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In Vivo Inhibition of Serine Protease Processing Requires a High Fractional Inhibition of Cathepsin C

Departments of Biochemistry and Molecular Biology (N.M., J.R., D.E., D.R., M.P.), Medicinal Chemistry (D.G., C.B., T.R.), and Comparative Medicine (K.O., S.W., D.N.), Merck Frosst Centre for Therapeutic Research, Kirkland, Quebec, Canada

Received January 25, 2008; accepted February 15, 2008

	Abstract

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Inhibition of cathepsin C, a dipeptidyl peptidase that activates many serine proteases, represents an attractive therapeutic strategy for inflammatory diseases with a high neutrophil burden. We recently showed the feasibility of blocking the activation of neutrophil elastase, cathepsin G, and proteinase-3 with a single cathepsin C selective inhibitor in cultured cells. Here we measured the fractional inhibition of cathepsin C that is required for blockade of downstream serine protease processing, in cell-based assays and in vivo. Using a radiolabeled active site probe and U937 cells, a 50% reduction of cathepsin G processing required 50% of cathepsin C active sites to be occupied by an inhibitor. In EcoM-G cells, inhibition of 50% of neutrophil elastase activity required 80% occupancy. Both of these serine proteases were fully inhibited at full cathepsin C active site occupancy, whereas granzyme B processing in TALL-104 cells was partially inhibited, despite complete occupancy. In vivo, leukocytes from cathepsin C^+/- mice exhibited comparable levels of neutrophil elastase activity to wild-type animals, even though their cathepsin C activity was reduced by half. The long-term administration of a cathepsin C inhibitor to rats, at doses that resulted in the nearly complete blockade of cathepsin C active sites in bone marrow, caused significant reductions of neutrophil elastase, cathepsin G and proteinase-3 activities. Our results demonstrate that the inhibition of cathepsin C leads to a decrease of activity of multiple serine proteases involved in inflammation but also suggest that high fractional inhibition is necessary to reach therapeutically significant effects.

Cathepsin C loss-of-function mutations have provided invaluable knowledge of its role in serine protease activation and in their role in inflammation. However, little information is available under circumstances where cathepsin C activity is partially reduced. We recently described potent reversible dipeptide nitrile inhibitors of cathepsin C that blocked the cathepsin C-mediated activation of Pr-3, CG, and NE in cells (Méthot et al., 2007). Here, we investigated the effects of partial cathepsin C inhibition on serine protease activation in cultured cells and in vivo. We used these reversible cathepsin C inhibitors in combination with a radiolabeled active site probe to measure the percentage of cathepsin C blocked by the inhibitors and the functional consequence on serine protease activation. Our results suggest that fractional inhibition requirements for processing of downstream serine protease vary with cell type and target serine protease but, in general, a high degree of cathepsin C inhibition is required for a functional effect. Cathepsin C fractional inhibition requirements were also investigated in vivo. Peripheral blood leukocytes of cathepsin C^+/- mice contained the wild-type levels of CG and NE activities, despite a 50% reduction in cathepsin C enzyme activity. These results strongly suggest that greater than 50% of cathepsin C inhibition will be needed for therapeutic success. Indeed, we could show that partial inhibition of CG, Pr-3, and NE was achieved in peripheral blood leukocytes of rats for long-term infusion with a cathepsin C inhibitor, but at the requirement of very high cathepsin C active site occupancy in the bone marrow.

	Materials and Methods

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Reagents. Gly-Phe-DMK (GF-DMK) was purchased from Valeant Pharmaceuticals (Costa Mesa, CA). Ala-4-I-Phe-DMK, Ala-4-¹²⁵I-Phe-DMK (Méthot et al., 2007), and dipeptide nitriles were synthesized at Merck Frosst (Kirkland, Quebec, Canada). A detailed description of their synthesis will be published elsewhere (D.G., manuscript in preparation). All dipeptide nitrile cathepsin C inhibitors are trifluoroacetic acid salts. Details on the properties of compound 1 have been published in Méthot et al. (2007). Details on the caspase inhibitor M867 can be found in Méthot et al. (2004). The gB inhibitor compound 20 (Willoughby et al., 2002) and the NE inhibitor L-694,458 (Davies et al., 1991) were obtained from Merck and Co. (Rahway, NJ). Aprotinin-agarose and β-estradiol were purchased from Sigma-Aldrich (St. Louis, MO). MeO-Suc-Ala-Ala-Pro-Val-AMC and Suc-Ala-Ala-Pro-Phe-pNA were obtained from Bachem AG (Bubendorf, Switzerland), and NH₂-Gly-Arg-AMC from NovaBiochem (Laufelfingen, Switzerland). (7-Methoxycoumarin-4-yl)acetyl-lysyl-2-(picolinoyl)-Tyr-Asp-Ala-L-ys-Gly-Asp-N-3-(2-4-dinitrophenyl)-1,2,3-diaminopropyonyl-NH₂ was custom-synthesized at AnaSpec Inc. (San Jose, CA). Methionine, glutamine-free RPMI 1640 medium, and mouse recombinant granulocyte-macrophage colony-stimulating factor were obtained from Invitrogen (Carlsbad, CA). Recombinant human IL-2 and IL-12 were purchased from eBioscience (San Diego, CA). RPMI 1640 medium, Iscove's modified Dulbecco's medium, sodium pyruvate, penicillin-streptomycin, and PBS solutions were from Mediatech Inc. (Herndon, VA), and FBS was from Hyclone (Logan, UT). [³⁵S]methionine (1000 Ci/mmol) was obtained from GE Healthcare (Chalfont St. Giles, Buckinghamshire, UK).

