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INFLAMMATION AND IMMUNOPHARMACOLOGY

Identification of a Potent and Selective Noncovalent Cathepsin S Inhibitor

Johnson & Johnson Pharmaceutical Research & Development, L.L.C., San Diego, California

Received for publication July 9, 2003
Accepted September 18, 2003.

	Abstract

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Cathepsin S is considered crucial for normal presentation of major histocompatibility complex (MHC) class II-restricted antigens by antigen presenting cells to CD4⁺ T cells. It is a key enzyme for the degradation of the class II-associated invariant chain, a process that is required for effective antigen loading of class II molecules. Here, we report a selective, orally available, high-affinity cathepsin S inhibitor, 1-[3-[4-(6-Chloro-2,3-dihydro-3-methyl-2-oxo-1H-benzimidazol-1-yl)-1-piperidinyl]propyl]-4,5,6,7-tetrahydro-5-(methylsulfonyl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazolo[4,3-c]pyridine. (JNJ 10329670), that represents a novel class of immunosuppressive compounds. JNJ 10329670 is a highly potent (K_i of 30 nM), nonpeptidic, noncovalent inhibitor of human cathepsin S, but it is much less active against the mouse, dog, monkey, and bovine enzymes. The compound is inactive against other proteases, including the closely related cathepsins L, F, and K. This selectivity makes JNJ 10329670 an excellent tool for exploring the role of cathepsin S in human systems. Treatment of human B cell lines and primary human dendritic cells with JNJ 10329670 resulted in the accumulation of the p10 fragment of the invariant chain (IC₅₀ of 1 µM). In contrast, inhibition of invariant chain proteolysis was much less effective in a human monocytic cell line, suggesting that other enzymes may degrade the invariant chain in this cell type. JNJ 10329670 was shown to block the proteolysis of the invariant chain in vivo by using immunocompromised mice injected with human peripheral blood mononuclear cells (PBMCs). Furthermore, this inhibitor blocks the presentation of tetanus toxoid and giant ragweed by human PBMCs. The properties of JNJ 10329670 make it a candidate for immunosuppressive therapy of allergies and autoimmune diseases.

Cathepsin S is a cysteine protease found in the lysosome of hematopoietic cells. It is a member of the papain superfamily and has 57% identity to cathepsins L and K. Unlike most other lysosomal proteases that are only active under acidic conditions, the activity of cathepsin S exhibits a broad pH optimum that extends to alkaline pH. In contrast to the housekeeping enzymes cathepsins B, D, and L, which are expressed ubiquitously, cathepsin S is expressed mainly in dendritic cells, B cells, and macrophages that act as the key antigen presenting cells of the immune system. The main substrate of cathepsin S in vivo is the invariant chain. The invariant chain binds to the MHC II heterodimers in the endoplasmic reticulum and is crucial for the stability and trafficking of MHC II molecules into the endosomal system. The invariant chain also prevents premature peptide loading by blocking the peptide binding groove of the MHC II molecules. When the MHC II-invariant chain complex reaches the endosomes, the invariant chain is degraded in a series of proteolytic steps. One of the key proteases involved in this process is cathepsin S (Chapman, 1998; Nakagawa and Rudensky, 1999; Villadangos et al., 1999; Villadangos and Ploegh, 2000). Cleavage by cathepsin S leaves only a short fragment of the invariant chain, class II-associated invariant chain peptide (CLIP), bound to MHC II. This fragment is removed through an interaction with the endosomal/lysosomal resident protein HLA-DM, which allows for the binding of antigenic peptides.

Mice deficient in cathepsin S are healthy and normal in most respects but exhibit defects in immune functions (Nakagawa et al., 1999; Shi et al., 1999). The cathepsin S-deficient mice show a decrease in invariant chain degradation in dendritic cells and B cells, but only a moderate effect in macrophages. These mice also display a diminished capacity to present antigens and show a reduced susceptibility to collagen-induced arthritis.

There have only been a few reports of cathepsin S inhibitors and most of these are covalent modifiers of the enzyme. Inhibitors have been shown to block both invariant chain processing and antigen presentation in cells and in vivo (Riese et al., 1996; Villadangos et al., 1997; Allen et al., 2001; Fiebiger et al., 2001; Podolin et al., 2001). Furthermore, in vivo treatment with cathepsin S inhibitors leads to the attenuation of the antibody responses in mice when immunized with ovalbumin (Riese et al., 1998), as well as smaller increases in IgE titer and less lung eosinophil infiltration in a mouse model of pulmonary hypersensitivity (Riese et al., 1998). The degree of inflammation was also decreased in both the rat adjuvant-induced arthritis model (Biroc et al., 2001) and the collagen-induced arthritis model in mice (Podolin et al., 2001). Cathepsin S inhibitors have also been shown to be effective in treating a murine model for Sjögren syndrome (Saegusa et al., 2002).

The published data suggest that inhibition of cathepsin S will block the degradation of the invariant chain and disrupt the presentation of antigens, thereby acting as an immunosuppressive agent. However, unlike most commonly used immunosuppressives, cathepsin S inhibitors will not directly target T cells and will be specific for CD4⁺ T cells. Such an approach should reduce the side effects commonly associated with immunosuppression. Here, we describe the first nonpeptidic and noncovalent inhibitor of cathepsin S that has high affinity and selectivity. The high degree of selectivity versus other cathepsins makes it an excellent tool for exploring the role of cathepsin S in humans. In addition, the high affinity for cathepsin S without relying upon covalent attachment to the active site thiol represents a major advance toward the development of small-molecule therapeutics targeting this protease.

