human tumours, including breast, lung, brain, gastrointestinal, prostate cancer, and melanoma [32]. The expression of lysosomal proteases, e.g. cathepsins B and L, have often been positively correlated with a poor prognosis for patients with a variety of malignancies, and antigen levels of cysteine cathepsins were measured as potential prognostic markers for several types of cancer. Cysteine proteases have been implicated in the progression of tumours from a premalignant to a malignant state and in various critical tumourbiological processes, including tu-

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mour cell hyperproliferation and apoptosis; tumour induced angiogenesis, as well as invasion of surrounding tissues and metastasis by malignant cells, suggesting that they are relevant drug targets for treating cancer [31]. Cysteine Cathepsins Influence the Balance of Cell Proliferation and Apoptosis An increased cell proliferation rate represents a key aspect of tumour biology. Cysteine cathepsins were recently discovered to influence the regulation of cell proliferation by several means. Strikingly, inhibition of cysteine cathepsins by JPM-ethyl ester has been found to significantly diminish tumour cell proliferation in the mouse model of pancreatic islet cell carcinoma (RIP1-Tag2) [33]. Using the same RIP1Tag2 model breed to several cathepsin knockout mouse strains revealed decreased BrdU-proliferation indices and significantly decreased tumour volumes in RIP1-Tag2 mice deficient for cathepsins B or L [34]. In line with these findings is the recently described function of cathepsin L in the nucleus, where a form of the enzyme devoid of a signal peptide promotes the cell cycle by proteolytic activation of the CDP/Cux transcription factor [35]. In contrast, the Ki67proliferation index was significantly increased in RIP1-Tag2 tumours deficient for cystatin C, most likely due to enhanced activity of cysteine cathepsins and loss of cystatin C activity in antagonizing cell proliferation [36]. However, the effects of cathepsins on cell proliferation seem to be quite tissue dependent, since mice deficient for cathepsin L show epidermal hyperplasia due to a hyperproliferation of basal keratinocytes that was shown to be caused by an enhanced recycling of growth factors from the endosomes to the keratinocyte plasma membrane, which result in sustained growth stimulation [37, 38]. On the other hand, defects in controlling programmed cell death (PCD) can extend the life span of cells, participating to tumour initiation and cancer formation. While the importance of caspases in the apoptotic process is firmly established, the role of the lysosomes and lysosomal enzymes in cell death has been unmasked only recently [39]. Yet, evidence has been accumulated for multiple scenarios of how cysteine cathepsins promote cell death. It is well established that massive lysosomal rupture can induce necrotic autolysis of cells, a process that is mediated by the lysosomal cathepsins and other “acidic” hydrolases. Models on the role of cathepsins in PCD are, however, based on a more selective release of lysosomal proteases from the acidic cellular compartment in response to certain death stimuli. Although still debated, two principle mechanisms for cathepsin release emerge. One mechanism could be direct damage of lysosomal membranes by toxic stimuli such as reactive oxygen species [40], bile salts [41], cold ischaemia–warm reperfusion [42], or microtubule stabilizing agents [43]. Secondly, lysosomal permeabilization could occur due to specific signalling processes as suggested for TNF -mediated apoptosis of murine hepatocytes [44], tumour cells [45, 46], and immortalized murine embryonic fibroblasts [47]. In TNF-mediated PCD of hepatocytes lysosomal permeabilization depends on activation of neutral sphingomyelinase (FAN) as well as on caspase 8 induced Bid activation [48]. Once released, cysteine cathepsins might initiate or enhance activation of caspases either directly [49], or by induction of mito-


chondrial cytochrome c release activation of Bid-cleavage or other mitochondrial factors [44, 50]. Since full caspase 3 activation is achieved under these conditions, the morphological and biochemical hallmarks of classical apoptosis are readily detectable. Alternatively, there is also evidence for a role of cathepsins in execution of PCD independent of caspase activation, which results in an apoptosis-like morphology [45]. Thus, both the rates of cell proliferation and apoptosis can be influenced by lysosomal cysteine proteases placing cathepsins as one of the critical control mechanisms for cellfate determinations. Remarkably, in most experimental settings cathepsin activity promotes cell proliferation as well as apoptosis. However, the in vivo functions of individual cathepsins are cell type and tissue specific, depending, among other factors, on cathepsin expression levels. Thus, careful investigations have to be performed to establish whether inhibition of an individual cathepsin decreases cell proliferation in a tumour without increasing PCD resistance in that particular cancer. Cysteine Cathepsins Affect Tumour Angiogenesis Angiogenesis is required for invasive tumour growth and metastasis and constitutes an important point in the control of cancer progression, since avascular tumours are severely restricted in their growth potential because of the lack of blood supply. Therefore, inhibition of angiogenesis is a valuable approach to cancer therapy [51]. Vascular remodelling during tumour growth is a multistep process requiring endothelial cell proliferation and tissue invasion by degradation of extracellular matrix. During neoplastic progression cancer-prone tissues induce an "angiogenic switch" by shifting the local balance of (i) proangiogenic factors, e.g. vascular endothelial growth factor (VEGF), and (ii) antiangiogenic factors, such as endostatin or angiostatin, towards the angiogenic site. Recent studies revealed that expression of cathepsin B from tumours correlates with angiogenesis and is thought to promote the remodelling of the extracellular matrix to permit capillary formation [52, 53]. Furthermore, overexpression of cathepsin B increases the intensity of angiogenesis in primary colon adenocarcinoma [54], whereas blockade of cathepsin B expression suppresses angiogenesis in human glioblastoma cells [55]. Similarly, blocking cathepsin activity affects tumour vascularization during RIP1-Tag2 tumourigenesis resulting in a reduction of vessel density and branching as compared to untreated tumours [33]. Cathepsin B and cathepsin S knockout mice bred to the RIP1-Tag2 tumour model revealed a significant reduction of angiogenic switch, while cathepsin L and cathepsin C deficient mice did not affect angiogenesis in the model [34, 56]. Moreover, a recent study on the cathepsin S knockout mouse implicated cathepsin S as an enhancer of angiogenesis associated with wound healing in the skin [57]. What are the mechanisms by which cathepsins, i.e. cathepsins B and S promote blood vessel formation? It is well established that proteolysis of the basement membrane is a critical step in vessel sprouting during angiogenesis [58], and notably, cathepsin B has been shown to directly degrade three major basement membrane components, laminin, col-