Serine-Protease Cell-Based Assays. The EcoM-G and U937 cell-based assays have been described in detail in Méthot et al. (2007). In brief, CG processing in U937 cells was measured by pulsechase and aprotinin-agarose binding. Exponentially growing U937 (American Type Culture Collection, Manassas, VA) cells were seeded at 2 x 10⁶ cells/ml in methionine and glutamine-free RPMI 1640 medium supplemented with 10% dialyzed FBS, and starved for 30 min in the presence of the cathepsin C inhibitors before the addition of [³⁵S]methionine (10 µCi/ml). The cells were pulsed for 30 min, washed twice, and reseeded in complete U937 culture media (RPMI 1640 medium containing 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, 1 mM sodium pyruvate, and 10 mM HEPES). Cathepsin C inhibitors were freshly added, and the cells were incubated for an additional 3 h. After the chase period, the cells were washed with PBS and lysed in lysis buffer A (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 1% NP40). Cell debris was removed by centrifugation at 15,000g for 10 min. The supernatant was mixed with 4 volumes of binding buffer A (50 mM Tris-HCl, pH 8.0, 1 M NaCl, and 1% NP40) and aprotinin-agarose beads (Sigma) and incubated for 1 h at room temperature with gentle rotation. The beads were pelleted by brief centrifugation at 750g then washed three times with binding buffer A and once with lysis buffer A. Aprotinin-agarose-bound proteins were resolved on 10 to 20% SDS-PAGE gels (Invitrogen). After fixation and drying, the gels were exposed to Kodak BioMax MR film (Carestream Health, Rochester, NY) with intensifying screens for 24 to 48 h. Densitometry on ³⁵S-labeled CG was performed using a calibrated densitometer (GS-800; Bio-Rad Laboratories, Hercules, CA) and QuantityOne software (Bio-Rad Laboratories).

All experiments using EcoM-G cells were performed with cultures less than 3 months old. EcoM-G clone EPS1 ER 3.3 (Sykes and Kamps, 2001) cells were grown in RPMI 1640, 10% FBS, 100 U/ml penicillin, and 100 U/ml streptomycin, 10 ng/ml recombinant murine granulocyte-macrophage colony stimulating factor, and 1 µM β-estradiol. To induce EcoM-G differentiation, the cells were washed with PBS and seeded at 0.5 x 10⁶ cells/ml in EcoM-G culture media without β-estradiol. The cathepsin C dipeptide nitrile inhibitors were added directly to the culture media 24 h later. Cells were harvested 48 h after seeding and lysed in lysis buffer B (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.2% NP40). Debris was removed by centrifugation at 15,000g for 10 min, and supernatants were kept. NE, CG, or Pr-3 enzymatic activities were measured as described in Méthot et al. (2007). Data were plotted using SigmaPlot 9.0 (Systat Software, Inc., San Jose, CA) and sigmoidal curve fitting was performed using the Hill 4-parameter equation.

The TALL-104 gB activation cell-based assay was performed as follows. TALL-104 cells (American Type Culture Collection) were propagated in Iscove's modified Dulbecco's medium supplemented with 20% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, and 100 U/ml IL-2. The cells were seeded at 3 x 10⁶ cells/ml in culture media in the presence of IL-12 (10 ng/ml) and cathepsin C inhibitors. The final DMSO concentration in each well was 0.5%. The cells were incubated at 37°C 5% CO₂ in a humidified incubator, harvested by centrifugation 24 h later, and washed twice in PBS before lysis with gB activity buffer (20 mM Tris-Cl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 5 mM dithiothreitol, and 0.5% NP-40). Caspase-3 inhibitor (1 µM M867; Méthot et al., 2004) and cathepsin C inhibitor (1 µM compound 1; Méthot et al., 2007) were also added to the gB activity buffer to prevent postlysis activation of gB and caspase-3-mediated cleavage of the synthetic substrate IEPD-AMC (Willoughby et al., 2002; Ewen et al., 2003). Cell debris was removed by centrifugation, and gB activity was determined in the lysate by addition of IEPD-AMC to a final concentration of 200 µM. Release of AMC was measured spectrophotometrically using a SpectraMax plate reader (Molecular Devices, Sunnyvale, CA), with excitation at 355 nm and emission at 460 nm, for 10 min. The kinetic rates were calculated from the linear portion of the reaction.

