Abstract

Cathepsin K (CatK) is a cysteine protease and drug target for skeletal disorders that is known for its potent collagenase and elastase activity. The formation of oligomeric complexes of CatK in the presence of glycosaminoglycans has been associated with its collagenase activity. Inhibitors that disrupt these complexes can selectively block the collagenase activity without interfering with the other regulatory proteolytic activities of the enzyme. Here, we have developed a fluorescence polarization (FP) assay to screen 4761 compounds for substrate-specific ectosteric collagenase inhibitors of CatK. A total of 38 compounds were identified that block the collagenase activity without interfering with the hydrolysis of active site substrates such as the synthetic peptide substrate, benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin, and gelatin. The identified inhibitors can be divided into two main classes, negatively charged and polyaromatic compounds which suggest the binding to different ectosteric sites. Two of the inhibitors were highly effective in preventing the bone-resorption activity of CatK in osteoclasts. Interestingly, some of the ectosteric inhibitors were capable of differentiating between the collagenase and elastase activity of CatK depending on the ectosteric site utilized by the compound. Owing to their substrate-specific selectivity, ectosteric inhibitors represent a viable alternative to side effect-prone active site-directed inhibitors.

Introduction

Cysteine cathepsins are found in all life forms and play an important role in mammalian intra- and extracellular protein turnover. They are members of the papain-like family (CA clan, C1 family) and have 11 proteases encoded in the human genome (cathepsins B, C, F, H, K, L, O, S, V, W, and X) [1]. Cathepsins K, V, and S are potent extracellular matrix (ECM) protein-degrading proteases and are powerful elastases [24]. In addition, cathepsin K (CatK) is a unique collagenase capable of cleaving at multiple sites within triple helical collagens and has been implicated in various cardiovascular and musculoskeletal diseases, including osteoporosis [58].

Since its first crystal structure reported in 1997 [9], major interest has been placed in developing CatK inhibitors as a potential treatment for osteoporosis [10]. However, all compounds developed thus far are active site-directed inhibitors that completely block the activity of the enzyme [11,12]. Because CatK is a multifunctional protease, it is likely that blocking its entire proteolytic activity will cause unwanted side effects. This may explain, in part, the failure of osteoporosis clinical trials using CatK inhibitors such as odanacatib and balicatib. Patients treated with these drugs experienced heightened risks of cardiovascular events and skin fibrotic phenotypes despite displaying a dramatic increase in bone mineral density [13,14].

Our previous studies have demonstrated that the degradation of ECM proteins, such as collagens and elastin by cysteine cathepsins, requires specific ectosteric sites [15,16]. These sites are either needed for the formation of collagenolytically active CatK oligomers in the presence of glycosaminoglycans (GAGs) [1719] or act as the secondary binding sites for ECM proteins such as elastin [15,16]. Blocking the formation of these collagen-degrading complexes, or secondary substrate-binding sites, with small molecules allows the selective inhibition of the collagenase and elastase activities of cathepsins without affecting the cleavage of other potentially regulatory substrates [16,20]. Thus, targeting the formation of CatK oligomers may serve as an anti-collagenase-specific approach for CatK inhibitors. It is expected that the blocking of the protein–protein interaction sites of the CatK oligomers as well as the CatK–GAG sites will prevent the enzyme's collagenase activity. We have recently demonstrated that specific collagenase inhibitors for CatK can be identified using a molecular docking approach targeting ectosteric site 1 [21].

In this study, we employed a high-throughput fluorescence polarization (FP) assay to identify compounds that selectively inhibit the collagenase activity of CatK by disrupting the oligomerization of the enzyme. FP is widely used in library screening and drug discovery to detect the potential disruption of interactions between ligands and proteins [22]. We have previously demonstrated the validity of this method by investigating the prevention of the formation of CatK/chondroitin 4-sulfate (C4-S) complexes by negatively charged peptides, which probably interfered with the CatK–GAG interaction [23]. Here, we use this technique to screen chemical libraries for CatK complex formation inhibitors. Active compounds were also screened using a fluorogenic peptide cleavage assay to exclude active site-directed inhibition. Compounds identified and validated in these assays were then tested in collagenase assays to specifically screen for their anti-collagenase activity. Next, their binding mode to the protease was assessed by computational molecular docking and the most active compounds were evaluated for their antiresorptive activity in osteoclast resorption assays (Figure 1). Finally, selected compounds were evaluated for their potential to separately inhibit the collagenase and elastase activities of CatK.

Experimental workflow for the identification of CatK collagenase inhibitors using FP and active site screening assays.

Figure 1.
Experimental workflow for the identification of CatK collagenase inhibitors using FP and active site screening assays.