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lagen IV, and fibronectin, at physiological pH [52]. Cathepsins have been shown to generate proangiogenic as well as antiangiogenic peptides from ECM macromolecules. Cathepsin S regulates the production of type-IV collagenderived anti-angiogenic peptides and the generation of bioactive pro-angiogenic gamma2 fragments from laminin-5 in RIP1-Tag2 tumour mice [56]. However, in this study neither cathepsin S deficiency nor increased cathepsin activity due to cystatin C deficiency showed a significant impact on endostatin levels in either sera or tumour extracts [56]. For endostatin it was shown by Felbor and co-workers that cathepsin L secreted from non-metastatic murine hemangioendothelioma cells is able to generate this antiangiogenic peptide from collagen XVIII in a moderately acidic extracellular milieu by cleavage of peptide bonds within the protease-sensitive hinge region of the C-terminal domain (NC1) [59]. In vitro studies on processing of recombinant human NC1 with proteases of all classes revealed that cathepsins L, B and K, as well as several matrix metalloproteases and pancreatic elastase can generate endostatin-like protein fragments, although with different efficiencies [60]. Moreover, some proteases, including cathepsins L, B, D, and K, degrade endostatin or NC1 fragments very efficiently [60]. Recently, it was suggested that endothelial cells produce proand antiangiogenic factors in a cathepsin B–regulated manner. According to this model cathepsin B maintains the endothelium in a nonangiogenic state by increasing endostatin generation while suppressing expression of vascular endothelial growth factor (VEGF) through degradation of hypoxiainducible factor-1 (HIF-1a), one of the regulators of VEGF transcription [61]. The classical tumour angiogenic process discussed above is characterized by proliferation, migration and capillary formation by local endothelial cells. However, bone-marrow derived circulating endothelial progenitor cells have been shown to promote neovascularization [62], and cathepsin L was identified as critical protease for endothelial progenitor cell tissue invasion in a mouse model of neovascularisation after hind limb ligation [63]. However, the contribution of endothelial progenitor cell cathepsins to new blood vessel formation in tumours still needs to be elucidated. Taken together, the overall effect of cathepsins on angiogenesis depends on their relative efficiency in nonspecific ECM degradation in order to open the way for migration of endothelial cells or endothelial cell progenitors and specific generation of proangiogenic factors as opposed to the generation of antiangiogenic, i.e. endostatin-like, peptides. Cysteine Cathepsins Contribute to Tissue Invasion and Metastasis of Cancer Cells There is increasing evidence that cysteine cathepsins promote invasion and metastasis by remodelling the extracellular matrix (ECM) in the tumour microenvironment. Cysteine cathepsins are clearly involved in the intracellular degradation of endocytosed ECM proteins [64, 65]. However, there is ample experimental support for extracellular functions of cysteine cathepsins in tumours. Increased secretion of cathepsin B proenzyme as well as of the mature proteases by human colorectal carcinoma cell lines and hepatomas, colorectal and lung cancers have been reported [6668]. Moreover, secretion of procathepsin B can occur from


cells that do not exhibit an increase in mRNA levels, indicating that this secretion is probably due to altered intracellular trafficking and distribution of this enzyme [69]. Another indication that tumour cells secrete cathepsin B is the increased serum level of this protease in patients with hepatocellular and ovarian carcinomas, prostate cancer and melanoma [70]. Cathepsin B has also been found in other body fluids surrounding tumours, such as bronchoalveolar lavage fluid of lung cancer patients or cerebrospinal fluid from patients with leptomeningeal metastasis [71, 72]. Besides being secreted, cathepsin B has been found as plasma membraneassociated protease in a number of cancer cells [73], and cathepsin B activities can be assayed on the surface of living cells in culture [74]. One mechanism of cathepsin B association with the cell surface is through an interaction with annexin II heterotetramers. Annexin II directs cathepsin B to caveolae [75], plasma membrane pits with high proteolytic capacity harbouring a wide variety of interdependent proteases, e.g. membrane bound matrix-metalloproteases and serine proteases of the plasminogen activator/ plasminogen system [76, 77]. Although the mechanism of cathepsin-B distribution to the basal plasma membrane remains to be elucidated, it occurs in late adenomas and early carcinomas, and is coincident with the activation of K-ras [78]. Recently, it was shown for HCT116 human colorectal-carcinoma cells that trafficking of cathepsin B to caveolae and its secretion is regulated by active K-ras [75]. Increased expression and translocation of active cathepsin B to the basal membrane is also observed during the progression of adenomas in Minmice [79]. An important role of plasma membrane associated cathepsin B for tumour invasion has been recently shown in primary breast carcinoma cells isolated from MMTVpolyoma middle-T (PyMT) transgenic mice [80]. Moreover, complete ablation of cathepsin B expression in cathepsin B knock-out tumour cells induced compensation by plasma membrane translocation of another cysteine peptidase, cathepsin X [80], providing additional evidence for the requirement of cysteine peptidase activity for tumour progression and metastasis. Once they are localized extracellularly, cathepsins may act on the contact sites of tumour cells with the basement membrane or interstitial stroma. These areas are often acidified by tumour cells, thereby generating conditions that are favourable for activation of secreted cathepsin precursors to active forms. Active cathepsins have been shown to be able to degrade the protein components of basement membranes and the interstitial connective matrix including laminin, fibronectin, elastin, tenascin and various types of collagen [53, 81]. On the other hand, cathepsin B indirectly enhances proteolysis by activating precursors of serine proteases to their active forms, such as pro-uPA (urokinase plasminogen activator) [82]. Pro-uPA is secreted as an enzymatically inactive proenzyme by tumour and stroma cells and bound to a high-affinity cell surface receptor (uPAR). In ovarian cancer cells, inhibition of cell surface cathepsin B prevents activation of pro-uPA, and subsequently, invasion of the carcinoma cells through artificial ECM (Matrigel) [83]. Cathepsin B as well as active uPA can subsequently convert plasminogen to plasmin, which is capable of degrading several components of tumour stroma, and may activate zymogens of matrix metalloproteases, the main family of ECM degrading proteases. In addition to indirect activation of MMPs via the plasminogen activator / plasmin cascade,