	Materials and Methods

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Materials. Anti-human invariant chain antisera was raised by immunization of rabbits using an invariant chain peptide-keyhole limpet hemocyanin (KLH) conjugate emulsified in complete Freund's adjuvant. The sequence of the invariant chain peptide was CVSKMRMATPLLMQA. KLH conjugation was carried out as suggested by the manufacturer (Pierce Chemical, Rockford, IL) using Imject maleimide-activated mariculture KLH. Bovine cathepsin S, human cathepsin B, human cathepsin D, and human cathepsin L were obtained from Calbiochem (La Jolla, CA). Human matrix metalloproteinase 13 (MMP-13) was purchased from Chemicon International (Temecula, CA). E-64 was purchased from Sigma-Aldrich (St. Louis, MO). The substrate for cathepsin Z was purchased from Enzyme System Products (Livermore, CA). Tetanus toxoid was purchased from the Massachusetts Biological Laboratories (Jamaica Plain, MA). Giant ragweed extract was purchased from Hollister-Stier Laboratories, L.L.C. (Minneapolis, MN). Phytohemagglutinin-M Phaseolus vulgaris was purchased from CalBiochem. SCID (C57Bl/6J) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Blood donors were obtained in collaboration with The Scripps Research Institute (San Diego, CA), and the procedures were approved by the Scripps Institutional Review Board.

JNJ 10329670 was prepared following the procedure recently disclosed (Butler et al., 2002). The free base of JNJ 10329670 has a molecular weight of 651.16. The monohydrochloride (mol. wt. 687.61) was used for most biological assays. Both forms are white, free-flowing powders with limited (<0.002 mg/ml) aqueous solubility in pH 2 or pH 7 buffers.

Cloning, Expression, and Purification of Cathepsin S. Recombinant human cathepsin S, cathepsin L2, cathepsin K, cathepsin F, cathepsin Z, cathepsin C, legumain, dog cathepsin S, monkey cathepsin S, and mouse cathepsin S were expressed using the baculovirus expression system. The cathepsin S proteins were purified using thio-propyl Sepharose chromatography as described previously (Baker et al., 2003). The other proteases were purified using cation exchange chromatography. Human cathepsin E was expressed in Drosophila cells and purified using pepstatin A affinity chromatography. Napsin was expressed as a glutathione S-transferase fusion protein and purified via a glutathione affinity column. ADAM-28 was expressed in the baculovirus expression system with a His-Tag, and purified using nickel-chelating columns.

Enzymatic Assays. In general, the assays were run using fluorescence resonance energy transfer-based substrates. For example, the cathepsin S, L, and L2 assays used the substrate (Aedens)EKARVLAEAA(Dabcyl)K-amide and cathepsin S cleaves between amino acids Leu-6 and Ala-7. The fluorescence of the aedens group is quenched by the dabcyl moiety in the intact peptide. Upon cleavage by cathepsin S, the quenching is released and the fluorescence of the aedens group can be measured.

The cysteine (cathepsins S, Z, F, C, L, L2, B, and legumain) and aspartic protease (cathepsins E, D, and napsin) assays were run in 100-µl volume with a buffer consisting of 100 mM sodium acetate, pH 5.0, containing 100 mM NaCl and 1 mM dithiothreitol, except for cathepsin Z, which used 10 mM dithiothreitol and cathepsins E, D, and napsin where no dithiothreitol was present. The MMP-13 assay was run in 50 mM Tris-HCl,pH 7.5, containing 5 mM MgCl₂, 200 mM NaCl, and 0.02% Brij35. The ADAM-28 assay was run in 10 mM HCl, pH 7.5, containing 0.02% Brij35. The enzymes were mixed with 7.5 ml of buffer and then 75 µl was added to a Dynax black Microfluor 2 plate. To this, 5 µl of a 20x solution of compound in 30% DMSO was added. This was followed by the addition of 20 µl of 5x substrate to initiate the reaction. In all cases, an 11 point 1:2 dilution of the compound was used at seven substrate concentrations (also 1:2). The increase in fluorescence was measured on a Cytofluor II (Applied Biosystems, Foster City, CA) with an excitation filter of 360/40 nm and an emission filter of 460/40 nm. A reading was made every minute for 20 to 60 min, depending on the assay, and the slope obtained from a linear regression of this time course was used as the reaction rate. The elastase assay was run according to the method of Adeyemi et al. (1990). ECE was run according to the method of Ahn et al. (1992). Cathepsin G was run according to the method of Nakajima et al. (1979). ACE was run according to the method of Hoorn and Roth (1993).

The inhibition constant, K_i, was calculated by fitting plots of the reaction rate () as a function of both the substrate concentration ([S]) and the inhibitor concentration ([I]) to the following equation.

(1)

Cathepsin S from all of the species exhibited substrate inhibition at high substrate concentrations. This can be taken into account by the following equation

(2)

These equations assume competitive inhibition and similar results were obtained when equations for mixed inhibition were used. The program GraFit (Erithacus Software Ltd., Surrey, UK) was used to carry out the nonlinear fits.