Enzyme Occupancy Assays. EcoM-G enzyme occupancy assays (EOA) were performed under conditions identical described for the serine protease cell-based assay, up to the lysis step. Before cell lysis, Ala-4-¹²⁵I-Phe-DMK (2000 Ci/mmol) was added directly to the culture to a final concentration of 0.5 nM. The labeling reaction proceeded for 15 min and was stopped by the addition of a 2000-fold excess of Ala-4-I-Phe-DMK. The cells were pelleted by centrifugation at 400g for 5 min washed with PBS containing 1 µM Ala-4-(I)Phe-DMK and lysed with lysis buffer C (20 mM MES, pH 6.0, 50 mM NaCl, and 0.5% NP40) for 15 min on ice, before centrifugation at 15,000g. Cytosolic extracts were denatured by addition of reducing Laemmli buffer and boiling. Ala-4-¹²⁵I-Phe-DMK-labeled proteins were resolved on 10 to 20% SDS-PAGE gels. The gels were fixed, dried, and exposed to film for 6 to 24 h. TALL-104 EOA were also carried out under conditions identical to the serine protease cell-based assay, up to the lysis step. Labeling with Ala-4-¹²⁵I-Phe-DMK and processing of the cell lysates was performed as described above for EcoM-G cells. U937 EOAs were performed under conditions similar to the pulse-chase assay. The cells were seeded in complete U937 culture media at 2 x 10⁶ cells/ml and incubated at 37°C with 5% CO₂ in a humidified tissue culture incubator in the presence of cathepsin C inhibitors. After 4 h, Ala-4-¹²⁵I-Phe-DMK (2000 Ci/mmol) was added to the culture to a final concentration of 0.5 nM for 15 min. The labeling reactions was stopped by the addition of a 2000-fold excess of Ala-4-I-Phe-DMK. The cells were pelleted by centrifugation at 400g for 5 min washed with PBS containing 1 µM Ala-4-I-Phe-DMK and lysed 15 min on ice with lysis buffer B. Debris was removed by centrifugation at 15,000g for 10 min and cytosolic extracts were treated as described for the EcoM-G EOA. Densitometric quantitations were carried out with the QuantityOne software and the Bio-Rad GS-800 densitometer. In all assays, care was taken to ensure the densitometric data were obtained with nonsaturated film exposures and within the pixel saturation curves of the densitometer. In some cases, multiple film exposures were obtained for comparison of densitometric data, and comparable results were obtained.

Western Blotting. Protein extracts were prepared as described for the EOA, separated by SDS-PAGE and transferred to nitrocellulose membranes. Nonspecific binding was reduced by incubating the membranes with blocking buffer (Tris-buffered saline supplemented with 0.1% Tween 20 and 5% powdered milk) for 1 h at room temperature, before probing with antibodies. For cathepsin C, a 500-fold dilution of rabbit anti-mouse cathepsin C (gift from C. Pham, Washington University School of Medicine, St. Louis, MO) was prepared in blocking buffer. Anti-human granzyme B (Millipore Bioscience Research Reagents) and anti-β-actin (Sigma) were prepared at 1 µg/ml and 3000-fold, respectively, in blocking buffer. Antigen-antibody complexes were revealed with anti-rabbit-IgG-HRP (GE Healthcare) diluted 5000-fold in blocking buffer and developed with SuperSignal West Femto chemiluminescence reagents (Pierce, Rockford, IL) on Hyperfilm ECL (GE Healthcare).

Animals. Male Sprague-Dawley rats (250-300 g; Charles River Laboratories, St-Constant, QC, Canada) and cathepsin C^+/+ and C^-/- mice (Pham and Ley, 1999) were housed in a 12-h light/dark cycle with free access to food and water. All procedures were carried out under appropriate Animal Care Committee approval in strict accordance to Merck and Co. animal care policies. Thioglycollate-induced mouse peritoneal macrophages were prepared as described by Boulet et al. (2004).