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cathepsin B was shown to activate some of the MMPs also directly, e.g. interstitial procollagenase (proMMP-3), prostromelysin-1 (proMMP-2), or by inactivation of tissue inhibitors of matrix metalloproteases TIMP-1 and TIMP-2 [84]. Cathepsin B, therefore, could be an important upstream regulator in the activation of pro-uPA plasminogen and proMMPs thus leading to dissolution of tumour matrix and basement membrane, which is a prerequisite for invasion and metastasis. Recent studies have suggested a role of proteases derived from stroma cells in promotion of tumour growth and metastasis (reviewed in [85]). Cathepsin B has been reported to be expressed in stromal fibroblasts and macrophages in carcinomas of breast, colon and prostate [86]. In some cases, macrophages at the invasive front of colon or prostate tumours were shown to express higher levels of cathepsin B than the tumour cells [86, 87]. Furthermore, studies in transgenic mouse tumour models have established a functional role for cysteine proteases in vivo. Cathepsin B activity from inflammatory cells was shown to be important during progression of pancreatic carcinomas in the RIP-Tag model [33]. Recent results on the PyMT mouse mammary cancer model using an experimental lung colonisation assay indicated that host cell-derived cathepsin B is potentially as important as the tumour-derived protease activity for metastatic growth. [80] Tumour-infiltrating macrophages were identified to induce cathepsin B expression upon recruitment to the tumour node [80], providing further evidence for functional involvement of proteolytic enzymes of tumourinvading bone marrow derived cells in cancer progression [88]. CYSTEINE CATHEPSINS IN THE IMMUNE SYSTEM AND VIRAL INFECTION MHC Class II Antigen Presentation MHC class II-restricted antigen presentation plays a central role in the immune response against exogenous antigens. In the MHC class II pathway two principal processes that involve cysteine cathepsins can be differentiated: (i) the generation of antigenic peptides by degradation of endocytosed ‘precursor’ proteins, and (ii) the formation and maturation of the major histocompatibility complex (MHC) [89]. Degradation of antigens in vitro and experiments using broad spectrum inhibitors established a role of cysteine proteases in antigen degradation and peptide generation [90]. However, antigen presenting cells (APCs) from knockout mice for the cathepsins in question did not yet reveal any processing defects of specific antigens such as ovalbumin, indicating a largely redundant function of cysteine cathepsins for MHC class II peptide generation [91]. On the other hand, specific cysteine cathepsins have been implicated in the processing of MHC class II invariant chain and, therefore, in maturation of the MHC class II complex. Interestingly, the different types of APC employ different cathepsins for invariant chain processing. Epithelial cells of the thymic cortex use cathepsin L for this processing, as revealed by the analysis of cathepsin L deficient mice [92]. Consequently impaired MHC class II loading with peptides and impaired positive selection of T lymphocytes has been


found in cathepsin L knockout mice. Thus a marked depletion of CD4+ T lymphocytes to about 20% of the normal level was observed in these mice, while CD8+ T cell counts were not affected [92]. However in humans, cathepsin V, a highly homologous enzyme to cathepsin L [93], was shown to be the dominant cysteine protease in cortical human thymic epithelial cells, while cathepsins L and S are not expressed in these cells [94]. In addition, recombinant cathepsin V was capable of converting Ii to class II-associated Ii peptide (CLIP) suggesting that cathepsin V is the protease that controls the generation of ß-CLIP complexes in the human thymus, in analogy to cathepsin L in mouse [94]. In contrast, cathepsin S deficiency impairs antigen presentation by bone marrow-derived APCs, but does not affect CD4+ T cell development [95, 96]. As such, B cells and DCs of mice deficient in cathepsin S did not convert the 10 kDa invariant chain fragment (Lip10) to CLIP and accumulate class-II-Lip10 complexes within endosomes [97, 98]. In addition, DCs developmentally regulate their capacity for antigen presentation by controlling the transport and surface expression of MHC class II molecules that have been shown to be regulated by the ratio of cystatin C to cathepsin S [99]. Interestingly, macrophages from mice deficient for both cathepsins S and L can process Ii and load peptides onto MHC class II dimers normally, whereas both processes were blocked by a cysteine protease inhibitor. Screening for cysteine proteases expressed in those cells revealed another cysteine protease, cathepsin F, shown to be as efficient as cathepsin S in CLIP generation. It has therefore has been proposed to be implicated in Ii degradation in macrophages [100]. However no defects in MHC class II maturation in splenocytes, bone-marrow derived and peritoneal macrophages attributable to cathepsin F could be detected in cathepsin F knock-out mice and from cathepsin F/S doublenull mice [101]. Another cysteine cathepsin involved in immunological response is cathepsin C that has been shown to activate granzymes, neutrophil elastase, and cathepsin G. Hence, it probably participates in cytotoxic lymphocyte granule-mediated cytotoxicity towards virally infected or malignant cells [102]. Expression of cathepsin C was shown to be upregulated in CD8 + T cells in the thymus and spleen [103], and further up-regulated in activated cytotoxic T lymphocytes [104]. However, studying patients with PapillonLefèvre syndrome, an autosomal recessive disease associated with loss-of-function mutations in the cathepsin C gene locus, has failed to detect a generalized T cell immunodeficiency phenotype [105]. In addition, cathepsin W might play a role in cytotoxic killing of abnormal cells, because of its selective expression in peripheral CD8+ T cells and natural killer cells [106]. However, cathepsin W knockout mice showed no gross phenotype or defects in cytotoxic immune cells [107]. Host Cathepsins in Viral Infections Cathepsins have been found to promote infection of cells by several virus types. Early studies suggest that specific endosomal proteases mediate disassembly of reovirus virions to infectious subviral particles (ISVPs) that are capable of penetrating membranes thereby delivering transcriptionally