Reversibility of the compound was tested by incubating cathepsin S with 100 µM of test compound for 15 min. The mixture was then dialyzed for 20 h at 4°C using a mini Slide-a-lyzer (Pierce Chemical) with a 10,000 molecular weight cut-off against 150 mM NaAc, pH 5.0, containing 1 mM dithiothreitol. The mixture was then assayed for cathepsin S activity as given above.

The pH dependence was determined using the same assay as given above except that different buffers were used for the different pH. The buffers used for the pH titrations were 100 mM sodium acetate for pH 3.5 to 5.5, 100 mM MES for pH 6 and 6.5, 100 mM HEPES for pH 7.0, and 100 mM Tris-HCl for pH 7.4 to 9.5. The reaction rates were determined as given above for 11 different inhibitor concentrations at each pH, and the percentage of inhibition was determined by comparison with the reaction rate in the absence of inhibitor. IC₅₀ values were determined by four parameter fits of plots of the percentage of inhibition versus inhibitor concentration using the program GraFit (Erithacus Software Ltd.).

Invariant Chain Degradation Assays. Human JY B cell line, human THP-1 monocyte cell line, and monkey SML B cell line were purchased from American Type Culture Collection (Manassas, VA). Human PBMC were purified from heparinized blood of healthy donors by Ficoll-Hypaque (Amersham Biosciences AB, Uppsala, Sweden) density gradient centrifugation. Purification of human dendritic cells was done using a MACS blood dendritic cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany).

For invariant chain degradation assays, cells were cultured in 24-well plates (2.5 x 10⁵ JY cells/well; 1 x 10⁶ THP-1 cells/well; 5 x 10⁵ SML cells/well; 5 x 10⁶ human PBMC/well; 5 x 10⁵ dendritic cells/well) in the presence of various concentrations of test compound for 24 h. Compound stock solutions (1000x) were prepared in DMSO, and the final DMSO concentration in cell culture was 0.1%. Cells were harvested and then lysed in 1% Triton X-100/1x phosphate-buffered saline with Complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). The accumulation of the invariant chain p10 fragment (p10Ii), which is the substrate for cathepsin S, was quantitated by Western blot using an invariant chain-specific antibody. The chemiluminescence intensity of the p10Ii bands was quantified on a Typhoon 8600 Imager (Amersham Biosciences Inc., Piscataway, NJ). The relative amount of p10Ii was determined by comparison to the maximum amount seen with 50 nM of a standard cathepsin S inhibitor, LHVS. IC₅₀ values were determined by four parameter fits of plots of the relative amount of p10Ii observed versus inhibitor concentration using the program GraFit (Erithacus Software Lt., Surrey, UK). For titrations of N-morpholinurea-homophenylalanyl-leucyl-vinylsulfonemethyl (LHVS), the values are given relative to the maximum accumulation of p10Ii.

Antigen Induced PBMC Proliferation. Blood donors were screened for reactivity to ragweed by radioallergosorbent test (RAST) test. DR typing of donors was done using the Olerup SSP DR low-resolution kit from Genovision (West Chester, PA). Human PBMCs were purified from heparinized blood of healthy donors by Ficoll-Hypaque (Amersham Biosciences AB) density gradient centrifugation. Purified PBMCs from either normal donors (n = 11) or allergic donors (n = 3) were cultured in duplicate or triplicate cultures in 96-well plates in the presence of antigen (1–10 µg/ml tetanus toxoid or 1:30,000 dilution of ragweed extract) for 7 days, or 5 µg/ml phytohemagglutinin for 4 days. [³H]Thymidine was added for the last 18 h of the culture. Cells were harvested onto filter plates, and [³H]thymidine incorporation was then determined using a TopCount (PerkinElmer Life Sciences, Boston, MA). Inhibitors were first titrated in DMSO and then a 1:15 dilution of DMSO stocks was made into 40% cyclodextrin in H₂O, which was then added into cell cultures (3 µl into 200 µl/well). Inhibitors were added at the same time as the antigens or phytohemagglutinin. The results given are the average of three to four determinations, and the error is given as the S.E.M.

In Vivo Invariant Chain Assay. Purified human PBMCs (10⁸ cells/0.5 ml/mouse) were injected intraperitoneally into SCID (C57Bl/6J) mice that received whole body irradiation (4 Gy) 1 day earlier. JNJ 10329670 administration (by subcutaneous injection) started 2 h before adoptive transfer of human PBMCs and was repeated every 6 h. Mice were euthanized 22 h after the first dose of compound. Peritoneal lavage was immediately performed using 5 ml of phosphate-buffered saline plus 3 mM EDTA. The level of p10Ii was determined by Western blot assay as described above.

Pharmacokinetic Analysis. Female Sprague-Dawley rats, mature male beagle dogs, and mature female cynomolgus monkeys were used in pharmacokinetics studies. Briefly, animals were fasted for 18 h before oral dosing. Oral dosing was carried out either by oral gavage for rats or via a stomach tube for dogs and monkeys. Intravenous (i.v.) dosing was done via tail vein (rats), or jugular or saphenous veins for dogs and monkeys. For oral dosing, the compound was prepared in 0.5% methylcellulose as a suspension; for i.v. dosing, the compound was dissolved in 10% solutol and the pH value was adjusted to 6. After dosing, blood samples were collected via tail vein (rats) or jugular or saphenous veins for dogs and monkeys into tubes containing heparin at the specific time points, which normally include 0.5, 1, 2, 4, 7, and 24 h for oral dosing and dosing 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 7 h for i.v. dosing. Plasma samples were then immediately prepared and compound concentrations were determined by LC-MS after acetonitrile extraction. Pharmacokinetic parameters were estimated using the program Winnonlin (Pharsight, Mountain View, CA).

