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.     

Neurotrophic factors regulate cathepsin S in macrophages and microglia: A role in the degradation of myelin basic protein and amyloid beta peptide.

BACKGROUND: Cathepsin S is a member of the family of cysteine lysosomal proteases preferentially expressed in macrophages and microglia and is active after prolonged incubation in neutral pH. Upon activation of macrophages by a number of inflammatory mediators, there is an increase in secreted cathepsin S activity accompanied by a decrease in cellular cathepsin S activity and protein content, as well as a decrease in cathepsin S mRNA. The decrease in cathepsin S mRNA and protein at the cellular level is in contrast to the response observed in some in vivo scenarios. MATERIALS AND METHODS: We investigated the effect of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF), two growth factors present during cell injury and inflammation but not known to activate macrophages and microglia, on the expression of cathepsin S, cathepsin B, and cathepsin L mRNAs in these cells, and on cathepsin S activity. We then tested the ability of cathepsin S to degrade myelin basic protein, and amyloid beta peptide at both acidic and neutral pH. RESULTS: Basic FGF and NGF treatment of macrophages and microglia significantly increased the levels of cathepsin S, B, and L mRNAs (2- to 5-fold). Basic FGF also increased cathepsin S activity intra- and extracellularly. Recombinant human cathepsin S was able to degrade myelin basic protein and monomeric and dimeric amyloid beta peptide at both acidic and neutral pH, as well as to process human amyloid precursor protein generating amyloidogenic fragments. CONCLUSIONS: These data suggest that bFGF and NGF may be the molecular signals that positively regulate the expression and activity of cysteine lysosomal proteases (cathepsin S in particular) in macrophages and microglia in vivo, and that there is an interplay between these factors and the activators of inflammation. Disruption of the balance between these two categories of signals may underlie the pathological changes that involve cysteine proteases. http://link.springer-ny.com/link/service/journals/00020/bibs /5n5p334. html     

The S2 subsites of cathepsins K and L and their contribution to collagen degradation

The exchange of residues 67 and 205 of the S2 pocket of human cysteine cathepsins K and L induces a permutation of their substrate specificity toward fluorogenic peptide substrates. While the cathepsin L-like cathepsin K (Tyr67Leu/Leu205Ala) mutant has a marked preference for Phe, the Leu67Tyr/Ala205Leu cathepsin L variant shows an effective cathepsin K-like preference for Leu and Pro. A similar turnaround of inhibition was observed by using specific inhibitors of cathepsin K [1-(N-Benzyloxycarbonyl-leucyl)-5-(N-Boc-phenylalanyl-leucyl)carbohydrazide] and cathepsin L [N-(4-biphenylacetyl)-S-methylcysteine-(D)-Arg-Phe-beta-phenethylamide]. Molecular modeling studies indicated that mutations alter the character of both S2 and S3 subsites, while docking calculations were consistent with kinetics data. The cathepsin K-like cathepsin L was unable to mimic the collagen-degrading activity of cathepsin K against collagens I and II, DQ-collagens I and IV, and elastin-Congo Red. In summary, double mutations of the S2 pocket of cathepsins K (Y67L/L205A) and L (L67Y/A205L) induce a switch of their enzymatic specificity toward small selective inhibitors and peptidyl substrates, confirming the key role of residues 67 and 205. However, mutations in the S2 subsite pocket of cathepsin L alone without engineering of binding sites to chondroitin sulfate are not sufficient to generate a cathepsin K-like collagenase, emphasizing the pivotal role of the complex formation between glycosaminoglycans and cathepsin K for its unique collagenolytic activity.     