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active core particles into the cytoplasm. The broad spectrum cysteine protease inhibitor E64 was shown to block reovirus disassembly [108, 109]. Moreover, mutant cells selected during persistent reovirus infection do not support reovirus disassembly despite internalizing and transporting virions to acidified, perinuclear compartments was shown to have defects in the activity of cysteine proteases cathepsin B and cathepsin L but not cysteine protease cathepsin H [110]. Treatment of L929 cells with cathepsin B and L inhibitors and using cathepsin L and B -deficient mouse embryo fibroblasts suggested that either cathepsin L or cathepsin B are required for reovirus entry into murine fibroblasts and indicate that cathepsin L is the primary mediator of reovirus disassembly [111]. Golden et al. identified many transformed and nontransformed cell lines as well as primary cells that restrict viral infection. In several of these restrictive cells, virion uncoating is inefficient or blocked, whereas addition of proteases to the cell culture medium generates ISVP-like particles and promotes viral growth in nearly all cell lines tested [112]. It was suggested that the acid dependency of reovirus infections of most other cell types may reflect the low pH required for the activities of lysosomal proteases rather than some other acid-dependent aspect of cell entry [113]. It was demonstrated that some reovirus strains do not infect P388D cells efficiently because a specific capsid protein, 3, is poorly susceptible to cathepsin S-mediated cleavage [113]. Hendra and Nipah viruses are highly related zoonotic paramyxoviruses, which cause fatal disease upon transmission to humans. Cathepsin L was recently shown to be involved in proteolytic processing of the Hendra virus fusion protein (F) [114]. Nonspecific and specific protease inhibitors ablated proteolytic processing of Hendra virus F, and cathepsin L shRNA-expressing Vero-cells transfected with a mammalian expression vector containing the Hendra virus F gene, significantly decreased cleavage of the Hendra virus F protein and membrane fusion activity, whereas coexpression of cathepsin L restored fusion activity. The data showed that multiple endosomal cathepsins can cleave Nipah F, but only cathepsin L specifically converts Nipah F to its mature and fusogenic form [114]. Ebola virus (EboV) rapidly causes fatal hemorrhagic fever in humans. Using selective protease inhibitors and protease-deficient cell lines, Chandran and co-authors demonstrated that EboV infection is mediated by cathepsins B and L by specific cleavage of the EboV glycoprotein-dependent entry-subunit (GP1) [115]. Cathepsin B and/or cathepsin L remove C-terminal sequences from GP1 to generate a Nterminal GP118K-like protein, which is than further digested by cathepsin B, an event triggering membrane fusion and virus entry [115]. Severe acute respiratory syndrome (SARS) is caused by an emergent corona virus (SARS-CoV), for which there is currently no effective treatment. It has been shown that inhibitors of cathepsin L prevent severe acute respiratory syndrome corona virus entry [116, 117] through activation of the viral envelope glycoprotein's membrane-fusion potential. Thus, Simmonse and co-authors suggest a mechanism for viral entry into target cells via a three-step process: angiotensin-converting enzyme 2 (ACE2) receptor binding and


induction of conformational changes in S glycoprotein followed by cathepsin L proteolysis within the endosomes [116]. Thus these data demonstrate that cathepsin L is an important activating protease for SARS-CoV infection proposing cathepsin L as a target for therapeutic intervention [116]. Recently a novel coronavirus causing human disease, HCoV-NL63 (HCoV-NL, HCoV-NH), has been described, which is a common, widely distributed pathogen associated with moderate respiratory illness in children, also utilizing the SARS-CoV receptor ACE2 to infect cells [118]. Notably, inhibition of endosomal cysteine proteases or expression of exogenous cathepsin L had substantially less effect on infection mediated by the HCoV-NL63 than on that mediated by the SARS-CoV indicating that despite utilizing a common receptor, two different corona viruses enter cells by distinct mechanisms [117]. Taken together all these findings indicate that lysosomal proteases might play an important role in host cell susceptibility for several types of viral infection, predetermine virus tropism and pathogenesis in infected hosts. CYSTEINE CATHEPSINS INVOLVED IN BONE RESORPTION Bone and Cartilage Turnover Bone development and homeostasis is a complex process, which is based on the balance between bone formation and resorption. The major effector cells of bone formation and resorption are the osteoblasts and osteoclasts [119]. Osteoclasts are specialized multinucleated cells that resorb bone in the remodelling cycle. At their site of attachment, osteoclasts generate an acidic microenvironment to dissolve bone minerals, thereby rendering the organic matrix available to proteases released from osteoclasts into the resorption lacunae and providing an optimal pH for proteolytic activity [120]. Initially, cysteine proteases were implicated in bone matrix degradation in the early 1980s by using various cysteine protease inhibitors such as leupeptin, antipain, and E-64 [121, 122]. Vacuoles in osteoclasts from cultured mouse bone explants treated with E-64 contained intact collagen fibrils, proposing an important role of cysteine cathepsins in digestion of phagocytosed collagen [123]. Primarily, cathepsins B and L have been detected in the bone tissue and their role in bone resorption related to the degradation of collagen molecules and gelatine, and procollagenase activation was proposed. However, the discovery of cathepsin K, which is abundantly and selectively expressed by osteoclasts and secreted into the resorption lacunae [124, 125], suggests that it is a key enzyme in the degradation of the organic bone matrix [126, 127]. Ninety percent of bone organic matrix and extracellular matrix of cartilage consists of type I and type II collagens, which both form triple helices with the exception of the single chain ends (telopeptides). Unspecific proteases including cathepsins L, B, and S cleave collagen molecules only in the telopeptides and not in the native triple helix, generating monomers of type I collagen [126]. In contrast, cathepsin K was shown to be the only protease that cleaves collagen I and II molecules both in the telopeptides and at