Chemical libraries were first screened using the FP assay to identify compounds that inhibit the formation of collagenolytically active complexes. Active compounds from the FP assay were then tested in vitro for their ability to block collagen degradation and their active site inhibitory activity. The most active compounds are further investigated in osteoclast resorption assays and molecular docking studies to predict their interactions with the enzyme.

Figure 1.
Experimental workflow for the identification of CatK collagenase inhibitors using FP and active site screening assays.

Chemical libraries were first screened using the FP assay to identify compounds that inhibit the formation of collagenolytically active complexes. Active compounds from the FP assay were then tested in vitro for their ability to block collagen degradation and their active site inhibitory activity. The most active compounds are further investigated in osteoclast resorption assays and molecular docking studies to predict their interactions with the enzyme.

Methods and materials

Catk/C4-S complex formation

The CatK/C4-S complex was generated by combining purified human CatK and C4-S in a 2 : 1 molar ratio in 100 mM sodium acetate buffer, pH 5.5, containing 2.5 mM dithiothreitol (DTT) (Sigma–Aldrich Canada, Oakville, Ontario, Canada) and 2.5 mM ethylenediametetraacetate (EDTA) (Sigma–Aldrich). Wild-type human CatK was expressed in Pichia pastoris and purified as previously described [24]. Fluoresceinamine-labeled C4-S (C4-S*) was prepared as previously described [23] and CatK/C4-S* complexes were generated in the same manner as the unlabeled complexes.

Screening for complex formation inhibitors using a fluorescence polarization

Library screening for complex formation inhibitors with the FP assay was performed as previously described using 20 nM fluorescently labeled C4-S and 40 nM CatK [23]. A library consisting of 1280 compounds from Sigma LOPAC, 1120 from Prestwick, 2000 from Microsource Spectrum, and 361 compounds from Biomol was screened. The compounds from Sigma LOPAC, Prestwick and Microsource Spectrum, here designed KD2 (Known Drugs 2), were screened using 384-well plates (Corning, U.S.A.) with an assay volume of 50 µl in a Synergy 4 multiplate reader. The Biomol compounds were screened under identical conditions using 96-well plates (Corning, U.S.A.) with an assay volume of 100 µl in a fluorescence polarimeter Fluostar optima (BMG LABTECH, Germany). The IC50 values were determined under the same conditions using 96-well plates with inhibitor concentrations ranging from 0.4 to 400 µM.

Benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin enzymatic assay

Active site inhibition of the compounds identified by FP was evaluated using benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin (Z-FR-MCA) as fluorogenic substrate (Bachem Americas, Inc, Torrance, California, U.S.A.). The enzymatic activity of CatK was monitored by measuring the rate of release of the fluorogenic group, amino-methyl coumarin at an excitation wavelength of 380 nm and an emission wavelength of 450 nm using a Molecular Devices SpectraMax Gemini spectrofluorometer. Each inhibitor was added prior to the measurement of enzyme activity and the assays were performed at 25°C at a fixed enzyme concentration (5 nM) and substrate concentration (5 μM) in 100 mM sodium acetate buffer, pH 5.5, containing 2.5 mM DTT and 2.5 mM EDTA.

Collagenase, gelatinase, and elastase assays

The collagenase inhibitory activities of the active compounds were measured using soluble bovine skin type I collagen (0.6 mg/ml) (Life Technologies) and 400 nM human CatK, in the presence or absence of 200 nM C4-S, in 100 mM sodium acetate buffer, pH 5.5, containing 2.5 mM DTT (Sigma–Aldrich) and 2.5 mM EDTA (Sigma–Aldrich). All inhibitors were dissolved in DMSO as a 10 or 20 mM stock solution. To minimize solvent effect on the activity of CatK, all reactions were kept below 1% DMSO, where inhibitory effect was negligible. After incubation at 28°C for 4 h, 10 μM E-64 was added to stop the residual activity of CatK. Collagen cleavage products were separated by SDS–PAGE and stained with Coomassie Blue before imaging using the ImageQuant LAS500 gel imaging system (GE Healthcare).

Gelatin (0.6 mg/ml) degradation assays were performed using 10 nM enzyme under the same buffer conditions as described for the collagenase assay and incubated at 37°C for 1 h before visualization using SDS–PAGE. Gelatin was produced by heating soluble bovine neck type I collagen for 30 min at 70°C.

Elastin-Congo red (Sigma–Aldrich) was used as an insoluble elastin substrate at a concentration of 10 mg/ml. Degradation was performed in the presence of 1 µM of recombinant CatK at 37°C overnight using the same buffer conditions as described above. The amount of degradation was quantified from the released degradation products in the supernatant and was measured using a UV–Vis spectrophotometer at 490 nm.