	Results

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A model of cathepsin S was generated using the coordinates from the X-ray crystal structure of the related cysteine protease cathepsin K (McGrath et al., 1997). A virtual screen of the Johnson & Johnson Pharmaceutical Research & Development compound database against the predefined active site of cathepsin S was carried out using DOCK software (Kuntz et al., 1982). Selected compounds were screened for cathepsin S inhibitory activity using recombinant human cathepsin S. This screen yielded two related compounds containing a tetrahydropyridyl[4,3-a]pyrazole pharmacophore that became the basis of a medicinal chemistry effort. Structure-activity relationship analysis indicated that three elements were optimal for cathepsin S inhibition: an aryl-bearing piperazine or piperidine, a 3–4 carbon linker, and a 3-aryl-4,5,6,7-tetrahydropyridyl[4,3-a]pyrazole (H. Cai, S. P. Meduna, D. J. Gustin, and J. P. Edwards, unpublished data). Further refinement showed that replacement of the arylpiperazine with a ketobenzimidazole piperidine resulted in a new series of potent noncovalent cathepsin S inhibitors (C. A. Sehon and J. P. Edwards, unpublished data). This led to the development of JNJ 10329670 (Fig. 1). Here, we describe the pharmacological characterization of JNJ 10329670.

View larger version (9K): [in this window] [in a new window]   Fig. 1. Structure of JNJ 10329670.

The inhibition of recombinant human cathepsin S by JNJ 10329670 was determined by measuring cleavage of a synthetic 11 amino acid peptide substrate. Figure 2A shows the reaction rate as a function of the substrate concentration for different concentrations of the inhibitor for one representative determination. It is evident from the curves that at high substrate concentrations either substrate inhibition or quenching was occurring. This can be accounted for by eq. 2 and a K_i value of 40 ± 5 nM can be calculated. If the substrate concentration range is limited (Fig. 2B), then the data can also be fit using the standard equation for competitive inhibition (eq. 1). This yields a K_i value of 37 ± 5 nM. Because the two methods yielded similar results, only the values from fits using eq. 1 were used. The average values from a number of different determinations are given in Table 1. Thus, JNJ 10329670 had a high affinity for human cathepsin S with a K_i of 34 ± 6nM(n = 11). Equations 1 and 2 assume competitive inhibition, and no improvement in the fits were obtained using equations for mixed or noncompetitive inhibition. The double-reciprocal plot in Fig. 2C emphasizes that the inhibitor was competitive versus the peptide substrate.

View larger version (20K): [in this window] [in a new window]   Fig. 2. JNJ 10329670 inhibition of human cathepsin S. Representative nonlinear fits to eqs. 2 (A) and 1 (B) of the reaction rate versus the substrate concentration for different concentrations of JNJ 10329670. The concentrations of inhibitor used were 5 µM (), 2.5 µM (), 1.25 µM ([), 0.62 µM (), 0.31 µM (), 0.16 µM (), 0.08 µM (), 0.04 µM (), 0.02 µM (), 0.01 µM (), 0.005 µM ([hexagon]), and 0 µM ([hexagonblack]). C, Double-reciprocal plot of 1/rate [min/relative fluorescence unit (RFU)] versus 1/[substrate] (micromolar-1) at six concentrations of JNJ 10329670. The concentrations of inhibitor used were 2.5 µM (), 1.25 µM (), 0.62 µM (), 0.31 µM (), 0.16 µM (), and 0 µM (). A Ki of 34 ± 6 nM was determined from 11 different replicates.

The reversibility of the inhibition of human cathepsin S was studied by incubating the enzyme with the inhibitor for 15 min followed by dialysis to remove the compound. Figure 3 shows the recovery of cathepsin S activity under these conditions. Before the dialysis, there was complete inhibition of cathepsin S activity by JNJ 10329670. After dialysis, the activity was restored to that of the DMSO control. In contrast, the activity was not recovered after dialysis of two irreversible inhibitors, LHVS (Riese et al., 1996) and E-64, even though the estimated final concentration does not yield any inhibition. These results indicate that the inhibition of cathepsin S by JNJ 10329670 was completely reversible.

View larger version (12K): [in this window] [in a new window]   Fig. 3. Reversibility of cathepsin S inhibition by JNJ 10329670. Cathepsin S was incubated for 15 min with DMSO, JNJ 10329670, LHVS, or E-64 each at 100 µM. Afterwards the compounds were dialyzed overnight at 4°C. Enzyme activity was determined before dialysis (gray bar), after dialysis (open bar), and after addition of compounds diluted because they would be during dialysis (black bar). The results shown are the average of two experiments and the error bars represent the S.E.M.

A kinetic analysis for JNJ 10329670 inhibition was carried out with cathepsin S from different species. As for the human isoform, all of the enzymes from the other species exhibited substrate inhibition at high substrate concentrations. However, fits with either eq. 1 or 2 yielded similar K_i values and only the ones from fits to eq. 1 are given in Table 1. The K_i values for mouse, dog, monkey, and bovine cathepsin S were dramatically higher than that for the human enzyme, with the dog homolog showing the closest activity. In all cases, the inhibition was competitive for the peptide substrate.