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multiple sites within the native triple helix [126, 127]. In addition, Li et al. confirmed that under physiologically relevant conditions, i.e. upon formation of a complex with boneand cartilage-resident glycosaminoglycans, cathepsin K represents the only lysosomal collagenolytic activity [29]. Inversely, inhibition of cathepsin K in vitro and in vivo reduces bone resorption [128]. Imbalance between bone formation and resorption can result in the formation of either too much bone (osteopetrosis / osteosclerosis) or too little bone (osteoporosis) (for review [129]). Osteoporosis Osteoporosis is a progressive skeletal disorder characterized by bone loss and microstructural deterioration resulting in skeletal fragility and an increased risk of bone fractures [129]. Overexpression of cathepsin K in osteoclasts was shown to accelerate the turnover of metaphysical trabecular bone in mice, which is indicative of osteoporosis [130]. Interestingly, the cathepsin K transgenic mice exhibit a marked increase in the number of osteoblasts. The rate of bone turnover, and the amount of mineralizing surface was observed, emphasizing an important role of cathepsin K in the bone remodelling cycle [130, 131]. Morko et al. showed that overexpression of cathepsin K gene by its genuine promoter in transgenic mice is sufficient to enhance osteoclastic bone resorption and results in osteopenia of metaphyseal trabecular bone and in increased porosity of diaphyseal cortical bone [132]. With ageing the excessive production of cathepsin K in these transgenic mice results in synovial hyperplasia and fibrosis and subsequent destruction of articular cartilage and bone [133]. Expression of cathepsin K is highly up-regulated under conditions of enhanced bone resorption, such as immobilization, and by bone resorbing agents [134, 135]. Moreover, the cathepsin K levels of patients with osteoporosis were significantly higher than in controls [136]. It was shown that the serum level of cathepsin K correlates with the incidence of nontraumatic fracture, bone-mineral density, markers of bone turnover (bone-specific alkaline phosphatase, osteocalcin, etc.), and has been proposed as a marker for fracture prediction and bone-mineral density [136]. Collectively, these data validated cathepsin K as a relevant target for diseases with excessive bone degradation such as osteoporosis [137, 138]. In addition to cathepsin K, a role for cathepsin L in osteoporosis was proposed by several studies. It was shown that the use of cathepsin L inhibitors reduced cortical bone mineral density and results in inhibition of bone resorption in thyroparathyroidectomized and ovariectomized rats [139]. In cathepsin L knockout mice loss of trabecular bone following ovariectomy occurred to a lesser extent in homozygote and heterozygote mice than in the wild-type mice [140]. Taken together these observations suggest that cathepsin L is also likely to have a role in controlling bone turnover during normal development and in pathological states. Osteosclerosis and Pycnodysostosis Inhibition of cathepsin K activity in vitro and in vivo reduces bone resorption resulting in an increased bone mass [128]. Pycnodysostosis is a rare, autosomal recessive trait characterized by osteosclerosis, short stature, acro-osteolysis


of the distal phalanges, bone fragility, clavicular dysplasia, and skull deformities with delayed suture closure. Gelb and co-workers used a positional cloning strategy to identify the cathepsin K gene as the cause of pycnodysostosis [141]. There are at least 15 known mutations in the cathepsin K gene resulting in cathepsin K deficiency and leading to pycnodysostosis [142]. Interestingly, one mutation (Y212C) causes appearance of a defective enzyme that loses its activity only against type I collagen, preserving its overall activity [143]. The Y212C mutation prevents formation of complexes between cathepsin K and chondroitin sulphate, which is essential for the enzyme to gain collagenolytic activity [143]. Mice deficient in cathepsin K have differentiated osteoclasts, which demineralize bone, but are unable to degrade organic bone matrix. [144, 145]. Histological analysis revealed that osteoclasts from patients with pycnodysostosis and from cathepsin K-deficient animals exhibit a similar morphology; they contain large cytoplasmic vacuoles with undigested collagen and, due to the lack of proteolytic activity, they form demineralized matrix fringes on the bone surface [144, 145]. Rheumatoid Arthritis (RA) RA is a chronic inflammatory joint disorder involving autoimmune processes that result in destruction of joint cartilage eventually leading to loss of joint function [146]. In RA, the synovial membrane proliferates to form a pannus that destroys adjacent bone and cartilage. Among the cells mainly responsible for cartilage damage are fibroblast-like synoviocytes (synovial fibroblasts) and activated macrophages that accumulate in the interface between pannus and cartilage [147]. Cathepsin B and, to a lesser extent, cathepsin L were found in the synovial fluids [148, 149] and synovial lining tissues of patients with RA [150]. Both enzymes have been detected at high levels in membranes of patients with RA, even at very early stages, while their levels were very low in normal synovium [151]. Keyszer and co-authors proposed post-transcriptional up-regulation of these enzymes in RA [152]. In addition, selective secretion of cathepsins B and L by synovial fibroblast-like cells upon cytokine induction was observed [153]. In experimental animal models increased levels of cathepsin B have been detected in joint tissues during the course of experimental arthritis [150, 154], while elevated levels of cathepsin L have been detected in the synovial lining of rabbits with antigen-induced arthritis [154]. Furthermore, involvement of cathepsins B and L in bone degradation was demonstrated by inhibition of bone resorption by selective inactivators of cysteine proteases [155]. Using retroviral gene transfer, ribozymes cathepsin L mRNA, specifically inhibiting the synthesis of cathepsin L, reduced cartilage destruction in vitro and in vivo using a mouse co-implantation model of RA [156]. In addition, expression of one of the most potent extracellular matrix-degrading mammalian enzymes, cathepsin K, was detected in synovial fibroblasts as well as in multinucleated giant cells from RA patients correlating with the severity of the disease [157]. Up to 90% of cells in RA lymphocytic infiltrates, but only 10-30% of the cells in a normal synovium, were found to express cathepsin K mRNA. Ca-