Specificity determinants of human cathepsin s revealed by crystal structures of complexes

Cathepsin S, a lysosomal cysteine protease of the papain superfamily, has been implicated in the preparation of MHC class II alphabeta-heterodimers for antigen presentation to CD4+ T lymphocytes and is considered a potential target for autoimmune-disease therapy. Selective inhibition of this enzyme may be therapeutically useful for attenuating the hyperimmune responses in a number of disorders. We determined the three-dimensional crystal structures of human cathepsin S in complex with potent covalent inhibitors, the aldehyde inhibitor 4-morpholinecarbonyl-Phe-(S-benzyl)Cys-Psi(CH=O), and the vinyl sulfone irreversible inhibitor 4-morpholinecarbonyl-Leu-Hph-Psi(CH=CH-SO(2)-phenyl) at resolutions of 1.8 and 2.0 A, respectively. In the structure of the cathepsin S-aldehyde complex, the tetrahedral thiohemiacetal adduct favors the S-configuration, in which the oxygen atom interacts with the imidazole group of the active site His164 rather than with the oxyanion hole. The present structures provide a detailed map of noncovalent intermolecular interactions established in the substrate-binding subsites S3 to S1' of cathepsin S. In the S2 pocket, which is the binding affinity hot spot of cathepsin S, the Phe211 side chain can assume two stable conformations that accommodate either the P2-Leu or a bulkier P2-Phe side chain. This structural plasticity of the S2 pocket in cathepsin S explains the selective inhibition of cathepsin S over cathepsin K afforded by inhibitors with the P2-Phe side chain. Comparison with the structures of cathepsins K, V, and L allows delineation of local intermolecular contacts that are unique to cathepsin S.     

The specificity of bovine spleen cathepsin S. A comparison with rat liver cathepsins L and B.

The peptide-bond-specificity of bovine spleen cathepsin S in the cleavage of the oxidized insulin B-chain and peptide methylcoumarylamide substrates was investigated and the results are compared with those obtained with rat liver cathepsins L and B. Major cleavage sites in the oxidized insulin B-chain generated by cathepsin S are the bonds Glu13-Ala14, Leu17-Val18 and Phe23-Tyr26; minor cleavage sites are the bonds Asn3-Gln4, Ser9-His10 and Leu15-Tyr16. The bond-specificity of this proteinase is in part similar to the specificities of cathepsin L and cathepsin N. Larger differences are discernible in the reaction with synthetic peptide substrates. Cathepsin S prefers smaller neutral amino acid residues in the subsites S2 and S3, whereas cathepsin L efficiently hydrolyses substrates with bulky hydrophobic residues in the P2 and P3 positions. The results obtained from inhibitor studies differ somewhat from those based on substrates. Z-Phe-Ala-CH2F (where Z- represents benzyloxycarbonyl-) is a very potent time-dependent inhibitor for cathepsin S, and inhibits this proteinase 30 times more efficiently than it does cathepsin L and about 300 times better than it does cathepsin B. By contrast, the peptidylmethanes Z-Val-Phe-CH3 and Z-Phe-Lys(Z)-CH3 inhibit competitively both cathepsin S and cathepsin L in the micromolar range.     

Insights from molecular modeling into the selective inhibition of cathepsin S by its inhibitor

Cathepsin S has been demonstrated to play a crucial role in the remodeling of extracellular matrix proteins such as elastin and collagen, which in turn contribute to the structural integrity of the cardiovascular wall. Atherosclerotic lesions, aneurysm formation, plaque rupture, thrombosis, and calcification are some of the cardiovascular disorders related to cathepsin S. A highly selective inhibitor of human as well as animal cathepsin S, RO5444101, was recently reported to attenuate the progression of atherosclerotic lesions. Here, we attempted to gain insight into the molecular mechanism of action of RO5444101 on cathepsin S by performing molecular docking and molecular dynamics (MD) simulation studies. The results of our studies correlate well with relevant reported experimental data and potentially explain the selectivity of this inhibitor for cathepsin S rather than cathepsin L1/L, cathepsin L2/V, and cathepsin K, which share conserved catalytic sites and have sequence similarities of 49%, 50%, and 55%, respectively, with respect to cathepsin S. In contrast to those closely related cathepsins, 20 ns MD simulation data reveal that the overall interaction of cathepsin S with RO5444101 is more stable and involves more protein–molecule interactions than the interactions of the inhibitor with the other cathepsins. This study therefore considerably improves our understanding of the molecular mechanism responsible for cathepsin S inhibition and facilitates the identification of potential novel selective inhibitors of cathepsin S.     