In Vivo Cathepsin C Inhibition. The ex vivo cathepsin C EOA was performed using rats (n = 3 per group) injected subcutaneously with compound 1 (10 mg/kg) or vehicle (100% PEG-200). The animals were euthanized by CO₂ inhalation 1 h after the injection. Femur bone marrow was flushed out with air, weighed, and processed immediately for an ex vivo EOA. The marrow was suspended with an equal volume (w/v) of RPMI 1640 supplemented with 10% FBS and 2 nM Ala-4-¹²⁵I-Phe-DMK. Labeling proceeded for 10 min and was stopped by the addition of 2 volumes of PBS containing 1 µM Ala-4-I-Phe-DMK. The cells were washed twice in PBS supplemented with 1 µM Ala-4-I-Phe-DMK and lysed in lysis buffer C as described in the cell-based EOA section. Protein concentration was measured using the Bio-Rad assay and bovine--globulin as standard, and 75 µg were resolved by SDS-PAGE for autoradiography or transferred to nitrocellulose for Western blotting. For the 2-week infusion experiment, rats were anesthetized with 2.5% isoflurane in oxygen and cannulated at the femoral vein by a small incision in the inguinal region. A silicone catheter (0.02''x 0.037''; Lomir, Notre-Dame-de-I'Î_le Perrot, QC, Canada) connected to a polyurethane catheter (3 French, 80 cm; Instech Laboratories, Plymouth Meeting, PA) was inserted into the vena cava, exteriorized at the nape of the neck, and clamped for the duration of the surgery. Enroflaxin (Baytril; 10 mg/kg) was administered on the day of the surgery and the following day. After 1 week of recovery with a sterile saline solution infusion, the rats were switched to solutions of either compound 1 (5 mg/kg/day in vehicle; n = 6) or vehicle (10% polyethylene glycol-200 and 0.9% NaCl; n = 7) at a flow rate of 1 ml/kg/hour. The animals were euthanized by CO₂ inhalation 2 weeks later, and blood and femur bone marrow were collected. Circulating leukocytes were prepared by lysing red blood cells with two consecutive hypotonic shocks in 0.2% NaCl. White blood cells were pelleted by centrifugation at 700g, washed once in PBS, and frozen. Cell pellets were lysed in 20 mM Tris-Cl, 100 mM NaCl, and 0.2% NP-40 for 15 min on ice, and debris were removed by centrifugation at 15,000g for 10 min. Protein content was measured using the Bio-Rad assay. Cytosolic extracts were subsequently used to measure NE, CG, and Pr-3 activities with their respective substrates, as described above, with modified assay buffer (20 mM Tris-Cl, pH 7.5, 1 M NaCl, and 0.2% NP40). To ascertain the specificity of the reaction, NE and CG assays were also carried out in the presence of their respective inhibitor [10 µM L-694,458 for NE (Davies et al., 1991) and 2 µM CG inhibitor I (Calbiochem) for CG]. The reported NE data correspond to the inhibitable portion of the MeOSuc-Ala-Ala-Pro-Val-AMC-cleaving activity, which typically amounted to 80% of the total. For CG, 100% of Suc-Ala-Ala-Pro-Phe-pNA cleavage was attributed to CG activity. Myeloperoxidase activity was determined in MPO assay buffer containing 50 mM K₂HPO₄, pH 6.0; 0.05% hexadecylmethylammonium bromide; 0.063 mg/ml o-dianisidine, and 0.4 mM H₂O₂. Cytosolic extracts were added to the MPO assay buffer and absorbance at 450 nM was determined kinetically over 5 min. Rates were calculated on the linear portion of the curve.

	Results

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The Cathepsin C Active Site Probe Detects Active Cathepsin C in Intact Murine Cells. Ala-4-¹²⁵I-Phe-DMK is a cathepsin C active site probe that binds covalently to multiple proteins in intact U937 cells, including the 23-kDa large subunit of cathepsin C (Méthot et al., 2007). To determine whether the other labeled peptides were related to cathepsin C, intact peritoneal macrophages, bone marrow suspension, and whole blood from cathepsin C^+/+ and C^-/- mice were exposed to Ala-4-¹²⁵I-Phe-DMK, lysed, and resolved by SDS-PAGE. Multiple proteins were labeled, with varying intensity and sizes, depending on the cell type. Only the 23-kDa protein, found in all cathepsin C^+/+ cell types examined, was consistently absent in cells originating from cathepsin C^-/- mice (Fig. 1A). Two-dimensional gel electrophoresis, Western blotting, and competition with selective cathepsin C inhibitors showed that these other labeled peptides are not cathepsins B, L, or S (data not shown).

View larger version (40K): [in this window] [in a new window]   Fig. 1. The active site probe Ala-4-125I-Phe-DMK binds to cathepsin C in intact murine cells. A, autoradiogram of Ala-4-125I-Phe-DMK-labeled proteins from cathepsin C+/+ (lanes 1, 3, 5, WT) and C-/- (lanes 2, 4, 6, KO) mice. Peritoneal macrophages (m; lanes 1 and 2), femur bone marrow (BM; lanes 3 and 4) and heparinated blood (lanes 5 and 6) were exposed to 0.5 nM Ala-4-125I-Phe-DMK for 15 min before quenching with Ala-4-I-Phe-DMK and resolution of lysates on SDS-PAGE. The 23-kDa protein was consistently absent in extracts from cathepsin C-/- mice. B, autoradiogram of Ala-4-125I-Phe-DMK-labeled proteins from EcoM-G cells. EcoM-G cells were undifferentiated (+β-estradiol, lanes 1 and 2) or differentiated (-β-estradiol; lanes 3 and 4) for 48 h, in the presence 10 µM compound 1 for the final 24 h of differentiation. Ala-4-125I-Phe-DMK was added for the last 15 min and the cells were processed as described under Materials and Methods. Compound 1 prevented labeling of the 23-kDa protein (arrow). C, Western blot of cathepsin C in EcoM-G cells. EcoM-G cells were undifferentiated (+β-estradiol, lanes 1 and 2) or differentiated (-β-estradiol, lanes 3-7) for 48 h, in the presence of the indicated amount of compound 1. Neither differentiation nor compound 1 changed the amount of the 23-kDa immunoreactive cathepsin C subunit (arrow). D, autoradiogram of Ala-4-125I-Phe-DMK-labeled proteins from EcoM-G cells. The cells were differentiated for 48 h and exposed to the indicated time and concentration of Ala-4-125I-Phe-DMK. The final conditions for the EOA label less than 5% of the total cathepsin C active sites (compare lanes 3 and 4 with saturated labeling in lanes 7 and 8).

View larger version (13K): [in this window] [in a new window]   Fig. 2. Structures of novel dipeptide nitrile cathepsin C inhibitors used in this study. The range of potencies against purified cathepsin C varies from 10 (compound 2) to 1000 nM (compound 5) and is shown in Table 1.