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thepsin K expression in primary cultures of synoviocytes was stimulated by proinflammatory cytokines, such as IL-1 and TNF- [157]. Furthermore, cathepsin K protein was localized in synovial fibroblasts and mononuclear macrophage-like cells at the sites of cartilage and bone degradation in RA [157, 158]. Inhibition of cathepsin K in RAderived synovial fibroblasts resulted in lysosomal accumulation of undigested type II collagen fibrils, whereas inhibition of cathepsins L, B, and S had no effect [158]. Interestingly, the physiological concentrations of chondroitin sulphate found in synovial fluids lead to an optimal stabilization of cathepsin K activity and subsequently to an increase of its collagenolytic activity [158]. Another potent proteoglycan-degrading cysteine peptidase, cathepsin S, was shown to be expressed in synovial macrophages in RA joints and efficient in hydrolyzing aggrecan at neutral and acidic pH. [159] Cathepsin S secretion from macrophages into the cartilage matrix was detected during the inflammatory process [160]. Furthermore, cathepsin S-deficient mice revealed decreased susceptibility to collagen-induced arthritis [95], and rats with adjuvantinduced arthritis displayed significant decrease in inflammation after oral administration of cathepsin S inhibitor LHVS supporting a specific role for this protease in autoantigen presentation during RA [161]. However, in contrast to cathepsin K, cathepsin S is a weak collagenase and thus unlikely to be involved in either direct type I (bone) collagen or type II (cartilage) collagen degradation [162]. The importance of neutrophil-derived cathepsin C in RA pathology was demonstrated by using an in vivo model of acute inflammatory arthritis. A strong protection against arthritis induction by anti-collagen antibodies was observed in cathepsin C knock-out mice compared to wild-type mice that developed clinically evident arthritis [157]. This finding was ascribed to the lack of activation of neutrophil-derived serine proteases by cathepsin C and subsequent impairment of cytokine production [157]. Osteoarthritis (OA) OA is characterized by dysregulation of chondrocyte function resulting in cartilage joint destruction [163]. A number of cysteine cathepsins have been suggested to participate in cartilage degeneration in OA [164, 165]. Moreover, a decrease in pH from 7.1 to 5.5 observed in the cartilage surface of the OA patients positively correlates to the extent of cartilage destruction and disease progression, favouring cysteine cathepsins over various metalloproteases as the major ECM degrading proteases in OA, at least in later stages of the disease [166]. Significant levels of cathepsins B and L were found immunocytochemically in macrophage-like cells of the synovial lining of the normal rat temporomandibular joint [167], whereas cultured synoviocytes have been shown to secrete pro-cathepsin L [168]. Remarkably, increased cathepsin B expression was detected during the initial cartilage damage, while it was declining in advanced degenerative stages. [169, 170] Near-infra-red-fluorescence-imaging using a cathepsin B activated probe showed a significant increase of proteolytic activity in the OA-joints as compared to the normal joints [171]. In addition, an alternative splicing of cathepsin B transcripts has been suggested to contribute to


this activation [172]. However, in an experimental OA Del1 mouse model, harbouring a short deletion in the type II collagen gene and predisposed to early onset osteoarthritis, no evidence for up-regulation of cathepsins B, H, L, or S on expressional level was found during the development of degenerative cartilage lesions [173]. Recent studies, however, point more to the cathepsin K. Cathepsin K expression was shown in synovial giant cells of OA patients and model animals [157, 173, 174]. Furthermore, the number of cathepsin K containing chondrocytes increased with the severity of the disease [173, 174]. On ageing, wild type animals also developed osteoarthritis, which was accompanied by increased cathepsin K expression [173]. Notably, a decrease in pH in cartilage of transgenic Del1 mice predisposed to early onset OA correlated with severity of the damage. [173]. A significant loss of subchondral bone in experimental dog OA was associated with an increase in the osteoclast population that stained strongly positive for cathepsin K in the early phase of the disease [174]. These observations, combined with the fact that cathepsin K is an acidic, lysosomal protease, suggest a physiological role of cathepsin K in cartilage turnover in addition to its role in bone resorption. This is supported with the finding that licofelone treatment reduced the level of synthesis of cathepsin K in both of these tissues [174], which might be ascribed to the inhibition of cytokine or growth factors synthesis since IL-1 and TNF- have been demonstrated to upregulate cathepsin expression [175]. Cathepsin K thus seems to be a relevant target also for the treatment of OA. DEVELOPMENT OF THERAPIES BASED ON INHIBITION OF CYSTEINE CATHEPSINS Major progress has been made in our current understanding of the role of cysteine cathepsins in various pathologies revealing a number of cathepsins in several pathological states, such as cancer as potential drug targets. However, only cathepsins K and S have been clearly validated as appropriate drug targets in osteoporosis (cathepsin K) and in immune disorders (cathepsin S in bronchial asthma, RA, psoriasis, …) with cathepsin K probably also being an appropriate target in both RA and OA. The majority of work on inhibitor development has focused on the development of active-site directed small molecule inhibitors [165, 176], although some other approaches, such as development of neutralizing antibodies [177], have also been tested. So what are the requirements for a successful inhibitor to become a drug? Since therapeutic inhibition of cathepsins S and K, and probably of other cathepsins, is linked to chronic diseases, irreversible inhibitors, although they were of tremendous help in elucidating the catalytic mechanism and specificities of individual cathepsins, are not suitable as therapeutic drugs. During prolonged administration, they could nonspecifically inhibit other proteases or trigger autoimmune response due to the generation of immunogenic haptens from covalently bound inhibitor-cathepsin adducts. Although the most suitable inhibitors would be noncovalent reversible inhibitors (high selectivity, low probability of an adverse immune response), the majority of work was done on the development of reversible covalent inhibitors using different thiol reactive warheads (nitriles, aldehydes, -ketoheterocycles, aliphatic ketones,…). The standard approach in de-