Selectivity of synthetic cathepsin inhibitors versus other proteases has been an issue. To address this, the inhibition of a number of other closely related proteases was investigated (Table 2). The compound did not inhibit the cysteine proteases cathepsin B, F, L, L2, K, Z, C, and legumain nor the aspartic proteases cathepsin E, D, or napsin at concentrations up to 50 µM. It also did not inhibit the metalloproteases MMP-13, ADAM-28, ECE, or ACE. There was no inhibition observed for the serine proteases elastase or cathepsin G. Therefore, JNJ 10329670 is highly selective for human cathepsin S with at least 7-fold selectivity over cathepsin S from other species and greater than 50-fold selectivity over a number of other proteases. As part of a general safety profiling, JNJ 10329670 has been assayed against 68 other targets representing major classes of biogenic amine receptors, neuropeptide receptors, ion channel binding sites, neurotransmitter transporters, kinases, and phosphatases. No inhibition greater than 40% was seen at 1 µM for the receptors, ion channels, and transporters and at 10 µM for the other enzymes (data not shown).

In contrast to other lysosomal proteases, cathepsin S is active over a fairly broad pH range (Fig. 4A). This may be important for the biological role of cathepsin S in invariant chain processing because a pH gradient exists in the endosomal/lysosomal compartment where the cleavage occurs. All of the inhibition studies cited above were carried out at pH 5.0. To explore the possibility that the inhibition could vary with pH, the IC₅₀ for JNJ 10329670 inhibition of cathepsin S activity was measured over the pH range of 4.5 to 7.5 (Fig. 4B). There are minor changes in the potency of the compound with it being the most potent over the range where the enzyme has the greatest activity (pH 5–6). On either extreme the IC₅₀ is increased 2- to 3-fold. This difference is not expected to have a great impact on the biological activity.

View larger version (12K): [in this window] [in a new window]   Fig. 4. pH profile of cathepsin S inhibition by JNJ 10329670. A, pH dependence of cathepsin S activity over the pH range of 3.5 to 8.5. The rate of substrate hydrolysis [relative fluorescence unit (RFU) per minute] is plotted as a function of pH. B, IC50 of JNJ 10329670 inhibition of cathepsin S at different pH values. At each pH an 11-point titration of compound was run, and the percentage of inhibition calculated from the reaction rate relative to the no compound control. The IC50 was calculated by a four-point fit to the plot of percentage of inhibition versus compound concentration. The results shown are the average of two experiments, and the error bars represent the S.E.M.

An in vivo substrate of cathepsin S is the invariant chain. The proteolysis of this protein can be followed in cells by Western blot analysis. When a human B cell line (JY) was incubated with JNJ 10329670, the proteolysis of the invariant chain was disrupted, and there was an accumulation of an intermediate product known as the p10 fragment that was detected by Western blot analysis using an antibody specific for invariant chain. A representative Western blot is shown in Fig. 5A with IC₅₀ plots shown in Fig. 5B. The irreversible cathepsin S inhibitor LHVS was very potent in this assay with an IC₅₀ of around 14 nM (Fig. 5B). Incubation of JY cells with JNJ 10329670 also led to the accumulation of the p10Ii fragment with an IC₅₀ of approximately 1 µM (Fig. 5B). We have also carried out the experiments with the human Raji B cell line and found similar results (data not shown). In contrast, JNJ 10329670 was not very potent in a monkey B cell line (SML), which is consistent with its lack of potency versus the purified monkey enzyme (Table 1). This data indicates that JNJ 10329670 can inhibit cathepsin S and thereby block the proteolysis of the invariant chain in human B cell lines.

View larger version (40K): [in this window] [in a new window]   Fig. 5. Inhibition of invariant chain proteolysis in cells. A, representative Western blot of JY cells treated with JNJ 10329670. The blot was probed with an antibody specific for invariant chain. In lane 1, cells were treated with 50 nM LHVS. Lanes 2 to 11 were treated with JNJ 10329670 starting at 10 µM (lane 2) and continuing with a 2-fold dilution. Lane 12 is DMSO treatment only. B, IC50 plots for the accumulation of the invariant chain p10 fragment was measured in various cells after incubation with either LHVS or JNJ 10329670. The amount of p10Ii fragment was normalized to the maximum amount seen with 50 nM LHVS treatment for the JNJ 10329670 titrations and to the maximum level of p10Ii accumulation for the LHVS titrations. The different cells are JY cells with JNJ 10329670 (, n = 10), human dendritic cells with JNJ 10329670 (, n = 2), SML cells with JNJ 10329670 (, n = 2), human PBMC treated with JNJ 10329670 (, n = 3), human dendritic cells with LHVS (, n = 1), JY cells with LHVS (, n = 2), and human PBMCs treated with LHVS (, n = 1). Shown are the average of several experiments, and the error bars represent the S.E.M.

Inhibition of cathepsin S can also block invariant chain degradation in primary cells. Dendritic cells are considered to play a major role in the presentation of antigens to CD4⁺ T cells. Both LHVS and JNJ 10329670 had potency in dendritic cells that matched their potency in the JY cells (Fig. 5B). Therefore, inhibition of cathepsin S can block invariant chain degradation in primary human dendritic cells. This assay was also run using purified human PBMCs. Here, LHVS had the same potency as it does for JY cells or dendritic cells, but the potency of JNJ 10329670 was shifted by at least 1 log. The major cell type in PBMCs is B cells and so the reason for the differences seen with the compound is unclear.