Cloning,expression, purification,and activity of dog (Canis familiaris) and monkey (Saimiri boliviensis) cathepsin S

Cathepsin S is the key protease responsible for the removal of the invariant chain from MHC class II molecules and, as such, has attracted much attention as a target for developing immunosuppressive drugs. To help in testing candidate compounds, the monkey (Saimiri boliviensis) and dog (Canis familiaris) cathepsin S cDNAs have been cloned. The monkey cDNA sequence encodes a 330 amino acid protein with 95% homology to human cathepsin S. The dog cDNA sequence encodes a 331 amino acid protein with 91% homology to human cathepsin S. The amino acid differences do not have a major effect on the hydrolysis of the substrate Z-VVR-AMC, but may affect the substrate specificity. As for human and bovine cathepsin S, activity against Z-VVR-AMC extends into the neutral pH range. These parameters are important for understanding the role of cathepsin S in different species and for testing inhibitors in animal models of autoimmunity.     

Cathepsin S from bovine spleen. Purification, distribution, intracellular localization and action on proteins.

Cathepsin S was detected in bovine kidney, spleen, lymph nodes and lung by immunochemical methods. The immunostaining of cathepsin S in kidney was concentrated to the cells of the proximal tubule, where the enzyme was present in cytoplasmic granules. The purification method for cathepsin S from bovine spleen involved (NH4)2SO4 fractionation, chromatography on CM-Sephadex C-50, gel filtration on Sephacryl S-200 and chromatofocusing (pH 8.0-6.0). The enzyme was partially destroyed by autolysis of the homogenate at pH 4.2. The isoelectric point of cathepsin S was 7.0. Cathepsin S was found to hydrolyse proteins at a similar rate to cathepsin L below pH 7.0. At pH values of 7.0-7.5 cathepsin S retained most of its activity, whereas cathepsin L was completely inactive.     

Cathepsin S. The cysteine proteinase from bovine lymphoid tissue is distinct from cathepsin L (EC 3.4.22.15).

Cathepsin S was purified from bovine spleen by acid autolysis, (NH4)2SO4 fractionation and chromatography on CM-Sephadex C-50, CM-cellulose and activated-thiol-Sepharose. Cathepsin L was isolated from lysosomal fractions of rat liver, rat kidney and bovine liver. Generally, cathepsin L was bound tightly to CM-Sephadex C-50. Preparations of cathepsin L from rat liver, rat kidney and bovine liver were shown to have kinetic constants for the substrate benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide in the same range (Km 2-3 microM). Benzyloxycarbonyl-Phe-Phe-diazomethane proved to be a sensitive irreversible inhibitor of cathepsin L from different species. Cathepsin S differed in all these characteristics from cathepsin L. A polyclonal antibody to cathepsin L from rat reacted with bovine cathepsin L but not with bovine cathepsin S.     

Synthesis of proline analogues as potent and selective cathepsin S inhibitors

Cathepsin S is a potential target of autoimmune disease. A series of proline derived compounds were synthesized and evaluated as cathepsin S inhibitors. We discovered potent cathepsin S inhibitors through structure–activity relationship studies of proline analogues. In particular, compound 19-(S) showed promising in vitro/vivo pharmacological activities and properties as a selective cathepsin S inhibitor.     

Selective inhibition of the collagenolytic activity of human cathepsin K by altering its S2 subsite specificity

The primary specificity of papain-like cysteine proteases (family C1, clan CA) is determined by S2-P2 interactions. Despite the high amino acid sequence identities and structural similarities between cathepsins K and L, only cathepsin K is capable of cleaving interstitial collagens in their triple helical domains. To investigate this specificity, we have engineered the S2 pocket of human cathepsin K into a cathepsin L-like subsite. Using combinatorial fluorogenic substrate libraries, the P1-P4 substrate specificity of the cathepsin K variant, Tyr67Leu/Leu205Ala, was determined and compared with those of cathepsins K and L. The introduction of the double mutation into the S2 subsite of cathepsin K rendered the unique S2 binding preference of the protease for proline and leucine residues into a cathepsin L-like preference for bulky aromatic residues. Homology modeling and docking calculations supported the experimental findings. The cathepsin L-like S2 specificity of the mutant protein and the integrity of its catalytic site were confirmed by kinetic analysis of synthetic di- and tripeptide substrates as well as pH stability and pH activity profile studies. The loss of the ability to accept proline in the S2 binding pocket by the mutant protease completely abolished the collagenolytic activity of cathepsin K whereas its overall gelatinolytic activity remained unaffected. These results indicate that Tyr67 and Leu205 play a key role in the binding of proline residues in the S2 pocket of cathepsin K and are required for its unique collagenase activity.     