Cathepsin C Active Site Occupancy and Inhibition of Downstream Serine Proteases in Cells. Next, we compared the occupancy of cathepsin C active sites present in EcoM-G with the percentage of NE inhibition, over a range of concentrations of cathepsin C inhibitors. Active site probes such as Ala-4-¹²⁵I-Phe-DMK have been used to detect cysteine, serine, and proteosome proteases in complex tissue extracts or living cells (Liu et al., 1999; Patricelli et al., 2001; Falgueyret et al., 2004; reviewed by Fonovi and Bogyo, 2007). As with cell-surface receptor occupancy studies, a tracer amount of probe should be used to minimize the number of sites occupied by the probe itself and prevent competition with the reversible inhibitor. Labeling times should also be short to limit dissociation of the reversible inhibitor (Farde et al., 1992; Kapur et al., 2001). Labeling conditions that minimally perturb cathepsin C-reversible inhibitor equilibrium in EcoM-G cells were established by treating cells with Ala-4-¹²⁵I-Phe with various concentrations, for different lengths of time. Increasing probe concentration or labeling time resulted in greater labeling intensity of all four polypeptides, including the cathepsin C p23 subunit (Fig. 1D). By densitometry, we estimate that less than 5% of the total cathepsin C active sites are labeled by 15 min of incubation with 0.5 nM probe, assuming labeling achieved with 50 nM probe is 100%. These conditions should therefore minimally affect binding equilibrium when a reversible inhibitor is present together with the probe. To evaluate how a range of cathepsin C active sites occupancy would affect serine protease processing, we compared fractional cathepsin C occupancy and serine protease activity in differentiating EcoM-G. The cells were seeded without β-estradiol to initiate differentiation, in duplicate. Cathepsin C inhibitors were added after 24 h, and incubated for another 24 h. In one set of assays, the cells were harvested, and NE activity was measured in lysates. In the duplicate assay, Ala-4-¹²⁵I-Phe-DMK (0.5 nM) was added to the cultures. Labeling was stopped with an excess of unlabeled Ala-4-I-Phe-DMK 15 min later. The cells were lysed and cytosolic extracts were resolved by SDS-PAGE. Figure 3 shows results obtained for a typical EcoM-G cathepsin C enzyme occupancy assay (EOA) with compounds 1 and 2 (Fig. 3, A and B, respectively). Cathepsin C active sites were accessible and labeled by Ala-4-¹²⁵I-Phe-DMK in the absence of inhibitor (DMSO control, lane 10). As the concentration of compound 1 increased, fewer cathepsin C active sites were available at equilibrium for capture by Ala-4-¹²⁵I-Phe-DMK, and none were detected at the highest concentration of compound 1 tested. In this particular experiment (one of three independent EOA), half of the total available cathepsin C active sites were occupied by compound 1 or compound 2 at concentrations of 0.044 and 0.038 µM, respectively. We then compared the EOA IC₅₀ with the inhibitor potencies for blockade of NE processing. The average IC₅₀ values for NE activation for compounds 1 and 2 were 0.16 and 0.15 µM, respectively (Méthot et al., 2007), 3- to 5-fold more than the EOA IC₅₀. Superimposition of the EOA and NE activation dose-response curves (Fig. 3, C and D) showed that inhibition by 50% of NE activation required 75 to 80% of cathepsin C active sites to be blocked. Several other cathepsin C inhibitors were tested. Those chosen (Fig. 2) are reversible and structurally similar but span a range of potencies on purified recombinant cathepsin C (10-1000 nM IC₅₀). We reasoned that the relationship between occupancy and serine protease processing should be similar, regardless of the inhibitor potency. The cathepsin C EOA IC₅₀ values were consistently lower than the NE processing IC₅₀ values, on average, by 3.8-fold (Table 1). Thus, in differentiating EcoM-G cells, an excess of cathepsin C activity must be blocked to curb NE processing. For CG processing inhibition, fractional inhibition requirements must be greater still, because the CG processing IC₅₀ values were always 3- to 5-fold greater than those for NE processing (Méthot et al., 2007).

View larger version (70K): [in this window] [in a new window]   Fig. 3. Cathepsin C EOA and NE processing inhibition in EcoM-G cells. A and B, autoradiography and densitometric data of Ala-4-125I-Phe-DMK-labeled EcoM-G cells treated with compound 1 (A) or compound 2 (B). Duplicate sets of EcoM-G cells were differentiated for 24 h, treated with the indicated concentration of cathepsin C inhibitor for an additional 24 h, and processed either for the NE activity assay or the cathepsin C EOA, as described under Materials and Methods. For both A and B, the autoradiogram is a representative example of three independent experiments each for compound 1 and compound 2. The percentage of cathepsin C active sites occupied by was calculated from densitometric data, with 0% occupancy set with the DMSO control. The concentration of inhibitor that blocked 50% of the cathepsin C active sites (IC50) is indicated in the lower right corner. C and D, overlap of cathepsin C EOA and NE activity inhibition for compound 1 (C) and compound 2 (D). Data from three individual EOA each for compound 1 and 2 were combined, whereas for NE activity, data from seven independent experiment (compound 1) or five experiments (compound 2) were combined. Error bars represent the S.E.M. A 50% inhibition of NE activity requires blockade of 80% of the cathepsin C active sites.