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signing cathepsin inhibitors was to attach a recognition sequence, sometimes mimicking the substrate, to the warhead. Since such inhibitors are peptidic in nature, this had to be reduced during the optimization procedures [138, 165, 176]. The major challenge of the inhibitor design still seem to be toxicity and bioavailability of the compounds, although specificity is still an issue, as cathepsins have relatively similar active site regions (see above). The selectivity of an inhibitor thus reflects cumulative contributions from multiple binding sites. The development of compounds targeting cathepsins K and S have already entered clinical studies, whereas all other work seems to be in early experimental phases. Therefore, we will focus only on the current advances in the development of cathepsin K and S inhibitors. Development of Cathepsin K Inhibitors: A New Approach in Osteoporosis Treatment The main current osteoporosis therapy is based on the use of biphosphonates, which prevent bone loss by inducing cell death of osteoclasts. However, biphosphonates have also several disadvantages, such as numerous side effects (osteonecrosis, upper gastrointestinal irritation, leukopenia, fever, pain, delayed fracture healing, ...) and an extremely long half-life in human body (>10 years), which is a serious concern [176]. Cathepsin K inhibitors therefore provide an alternative, and the recent results from clinical studies suggest that cathepsin K inhibitors would be more beneficial than biphosphonates because they have also a positive effect on bone reformation in addition to the inhibition of bone resorption and their more rapid onset of action. The development of cathepsin K inhibitors started as soon as cathepsin K was discovered as the major cysteine protease in osteoclasts [138, 176]. One of the problems in designing cathepsin K inhibitors was also associated with the development of appropriate biological testing method. Although the amino acid sequences of rodent and human cathepsin K are highly homologous, there are important differences in several critical residues in the active site cleft, involved in substrate and/or inhibitor binding. Consequently, the compounds developed against human enzyme, would have substantially lower biological activity in a rodent disease model [178]. Two major ways to overcome this problem have been described in literature. The first is based on the use of different human-derived cellular models and is widely used today [179, 180], whereas the second is based on the use of ovariectomized cynomolgus monkeys. The latter model is based on the findings that human and monkey cathepsin K sequences are identical and that the expressed proteins exhibit indistinguishable enzymatic properties, and was introduced by the researchers from GlaxoSmithKline [181]. However, rodent models are still in use [182, 183]. Three of the major companies involved in cathepsin K inhibitor design, Novartis, GlaxoSmithKline and Merck (a joint program with Celera) brought their inhibitors into clinical trials. Novartis, although published least among the three companies mentioned, has even two compounds in clinical trials: AAE-581 (balicatib) and AFG-495. AAE-581 was reported to have successfully progressed through Phase II trials in 2005 and Phase III trials are expected in 2006 (www.novartis.com). AAE-581 is a peptidic nitrile and was


shown to be a very potent inhibitor of human cathepsin K with a Ki value of 0.7 nM, whereas inhibition of rat cathepsin K was considerably weaker (Ki = 57 nM). The compound exhibited very high selectivity versus related human cathepsins B, L and S (>2000-fold), however, the selectivity was lower for the rat cathepsins. Oral administration of the compound for 18 month at 10-30 mg/kg in ovariectomized cynomolgus monkey model resulted in substantial reduction of the markers of bone resorption and in a stimulatory effect on periosteal bone formation (Misbach, 20051; Jerome, 20052). In a report disclosing the results of a Phase II trial in 140 postmenopausal women, AAE-581 when administered once a day orally (10-50 mg per day) exhibited dual action by successfully reducing bone resorption and improving bone formation. The compound was well tolerated and no adverse effects were reported. Recently, researchers from Merck reported that basic inhibitors such as AAE-581 and their compound CRA-013783, were lysosomotropic and accumulated in lysosomes, which resulted in nonspecific offtarget effect, thereby diminishing in vivo efficiency of the inhibitor 10-100-fold as measured in cell-based enzyme occupancy assays. One such example was a weak effect on the functional cathepsin S antigen presentation assay [184]. The second compound, AFG-495, has entered phase I trials, however, no data were disclosed yet. In addition, Novartis is also developing noncovalent inhibitors such as arylaminoethyl amides [185, 186], but they are all in experimental phases. However, Grabowska et al. (2005) reported that inhibition of cathepsin K by aminoethylamides could be a result of the presence of an irreversible component, which would be a major drawback. In 2002, GlaxoSmithKline announced that they have started Phase I clinical trials with their compound SB-462795, however, no details about the compound were disclosed. The compound is most likely an -heteroatom cyclic ketone related to the compound SB-357114, which showed good results in ovariectomized cynomolgus monkeys. The compound is very potent (Ki=0.17 nM), although not that selective inhibitor of cathepsin K, as it also inhibits cathepsin L with a Ki value of 2.2 nM [181]. Researchers at GlaxoSmithKline published a great deal of information about their preclinical research, suggesting that they were the most active in the field. They have been exploring largely all the subsites and different warheads [138, 182, 187-197]. The major conclusion was that the most promising compounds are the ketones and the nitriles [138]. In summer 2004 Merck announced, that they have also brought a compound, resulting from their joint program with Celera Genomic into Phase I. No preclinical data were disclosed, however, it is likely that the compound is from the same series as CRA-013783 [198]. CRA-013783 is a tri ring P3 benzamide-containing aminonitrile, which is a very potent and selective cathepsin K inhibitor (Ki = 0.25 nM; Ki >