Characterization of Förster resonance energy transfer in a botulinum neurotoxin protease assay

Using the cell-permeable, radioiodinated, irreversible inhibitor BIL-DMK, we probed active cysteine cathepsins in blood. Incubation of the probe in human whole blood followed by separation of white blood cells by dextran sedimentation led to the labeling of one major band at 24 kDa. Two-dimensional gel electrophoresis showed that the band resolved in a single protein spot and corresponded to cathepsin S based on its molecular mass, isoelectric point, and Western blot analysis using anti-human cathepsin S antibodies. Cathepsin S activity in human whole blood was dependent on the time of blood collection, suggesting that cathepsin S activity is subject to circadian variations. Separation of white blood cell populations using a magnetic cell sorter and further characterization by FACS (fluorescent-activated cell sorting) analysis demonstrated that the majority of active cathepsin S resided in the monocyte and neutrophil populations, whereas on a cell basis cathepsin S activity in granulocytes is 10-fold lower than that in monocytes. A whole blood cathepsin S assay was developed and used to measure cathepsin S inhibition in both in vitro and ex vivo conditions. To determine the correlation between the in vitro and ex vivo assays, a reversible cathepsin S inhibitor was dosed intravenously to a rhesus monkey. The inhibitor concentration required to inhibit 50% of the cathepsin S activity ex vivo correlated well with the concentration required to inhibit the enzyme in rhesus monkey whole blood in vitro. The results reported here demonstrate the utility of the activity-based probe BIL-DMK for the ex vivo assessment of cathepsin S inhibition.     

Cathepsin B mediates tumor necrosis factor-induced arachidonic acid release in tumor cells

Arachidonic acid (AA) generated by cytosolic phospholipase A2 (cPLA2) has been suggested to function as a second messenger in tumor necrosis factor (TNF)-induced death signaling. Here, we show that cathepsin B-like proteases are required for the TNF-induced AA release in transformed cells. Pharmaceutical inhibitors of cathepsin B blocked TNF-induced AA release in human breast (MCF-7S1) and cervix (ME-180as) carcinoma as well as murine fibrosarcoma (WEHI-S) cells. Furthermore, TNF-induced AA release was significantly reduced in cathepsin B-deficient immortalized murine embryonic fibroblasts. Employing cPLA2-deficient MCF-7S1 cells expressing ectopic cPLA2 or cPLA2-deficient immortalized murine embryonic fibroblasts, we showed that cPLA2 is dispensable for TNF-induced AA release and death in these cells. Furthermore, TNF-induced cathepsin B-dependent AA release could be dissociated from the cathepsin B-independent cell death in MCF-7S1 cells, whereas both events required cathepsin B activity in other cell lines tested. These data suggest that cathepsin B inhibitors may prove useful not only in the direct control of cell death but also in limiting the damage-associated inflammation.     

Immunochemical localisation of cathepsin S, cathepsin L and MHC class II-associated p41 isoform of invariant chain in human lymph node tissue

Antigen presentation by MHC class II molecules requires cysteine proteases (CP) for two convergent proteolytic processes: stepwise degradation of the invariant chain (Ii) and generation of immunogenic peptides. Their activity is controlled by intracellular CP inhibitors, including presumably the p41 isoform of invariant chain (p41 Ii), which is in vitro a potent inhibitor of cathepsin L but not of cathepsin S. In order to evaluate the inhibitory potential of p41 Ii in antigen-presenting cells (APC), these three proteins were stained in lymph node tissue using specific monoclonal and polyclonal antibodies. The most abundant labelling was observed in subcapsular (cortical) and trabecular sinuses of the lymph node. In this area the most frequent APC were macrophages, as confirmed by the CD68 cell marker. Using confocal fluorescence microscopy, co-localisation of p41 Ii with cathepsin S, but not with cathepsin L was found in these cells. Our results are consistent with the hypothesis that cathepsin S participates in degradation of the invariant chain, but they do not support the association between cathepsin L and p41 Ii in APC.     