The EOA and serine protease activation dose-response curves were compared in other cell types. U937 cells constitutively synthesize and process CG in a cathepsin C-dependent manner (Méthot et al., 2007). For all dipeptide nitriles tested, the EOA and CG activation IC₅₀ values were very similar; overall, there was no significant shift between the two assays (Table 2). Based on these results, we conclude that processing of the constitutively expressed CG in U937 cells does not require a high cathepsin C fractional inhibition.

Finally, we compared EOA and serine protease processing in the CD8+ T cell line TALL-104, which inducibly expresses gB when treated with IL-12 (Cesano et al., 1993). Granzyme B was reported to require cathepsin C-mediated processing for activation (Thiele et al., 1997; Pham and Ley, 1999). Cleavage of the gB substrate IEPD-AMC was increased by 2.5-fold in lysates from TALL-104 cells treated with IL-12 for 24 h (Fig. 4A). This increase was enhanced if the cells were exposed to a caspase inhibitor (M867; Méthot et al., 2004). In contrast, a gB inhibitor (compound 20; Willoughby et al., 2002) reduced the IEPD-AMC cleavage. The irreversible cathepsin C inhibitor Gly-Phe-DMK (McGuire et al., 1993) also reduced gB activity but did so indirectly because it did not inhibit purified gB enzyme (data not shown). Western blotting demonstrates that none of the inhibitors affected the total amount of gB protein (Fig. 4B). Furthermore, a slight decrease in gB protein mobility was often observed, possibly from the presence of two extra amino acids at the N terminus that would be expected if cathepsin C activity was blocked. These data indicate that cathepsin C affects gB processing in these cells.

View larger version (45K): [in this window] [in a new window]   Fig. 4. Cathepsin C EOA and granzyme B processing inhibition in TALL-104 cells. gB enzymatic activity (IEPD-AMC cleavage) in lysates from TALL-104 cells treated for 24 h with IL-12 and the indicated compound. B, granzyme B Western blot of TALL-104 lysates used to generate enzyme activity data in A. The gB inhibitor compound and the cathepsin C inhibitor GF-DMK reduced gB activity without affecting gB protein levels. Note the slight decrease gB mobility in lysates from GF-DMK-treated cells. C and D, autoradiography and densitometric data of Ala-4-125I-Phe-DMK-labeled TALL-104 cells treated with compound 1. Duplicate sets of TALL-104 cells were treated with IL-12 and compound 1 for 24 h and processed either for the gB activity assay or the cathepsin C EOA, as described under Materials and Methods. The autoradiogram is a representative example from two independent experiments. The percentage of cathepsin C active sites occupied by was calculated from densitometric data, with 0% occupancy set with the DMSO controls. The concentration of compound 1, which blocked 50% of the cathepsin C active sites (IC50) is indicated in the lower right corner. D, overlap of cathepsin C EOA and gB activity inhibition for compound 1, with combined data from two independent EOA and seven independent gB activity assays. Error bars represent SEM.

High Cathepsin C Fractional Inhibition Is Required in Vivo for Blockade of Neutrophil Serine Proteases. A comparison of IC₅₀ values for EOA and inhibition of serine protease activity in cells pointed to various cathepsin C fractional inhibition requirements, depending on the cell type and the serine protease. Because granulocytes would be a major site of intervention for pharmaceutical cathepsin C inhibition, we measured NE and myeloperoxidase (MPO) activities in freshly isolated circulating leukocytes from cathepsin C^+/+, C^+/-, and C^-/- mice. No cathepsin C enzymatic activity was detected in leukocytes from cathepsin C^-/-, while approximately half of the wild-type level was measured in cathepsin C^+/- cells (Fig. 5A). In contrast, equal levels of NE activity were measured in leukocytes from cathepsin C^+/- and C^+/+ mice. As expected (Adkison et al., 2002), cathepsin C^-/- leukocytes contained less than 5% of wild-type NE activity levels (Fig. 5B), and MPO activity was equal regardless of the genotype (Fig. 5C). Based on this, we expect that inhibition of serine protease activities via the pharmacological inhibition of cathepsin C in vivo will require greater than 50% active site occupancy.

View larger version (36K): [in this window] [in a new window]   Fig. 5. High fractional inhibition of cathepsin C required for inhibition of serine protease processing in vivo. Cathepsin C (A), NE (B), and MPO (C) enzymatic activities in circulating leukocyte lysates from cathepsin C+/+, C+/-, and C-/- mice. The data shown is the average from four individual animals ± S.E.M. D, autoradiograph of Ala-4-125I-Phe-DMK-labeled bone marrow obtained from compound 1 (n = 3) or vehicle (n = 3) injected rats. The samples were processed as described under Materials and Methods section. The labeled cathepsin C p23 subunit is indicated by an arrowhead. E, cathepsin C Western blot of extracts shown in D. Ala-4-125I-Phe-DMK failed to label active cathepsin C in the presence of compound 1 (D) despite the presence of cathepsin C protein, indicating bona fide active site occupancy by compound 1. F, neutral serine proteases (NE, CG, and Pr-3) and MPO enzymatic activities in lysates of circulating leukocytes from rats treated for 2 weeks with compound 1 (n = 6) or vehicle (n = 7). The reported NE data correspond to the L-694,458-inhibitable portion of the MeOSuc-Ala-Ala-Pro-Val-AMC-cleaving activity, which typically amounted to 80% of the total. For CG, 100% of Suc-Ala-Ala-Pro-Phe-pNA cleavage was attributed to CG activity (see Materials and Methods for details). Statistically significant reductions of NE, CG, and Pr-3 but not MPO activities were measured in the compound 1-treated rats. Error bars represent SEM.