It has been reported that in the mouse, the processing of the invariant chain in macrophages is less dependent on cathepsin S than it is in B cells and dendritic cells (Nakagawa et al., 1999; Shi et al., 1999). To address this issue in humans, we have studied the invariant chain processing in a human monocytic cell line THP-1, which was treated overnight with IFN to induce differentiation to a macrophage-like phenotype. Figure 6A shows that LHVS inhibited the proteolysis of the invariant chain and led to the accumulation of the p10Ii fragment in these cells. However, the IC₅₀ was higher than in dendritic cells or B cells, which implies that another enzyme was responsible for this degradation (Fig. 6B). The requirement for a different protease was clearly shown with the cathepsin S-specific compound JNJ 10329670 (Fig. 6A) where no accumulation of the invariant chain p10 fragment was observed. Therefore, as seen in mice, it seems that human macrophages use another enzyme in addition to cathepsin S for proteolysis of the p10Ii fragment.

View larger version (38K): [in this window] [in a new window]   Fig. 6. Inhibition of invariant chain proteolysis in human macrophages. A, representative Western blot of THP-1 cells treated with JNJ 10329670 or LHVS and probed with invariant chain antibody. In lanes 1 to 10, cells were treated with LHVS starting at 10 µM (lane 1) and continuing with a half-log dilution. Lane 11 is DMSO treatment only. In lane 13, cells were treated with 50 nM LHVS. Lanes 14 to 23 were treated with JNJ 10329670 starting at 10 µM (lane 14) and continuing with a 2-fold dilution. Lane 24 is DMSO treatment only. The accumulation of the p10 fragment of the invariant chain (the band at approximately 10 kDa) can be seen for the LHVS-treated cells but not with the cells treated with JNJ 10329670. B, IC50 plots for the accumulation of the invariant chain in LHVS-treated THP-1 cells (, n = 6) compared with JY cells (, n = 2). Shown are the average of several experiments, and the error bars represent the S.E.M.

Inhibition of invariant chain degradation should block antigen presentation to CD4⁺ T cells. To test this we have used a human ex vivo system. When PBMCs derived from blood of allergic human donors are incubated with allergen extracts, there is a proliferation of antigen-specific memory CD4⁺ T cells. This proliferation is dependent on the uptake of the allergen by antigen presenting cells and the presentation to T cells. This is similar to the process that occurs in vivo. Blockage of cathepsin S should block the invariant chain degradation and reduce the amount of allergen that is presented. Figure 7 shows this for two different antigens, tetanus toxoid and giant ragweed. JNJ 10329670 was able to block proliferation of T cells in response to both antigens, but less so to the nonspecific T cell mitogen phytohemagglutinin. This indicates that inhibition of cathepsin S blocks the presentation of these antigens to T cells. We have carried this assay using PBMCs purified from donors representing many different MHC II haplotypes. Eleven donors with reactivity to tetanus toxoid and three donors with reactivity to both tetanus toxoid and giant ragweed were used. These results are given in Table 3 for both antigens. Regardless of the haplotype, inhibition of cathepsin S was able to block the presentation of these antigens.

View larger version (18K): [in this window] [in a new window]   Fig. 7. Effect of cathepsin S inhibition on antigen presentation. The effects of JNJ 10329670 on the PBMC proliferation induced by tetanus toxoid (A), giant ragweed (B), both in black columns, or a nonspecific T cell mitogen, phytohemagglutinin, in gray columns. The columns represent the average from different replicates (n = 4 for tetanus toxoid and n = 3 for giant ragweed), and the error bars represent the S.E.M.

Due to the species difference in the cathepsin S inhibition by JNJ 10329670, it was not possible to carry out in vivo studies in mice. This issue has been circumvented by using SCID mice (C57Bl/6) that were injected intraperitoneally with PBMCs isolated from human blood. The mice were dosed subcutaneously at 60 mg/kg of JNJ 10329670 every 6 h. Even though JNJ 10329670 was orally available in several species (cf. Table 4), the compound was dosed subcutaneously due to ease of administration and the ability to achieve higher peak plasma levels with lower doses. After 22 h, the animals were sacrificed and the human PBMCs were harvested by peritoneal lavage. As for the cell-based studies, the accumulation of the p10 fragment of the invariant chain was quantitated by Western blot analysis. The results (Fig. 8) show that there was significant accumulation of the p10Ii fragment when the animals were treated with JNJ 10329670. The plasma concentration of JNJ 10329670 was predicted to be between 2 and 7 µM throughout the experiment. At the time of euthanasia (4 h after the last dose), the average plasma concentration of the compound was 5.7 ± 0.8 µM. These results show that JNJ 10329670 can block the proteolysis of the invariant chain in vivo.

View larger version (12K): [in this window] [in a new window]   Fig. 8. In vivo accumulation of the p10 fragment of the invariant chain in mice. Humanized SCID mice dosed with JNJ 10329670. The accumulation of human p10Ii from recovered human PBMCs is shown. The results were judged to be significant (p value < 0.05 using a Student's t test). Data for each individual mouse is shown () along with the average (—).