N-Sulfonyl dipeptide nitriles as inhibitors of human cathepsin S: In silico design,synthesis and biochemical characterization

A library of cathepsin S inhibitors of the dipeptide nitrile chemotype, bearing a bioisosteric sulfonamide moiety, was synthesized. Kinetic investigations were performed at four human cysteine proteases, i.e. cathepsins S, B, K and L. Compound 12 with a terminal 3-biphenyl sulfonamide substituent was the most potent (K_i = 4.02 nM; selectivity ratio cathepsin S/K = 5.8; S/L = 67) and 24 with a 4′-fluoro-4-biphenyl sulfonamide substituent the most selective cathepsin S inhibitor (K_i = 35.5 nM; selectivity ratio cathepsin S/K = 57; S/L = 31). In silico design and biochemical evaluation emphasized the impact of the sulfonamide linkage on selectivity and a possible switch of P2 and P3 substituents with respect to the occupation of the corresponding binding sites of cathepsin S.     

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.     

The alpha1/2 helical backbone of the prodomains defines the intrinsic inhibitory specificity in the cathepsin L-like cysteine protease subfamily

Proregions of papain-like cysteine proteases are potent and often highly selective inhibitors of their parental enzymes. The molecular basis of their selectivity is poorly understood. For two closely related members of the cathepsin L-like subfamily we established strong selectivity differences. The propeptide of cathepsin S was observed to inhibit cathepsin L with a K(i) of 0.08 nM, yet cathepsin L propeptide inhibited cathepsin S only poorly. To identify the respective structural correlates we engineered chimeric propeptides and compared their inhibitory specificity with the wild-types. Specificity resided in the N-terminal part, strongly suggesting that the backbone of the prodomain was the underlying structure.     

Synthesis and cathepsin D inhibition of peptide-hydroxyethyl amine isosteres with cyclic tertiary amines

Summary Cathepsin D, a lysosomal aspartic protease, has been suggested to play a role in the metastatic potential of several types of cancer. A high activated cathepsin D level in breast tumor tissue has been associated with an increased incidence of relapse and metastasis. High levels of active cathepsin D have also been found in colon cancer, prostate cancer, uterine cancer and ovarian cancer. Hydroxyethyl isosteres with cyclic tertiary amine have proven to be clinically useful as inhibitors of aspartyl proteases similar to cathepsin D in activity, such as the HIV-1 aspartyl protease. The design and the synthesis of (hydroxyethyl)amine isostere inhibitors with cyclic tertiary amines is described. The IC₅₀ and K_i(app) values for the six cathepsin D inhibitors and pepstatin are reported. Compounds7b,3(S)-[Acetyl-L-valyl-L-phenylalanylamino]-4-phenyl-1-N-piperidine-2(S)-butanol, and7c, 3(S)-[Acetyl-L-leucyl-L-phenylalanylamino]-4-phenyl-1-N-piperidine-2(S)-butanol, showed the most potent inhibition of cathepsin D hydrolysis of hemoglobin with IC₅₀ values of 3.5 nM and 4.5 nM, respectively.     

Peptidomimetic 2-cyanopyrrolidines as potent selective cathepsin L inhibitors

Cathepsins have been found to have important physiological roles. The implication of cathepsin L in various types of cancers is well established. In a search for selective cathepsin L inhibitors as anticancer agents, a series of 2-cyanoprrolidine peptidomimetics, carrying a nitrile group as warhead, were designed. Two series of compounds, one with a benzyl moiety and a second with an isobutyl moiety at P(2) position of the enzyme were synthesized. The synthesized compounds were evaluated for inhibitory activity against human cathepsin L and cathepsin B. Although, none of the compounds showed promising inhibitory activity, (E)N-{(S)1-[(S)2-cyano-1-pyrrolidinecarbonyl]-3-methylbutyl}-2,3-diphenylacrylamide (24) with an isobutyl moiety at P(2) was found to show selectivity as a cathepsin L inhibitor (Ki 5.3 microM for cathepsin L and Ki > 100 microM for cathepsin B). This compound could act as a new lead for the further development of improved inhibitors within this inhibitor type.