We then tested whether the requirement for high fractional inhibition precludes cathepsin C inhibition as a strategy to reduce the enzymatic activity of multiple serine proteases in vivo. NE, CG, and Pr-3 are expressed and activated in the bone marrow during the promyelocyte stage (day 2-3 of neutrophil differentiation; Walker and Willemze, 1980; Fouret et al., 1989; Garwicz et al., 2005). Mature neutrophils are released from the bone marrow into the circulation after approximately 11 to 14 days of maturation and, if left unstimulated, die after approximately 10 h (Walker and Willemze, 1980). We therefore reasoned that cathepsin C inhibition must take place in the bone marrow to be effective. To determine whether compound 1 can access cathepsin C in the bone marrow, an ex vivo whole-cell EOA was performed. Rats (n = 3/group) were dosed with compound 1 (10 mg/kg) or vehicle, and marrow from both femurs was recovered 1 h after dosing. Drug levels were measured in marrow from one femur, whereas marrow from the other was rapidly suspended in a minimum volume of culture media containing Ala-4-¹²⁵I-Phe-DMK and incubated for 10 min. Labeling of the 23-kDa cathepsin C subunit was evident in vehicle-treated animals but significantly (>90%) reduced in compound 1-treated rats (Fig. 5D). A cathepsin C Western blot shows similar levels of 23-kDa subunit in all samples (Fig. 5E). The concentration of compound 1 in the bone marrow was approximately 1.4 µM. We conclude that compound 1 blocks cathepsin C active sites in vivo in the bone marrow of rats. To test for the effect of compound 1 on serine protease activation in vivo, rats were cannulated in the femoral vein and infused with compound 1 (5 mg/kg/days) or vehicle. NE, CG, Pr-3, and MPO activities were measured in blood leukocytes after 2 weeks of infusion, at which time mature neutrophils that had been exposed to the cathepsin C inhibitor throughout their development should have entered the circulation. Compound 1 did not significantly affect the percentage of lymphocytes, monocytes, and granulocytes in blood (not shown), and left MPO activity unchanged (Fig. 5F). In contrast, NE, Pr-3, and CG activities were significantly reduced (p < 0.05) by 80, 70, and 50%, respectively. The average bone marrow level of compound 1 after 2 weeks' dosing was 3.3 ± 0.5 µM. Thus, the simultaneous reduction of NE, CG, and Pr-3 activities in granulocytes is achievable in vivo with a cathepsin C inhibitor despite a need for high enzyme occupancy.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The inhibition of cathepsin C has the potential to reduce the activity of multiple pro-inflammatory serine proteases, and is therefore of interest for diseases with a high neutrophil burden, such as COPD and cystic fibrosis. We developed selective, reversible and nontoxic cathepsin C inhibitors, and showed that it is possible to inhibit simultaneously more than 90% of NE, CG and Pr-3 enzyme activities in the neutrophil cell line EcoM-G (Méthot et al., 2007). In this article, we investigated further the serine protease inhibition requirements by developing cell-based EOA using the cathepsin C active site probe Ala-4-¹²⁵I-Phe-DMK. We quantified the percentage of active site that must be blocked to affect serine processing in EcoM-G and U937 cells and extended our analysis to a novel cell-based assay that measures gB activation in the CD8+ TALL-104 cell line. In EcoM-G cells, which inducibly express NE, Pr-3, and CG, occupancy of 80% of the cathepsin C active sites reduced NE activity by half, whereas the same 50% inhibition of CG processing in U937 cells was obtained when 50% of the cathepsin C active sites were occupied. In TALL-104 cells, IC₅₀ values for gB inhibition were 3- to 5-fold greater than that for cathepsin C active site occupancy. Thus, the cathepsin C fractional inhibition requirements for inhibition of downstream serine protease processing vary with cell line and target serine protease, but two examples point to a requirement for relatively high cathepsin C fractional inhibition. Although cathepsin C inhibitors significantly reduced gB activity in IL-12-treated TALL-104 cells, the dose-response relationships were shallow compared with those obtained with EcoM-G and U937 cells, and the maxima of inhibition rarely surpassed 80%, despite complete cathepsin C active site occupancy. Granzyme B inhibition could not be maintained beyond 24 h as the enzyme activity gradually increased over time, without an increase in gB protein (J.R., unpublished observations). This contrasts with NE, CG and Pr-3 inhibition in EcoM-G cells, which was fully maintained for at least 3 days after the addition of compound 1 (Méthot et al., 2007). These data suggest that cathepsin C inhibition would not be an appropriate strategy to block granzyme B in vivo. Recent results from Meade et al. (2006) and Sutton et al. (2007) also indicate that gB activation is not fully dependent on cathepsin C.