An ideal therapeutic cathepsin S inhibitor should be orally bioavailable. To address this, single-dose pharmacokinetics of JNJ 10329670 has been evaluated in rats (n = 4), dogs (n = 3), and monkeys (n = 3). The compound was administered orally at 30 mg/kg for rats and 10 mg/kg for dogs and monkeys. To determine the bioavailability (%F), the compound was also dosed intravenously at 3 mg/kg for rats and 1 mg/kg for dogs and monkeys. LC-MS analysis was used to quantitate the plasma levels. After oral administration, JNJ 10329670 was orally available in all species with an absolute oral availability of 40 to 75% and a half-life of 4 to 14 h (Table 4).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Many of the current treatments for autoimmune diseases and allergies are centered on controlling the inflammatory response and have a fairly broad specificity. It would be of benefit to have more specific treatments that target the immune response that is thought to initiate these diseases. One point of intervention is the antigen presentation pathway. Here, we describe a novel compound that targets cathepsin S, one of the key enzymes in this pathway.

JNJ 10329670 is a potent inhibitor of cathepsin S that represents a new class of cysteine protease inhibitors. To date, most of the cathepsin S inhibitors described in the literature or in patents are covalent modifiers of the active site cysteine. JNJ 10329670 does not have the capacity for covalent modification, yet it has an affinity for the enzyme in the low nanomolar range. This is the first potent noncovalent cathepsin S inhibitor described.

Although JNJ 10329670 is very potent versus the human enzyme, the potency drops dramatically when cathepsin S from other species are analyzed. The compound is the most potent versus dog cathepsin S where the K_i value is 7-fold higher than for the human isoform. Versus the mouse, monkey, and bovine enzyme the K_i values are 10- to 1000-fold higher than for the human enzyme. This is somewhat surprising because the enzyme is fairly well conserved among the different species with the homology ranging from 88 to 96% (Baker et al., 2003). However, minor differences could account for the changes in potency. As expected, the catalytic triad of C139, H279, and N299 is conserved among all of the species as it is for all thiol cathepsins. Also, the key oxyanion hole residues of Q133 and W301 are conserved. There are, however, some differences in the active sites. The S2 pocket is thought to be the primary determinant of specificity, and only the human form has a tyrosine at residue 326 that forms the bottom of the pocket, whereas all of the other species have a phenylalanine at this position. Another potentially important change is in the S1' pocket where the human form has an arginine at position 256, where it is serine in all other species. The charged residue at this position is unusual because both cathepsin K and L have a glycine at this position. Another interesting difference is at position 188, where both the human and dog enzyme contain a threonine, but the mouse and monkey have glutamic acid at this position. This residue is adjacent to the activity site but not a part of the traditional substrate-binding pocket.

In antigen presenting cells such as B cells, dendritic cells, and macrophages, the invariant chain must be released from the MHC II molecules before peptides can be loaded. This occurs by a series of proteolytic events that cumulates in only a small fragment, CLIP, remaining in the peptide-binding groove. This fragment is then removed by interaction with HLA-DM by mechanisms that are still unclear. The exact identity and number of proteases involved in the invariant chain degradation are unknown, but it is clear that in some cell types cathepsin S is responsible for the final proteolytic step that generates CLIP. We have used the accumulation of the precursor for CLIP, the invariant chain p10 fragment (p10Ii), as a readout of cathepsin S activity in cells. Both LHVS and JNJ 10329670 are able to block the cleavage of p10Ii in B cell lines and in primary human dendritic cells. Because LHVS is not completely specific for cathepsin S, the use of the cathepsin S-specific compound JNJ 10329670 convincingly demonstrates that cathepsin S is the major p10Ii-degrading enzyme in these cells.

In mice, it has been reported that cathepsin S is not essential for the degradation of the invariant chain in macrophages. The human monocytic cell line THP-1 differentiates to a macrophage-like phenotype after IFN treatment, and we used these differentiated cells to investigate whether human macrophages are dependent on cathepsin S for invariant degradation. Without treatment with IFN, there was no expression of invariant chain, but cathepsin S was still expressed (data not shown). Upon differentiation the levels of both the invariant chain and cathepsin S increased (data not shown). LHVS could block the degradation of the invariant chain, albeit with a shift in potency compared with B cells or dendritic cells, which suggest that a protease different from cathepsin S can perform this task. More definitive evidence for this can be seen with our selective cathepsin S inhibitor where there was no accumulation of p10Ii. This shows that in these cells, cathepsin S is not essential for the degradation of the invariant chain. The shift in potency with LHVS, along with the lack of activity of JNJ 10329670, suggests that other proteases such as cathepsin L may be the responsible for the invariant chain degradation in these cells. Cathepsin F is also a candidate, but the affinity of LHVS for cathepsin F is similar to that of cathepsin S. Therefore, if cathepsin F is involved, one would expect LHVS to be as potent in THP-1 cells as it is in JY cells, which was not observed.

One interesting finding is that JNJ 10329670 had a reduced potency in human PBMCs compared with B cell lines or dendritic cells. This shift was not seen with LHVS. B cells make up 15% of PBMCs, and one would expect the potency of a cathepsin S inhibitor to be similar to that in JY or Raji cells. One possible explanation is that another protease exists in these primary B cells that can substitute for cathepsin S. This explanation would require that the unknown protease and cathepsin S are equally sensitive to LHVS, which suggests a role for cathepsin F. As mentioned, LHVS has a similar potency for cathepsins S and F, whereas our compound does not inhibit cathepsin F.