1

Misbach M, Altmann E, Betschart C, Buhl T, Gamse R, Gasser JA, et al. AAE581, a potent and highly specific cathepsin K inhibitor, prevents bone resorption after oral treatment in rat and monkey. Annual Meeting of ASBMR 2005: www.asbmr.org 2 Jerome C, Misbach M, Gamse R. AAE581, a Novel Cathepsin K Inhibitor, Protects Against Ovariectomy-Induced Bone Loss in Non-Human Primates, in Part by Stimulation of Periosteal Bone Formation. Annual Meeting of ASBMR 2005: www.asbmr.org.


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1 μM for cathepsins B, L and S). The compound has good oral bioavailability in rats and significantly reduced collagen breakdown in ovariectomized rhesus monkeys (~75 %) when administered orally once a day for seven days. However, the compound is lysosomotropic and displayed reduced selectivity in vivo (see above; [184]). A number of other companies are active in this area, among them Medivir AB, who in April 2005 announced the selection of a candidate drug [176]. Development of Cathepsin S Inhibitors Although the development of cathepsin S inhibitors started almost simultaneously with the development of cathepsin K inhibitors, the progress was slower. The discovery of dipeptidyl vinyl sulfones, such as LHVS (N-morpholinourealeucine-homophenylalaninephenylvinylsulfone) by the researchers from Khepri Pharmaceuticals (now Celera Genomics), had a major impact on the development of cathepsin S inhibitors [199]. Although the inhibitors were irreversible, they were highly selective over cathepsins L, K and B and their use validated cathepsin S as a relevant drug target in various experimental disease models, such as arthritis [200]. Celera continued working on the development of cathepsin S inhibitors and in September 2005 they announced the initiation of Phase I clinical testing for their cathepsin S inhibitor, CRA-028129 (no structural data disclosed), for the treatment of psoriasis. Psoriasis is believed to affect 1-3% people worldwide. The inhibitor is claimed to be highly potent and selective in picomolar range in vitro and in cell systems in the nanomolar range (www.celera.com). Celera Genomics, which acquired the extensive Axys Pharmaceuticals cathepsin program, has also filed a number of patents for cathepsin S inhibitors using different chemical templates [200]. Celera has also announced that they have additional cathepsin S inhibitors in preclinical evaluation (www.celera. com). Since Celera was involved in partnership with Aventis (now Sanofi Aventis), the latter will probably receive exclusive rights to the treatment of bronchial asthma, rheumatoid arthritis and some other diseases [165]. In February 2006 Medivir AB announced that they are taking over the cathepsin S program, which they have developed together with Peptimmune (http://www.medivir.se). An IND application and the beginning of clinical testing are expected in 2006. The compound (MV57471), which is an orally active reversible inhibitor (possibly an aliphatic ketone), showed good results in rodent models of type II collagen induced arthritis and multiple sclerosis. The compound was also reported to have no adverse effects during the experimental period ([165, 200]; http://www.peptimmune.com). Also Johnson&Johnson has an active cathepsin S program. They have, however, focused on the development of noncovalent reversible inhibitors. Using virtual screening based on the cathepsin K structure they identified several potential hits, which were further optimized to the selective cathepsin S inhibitor (N-arylpiperazine type) with an IC50 value of ~20 nM [201]. Further optimization of the compound led to the ketobenzimidazole piperidine moiety, and the lead compound JNJ 10329670 is the first nonpeptidic noncovalent orally available cathepsin S inhibitor (Ki ~30


nM), which showed good efficacy in cellular and in a mouse model [202, 203]. However, the compound showed lower affinity towards mouse, dog monkey and bovine cathepsin S variants, although no cross reactivity with other human cathepsins was observed even at high concentration (Ki > 50 μM; [203]). There are several other companies working on cathepsin S, among them Boehringer Ingelheim Pharmaceuticals, Amura, Novartis, GlaxoSmithKline and Astra Zeneca, however, the work is still in experimental phase.

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CONCLUSIONS In conclusion, cysteine cathepsins are involved in regulation of critical steps in cancer biology, immunological responses, degradation of the articular cartilage matrix and other pathological processes, playing a major role both in intra- and extra-cellular protein turnover. They are therefore considered as highly relevant targets in several pathological states, such as immune disorders, osteoporosis, rheumatoid and osteoarthritis. New emerging areas where cathepsins seem to be relevant targets are among others also cancer, atherosclerosis and viral infections. The development of drugs based on inhibition of cysteine cathepsins has advanced into clinical testing with compounds targeting cathepsins S and K, and the cathepsin K inhibitor AAE-581 (balicatib) as the most advanced of them is expected to enter Phase III clinical trials in 2006. ACKNOWLEDGEMENTS We would like to thank Roger Pain for critical reading of the manuscript. This work was supported by grants from Slovene Ministry of Higher Education, Science and Technology and by the European Union Framework Programme 6 Project LSHG-CT-2006-018830 CAMP to B.T., D.T. and V.T. and by grants from Deutsche Forschungsgemeinschaft (Re1584 grants 2-1, 2-2, and 3-1), Deutsche Krebshilfe (Re106977), Ministerium für Wissenschaft und Kunst, Baden-Württemberg (grant 23-7532.22-33-11/1), the European Union Framework Programme 6, Project LSHC-CT-2003503297, CancerDegradome to C.P. and T.R.

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