The active site probe also permitted several other interesting observations. For example, compared with U937 and EcoM-G cells, 7- to 20-fold greater concentrations of compound 1 were necessary to block 50% of the cathepsin C active sites in TALL-104 cells. Compound 1 was equally stable under all three culture conditions, excluding compound degradation as an explanation. The number of cathepsin C active sites per cell (approximately 3000 for both U937 and TALL-104) and per assay (between 0.8 and 1.5 fmol for both assays) were similar and were 100-fold lower than the number of active Ala-4-¹²⁵I-Phe-DMK molecules present in the assay (J.R., C.B., unpublished data). We speculate that the difference of EOA IC₅₀ values between TALL-104 and the neutrophil-like cell lines is a reflection of cellular permeability to compound 1 or the accumulation of compound 1 in lysosomes of a different nature. Compared with the intrinsic potency against purified cathepsin C, the EOA results were more predictive of the compound efficacy on serine protease processing. Potent cathepsin C inhibitors that suffered from low chemical stability were poor at blocking serine protease processing in cells and accordingly showed a high EOA IC₅₀ value (J.R., unpublished data).

The biology of neutrophil maturation and serine protease activation implies that cathepsin C inhibition must occur in bone marrow to be therapeutically effective. Using an ex vivo EOA, we could show that cathepsin C active sites in the bone marrow of rats treated with compound 1 were almost completely blocked, with a compound 1 concentration of 1.4 µM in the femur marrow. In rats continuously infused for 2 weeks with compound 1, the bone marrow levels reached 3 µM (approximately 130 times the EcoM-G cell cathepsin C EOA IC₅₀ value), and the net effect on the activities of NE, CG, and Pr-3 was an 80, 50, and 70% reduction, respectively. It is not known presently whether these percentages of inhibition are the maxima achievable or if further inhibition is possible. Unfortunately, the chemical properties of compound 1 are not suitable to answer this question as more potent cathepsin C inhibitors would be needed. Compound 1 could not be dosed at higher levels as a result of its limited solubility in the intravenous vehicle. Nevertheless, to our knowledge, this is the first in vivo demonstration of the feasibility of inhibiting multiple serine proteases with a single cathepsin C inhibitor. The results also suggest that in vivo efficacy will require a very high fractional inhibition. It is noteworthy that, similarly to what was observed in the EcoM-G cell-based assay (Méthot et al., 2007), in vivo leukocyte CG activity inhibition was also more difficult than NE and Pr-3 inhibition. Cathepsin C deficiency in humans causes PLS (Hart et al., 1999; Toomes et al., 1999). More than 41 mutations in the human cathepsin C gene have been documented (Selvaraju et al., 2003) and most patients with PLS tested exhibit an almost complete (>95%) loss of cathepsin C activity (de Haar et al., 2004; Hewitt et al., 2004; Pham et al., 2004; Nitta et al., 2005). In two published cases, relatives of PLS showed reduced cathepsin C activity but were asymptomatic (Hewitt et al., 2003; Nitta et al., 2005). One of these cases is particularly interesting, with a symptom-less sibling of a PLS patient having only 13% of the normal cathepsin C activity (Hewitt et al., 2003). The latter example supports the notion that very high cathepsin C fractional inhibition will be required for successful therapeutic intervention in humans.

	Acknowledgements

 
We thank Christine Pham for the cathepsin C-null mice and the cathepsin C antibody. We are grateful to Roberta Rasori and Cindy Jones for their expert assistance with all in vivo and diagnostic work. We are indebted to Paul Tawa for two-dimensional electrophoresis, Herb Bull for the gift of compound 20, and Rick Mumford for the gift of L-694,458 (both donors from Merck Research, Rahway, NJ).

	Footnotes

 
ABBREVIATIONS: PLS, Papillon-Lefèvre syndrome; CG, cathepsin G; NE, neutrophil elastase; Pr-3, proteinase-3; gB, granzyme B; GF-DMK, Gly-Phe-DMK; DMK, diazomethyl ketone; M867, (3S)-3-({(2S)-2-[5-tert-butyl-3-{[(4-methyl-1,2,5-oxadiazol-3-yl)methyl]amino}-2-oxopyrazin-1(2H)-yl]butanoyl}amino)-5-[methyl(pentyl)amino]-4-oxopentanoic acid; L-694,458, N-(1-(1,3-benzodioxol-5-yl)butyl)-3,3-diethyl-2-(4-((4-methyl-1-piperazinyl)carbonyl)phenoxy)-4-oxo-1-azetidinecarboxamide; AMC, aminomethyl coumarin; PBS, phosphate-buffered saline; NP40, nonidet P40; IL, interleukin; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis; IEPD, Ile-Glu-Pro-Asp; EOA, enzyme occupancy assay; MPO, myeloperoxidase; MES, 2-(N-morpholino)ethanesulfonic acid.

Address correspondence to: M. David Percival, Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, P.O. Box 1005, Pointe-Claire-Dorval, Quebec, Canada. H9R 4P8. E-mail: dave_percival{at}merck.com

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