Because JNJ 10329670 has very little activity versus the mouse cathepsin S, we resorted to a human/mouse chimera model for in vivo studies. In this model, human PBMCs were injected intraperitoneally into SCID mice, and the compound was administered subcutaneously. PBMCs were subsequently harvested and the p10Ii fragment quantitated. Although this system is obviously highly artificial, it nonetheless demonstrates that the compound is able to block cathepsin S activity in vivo.

Blocking cleavage of the invariant chain should inhibit the loading of peptides onto MHC II molecules and block CD4⁺ T cell activation. This can be mimicked in vitro by treating PBMC from sensitized patients with antigen and measuring CD4⁺ T cell proliferation. We show that JNJ 10329670 blocked the proliferation in response to both tetanus toxoid and giant ragweed. These results are somewhat difficult to analyze quantitatively because the inhibition will depend on many factors such as antigen dose and the initial number of specific T cells. Therefore, we have not attempted to calculate IC₅₀ values from these results. However, a qualitative assessment for both antigens indicates that the potency of the compound is consistent with the potency for inhibiting invariant chain degradation in dendritic cells. It is also noted that the compound has effects on the phytohemagglutinin-induced proliferation. Phytohemagglutinin-induced proliferation should not depend on cathepsin S activity because it is antigen independent and works by crosslinking receptors on the T cells. This is complicated, however, by the fact that the presence of antigen presenting cells actually amplifies the phytohemagglutinin response, perhaps by providing other costimulatory molecules. Nevertheless, even when JNJ 10329670 is incubated with phytohemagglutinin and T cells alone, there is inhibition of proliferation at concentrations above 10 µM (data not shown). This indicates that the compound may have another target or some nonspecific activity (for example, it accumulates readily in membranes due to its hydrophobic nature). However, there is a separation between the concentration needed to inhibit antigen presentation and that which inhibits phytohemagglutinin-induced proliferation. Although it is difficult to prove complete selectivity, JNJ 10329670 has no activity in vitro versus many other targets. These data point very strongly to the role of cathepsin S in the presentation of these antigens.

In mice, it is known that the affinity of different MHC II molecules for the invariant chain varies. This means that in some haplotypes invariant chain degradation is not essential for peptide loading to MHC II. Thus, cathepsin S-deficient B cells and dendritic cells from H-2^b mice (where invariant chain has high affinity for H2-A^b) are not able to present antigens, whereas cells from H-2^q mice are, in line with the fact that H2-A^q binds invariant chain less well (Nakagawa et al., 1999). This finding led us to evaluate antigen-induced proliferation assays using PBMCs from donors with a variety of haplotypes (Table 3). Somewhat surprisingly, we found that regardless of the haplotype the antigen presentation was blocked by inhibition of cathepsin S.

Together, the data presented here and elsewhere suggest that inhibitors of cathepsin S may be useful in treating T cell-mediated diseases in a large population of patients. Indeed, the fact that cathepsin S inhibitors can block the T cell proliferation induced by giant ragweed suggests that allergic conditions may be possible clinical indications. Other obvious indications are autoimmune diseases such as rheumatoid arthritis or multiple sclerosis where autoreactive T cells are considered the root cause of the pathogenic conditions. The limited knowledge of relevant antigens in these diseases makes target validation difficult, and it is likely that the usefulness of cathepsin S inhibitors for the treatment of autoimmune diseases will have to be demonstrated in the clinic.

In conclusion, we have identified a novel potent and noncovalent cathepsin S inhibitor that displays high selectivity for human cathepsin S over other proteases. In vitro the compound blocks invariant chain proteolysis in B cells and dendritic cells, as well as antigen-induced T cell proliferation. The noncovalent nature of JNJ 10329670 as well as its selectivity versus other cathepsins makes it an ideal tool for exploring cathepsin S biology. Furthermore, because this compound is bioavailable in several species and shows activity in an in vivo model system, we propose that it may useful for the treatment of allergies or autoimmune diseases.

	Acknowledgements

 
We thank Juli DeGraw for the DR typing of PBMC samples, Bill Hageman and Jefferey Hall for pharmacokinetics studies in dogs and monkeys, Kenway Hoey and Mark Feisnstein for LC-MS analysis of pharmacokinetic plasma samples and also for the synthesis of the peptide substrates, Fawn Coles and Anne Fourie for running the MMP 13 and ADAM-28 assays, and Jon Blevitt for expression and purification of napsin. We are also indebted to Shelly Courtney and Julie Culver for helping with compound administration in vivo.

	Footnotes

 
DOI: 10.1124/jpet.103.056879.

ABBREVIATIONS: MHC II, major histocompatibility complex class II; CLIP, class II-associated invariant chain peptide; MMP, matrix metalloproteinase; p10Ii, p10 fragment of the invariant chain; DMSO, dimethyl sulfoxide; ECE, endothelin-converting enzyme; ACE, angiotensin-converting enzyme; MES, 4-morpholineethanesulfonic acid; PBMC, peripheral blood mononuclear cell; LHVS, leucine homophenylalanine vinyl sulfone; LC-MS, liquid chromatography-mass spectrometry; IFN, interferon-; JNJ 10329670, 1-[3-[4-(6-Chloro-2,3-dihydro-3-methyl-2-oxo-1H-benzimidazol-1-yl)-1-piperidinyl]propyl]-4,5,6,7-tetrahydro-5-(methylsulfonyl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazolo[4,3-c]pyridine.

Address correspondence to: Dr. Robin L. Thurmond, 3210 Merryfield Row, San Diego, CA 92121. E-mail: rthurmon{at}prdus.jnj.com

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