Human osteoclast cultures and bone resorption analysis

Osteoclasts were generated from mononucleated cells isolated from human bone marrow tissue (Lonza, Walkersville, MD). The bone marrow cells were centrifuged at 400g (5 min) and the pellet was re-suspended in 10 ml α-MEM (α-Minimal Essential Media) and layered onto 10 ml of Ficoll-Paque media solution. Centrifugation at 500g was performed for another 30 min and the white interface containing the monocytes was harvested and washed twice with α-MEM. Cells were cultured in α-MEM containing 10% FBS and 25 ng/ml M-CSF for 24 h and then cultured in 25 ng/ml RANKL (R&D Systems, Minneapolis, MN) and 25 ng/ml M-CSF (R&D Systems) for 7 days. Differentiated osteoclasts (100 000cells per slice) were seeded on each bone slice (5.5 mm diameter, 0.4 mm thickness) with and without inhibitors and incubated at 37°C for 72 h at 5% CO2 with 0.1% DMSO. The inhibitor concentration range tested varied between 200 nM and 3 µM.

To compare the effects of the compounds on cell survival, the metabolic activity of the osteoclasts was determined using the CellTiter-Blue Viability Assay (Promega, Madison, WI, U.S.A.). Bone slices from each condition (inhibitor-treated and control groups) were fixed in 4% formaldehyde and subsequently stained for tartrate-resistant acid phosphatase (TRAP) activity [Acid Phosphatase, Leukocyte (TRAP) Kit; Sigma–Aldrich]. Aliquots taken from cell culture media were used to determine the CTx-1 concentration (MyBiosource ELISA kit, San Diego, CA). CTx-1 is a CatK-specific C-terminal cleavage product of triple helical type I collagen. The total number of osteoclasts per bone slice was determined after TRACP staining. Cells with ≥2 nuclei were considered as osteoclasts. After 72 h, bone slices from each condition were incubated in filtered water to induce cell lysis and cell debris was removed using a cotton stick. The resorption cavities were stained with toluidine blue and observed using light microscopy. The number of resorption events and the eroded bone surface area were determined as previously described [25]. We discriminated bone resorption events based on the total eroded area (resorption pits and trenches) and the trench formation alone. Resorption pits are normally shallow and small roundish excavations on the bone surface that primarily represent demineralization events of stationary osteoclasts. In contrast, trench-like excavations are formed by collagenolytically active and moving osteoclasts [25]. This is mostly due to the CatK activity. All light microscopic analyses were performed using a Nikon Eclipse LV100 microscope and a Nikon Eclipse Ci microscope.

Molecular docking of identified collagenase inhibitors

The appropriate three-dimensional structures for the anti-collagenase compounds were generated using LigPrep (Maestro) and OPLS3 force fields and ionization states generated at pH 5.5 to mimic the assay conditions [26]. Geometric rotamers generated for each compound were limited to ten per ligand and were exported as SDF files prior to docking. The enzyme molecule used for docking was the inhibitor-free CatK structure we previously determined (PDBID: 5TUN). The enzyme molecule was pre-processed in Maestro and the heteroatoms as well as the water molecules were removed prior to energy minimization. The appropriate receptor grid for ectosteric site 1 was generated as previously described [21]. For the C4-S-binding site, the grid was generated using the position of C4-S from the C4-S/CatK bound structure (PDBID: 3C9E). The prepared ligands were docked to the enzyme using Glide (Maestro) in the extra precision (XP) mode with flexible ligand sampling. Post-dock minimization was performed and a maximum of twenty poses were generated for each compound [26]. The final poses were visually examined in Pymol (Version 1.8) and ranked by Glidescore.

Results

Fluorescence polarization screening

A collection of 4400 screening compounds from the Sigma LOPAC, Prestwick, and Microsource Spectrum (here termed KD2 library) was screened in the FP assay using fluorescently labeled C4-S to detect disruption to the formation of collagenolytic complexes. The assay was found to be robust, with a Z′ value range of 0.60–0.82 and an average of 0.71 for the 16 tested plates, suggesting minimal overlap between the positive and negative controls. Of the 4400 compounds tested at 10 µM, 59 disrupted the formation of CatK/C4-S complexes by more than 40% (Figure 2A). All 59 compounds (10 µM) were analyzed in a secondary screening assay using Z-FR-MCA to exclude active site inhibition. A total of 26 FP-active compounds without active site-directed inhibitory activity remained. Two main classes of compounds were identified: negatively charged (6 compounds) and polyaromatic compounds (16 compounds) (Supplementary Table S1). Six representative and commercially available compounds were selected for further study: aurintricarboxylic acid (ATC), ellipticine, sanguinarine chloride (SGC), suramin, reactive blue 2, and sepiapterin.

The distribution of potential inhibitor hits from KD2 and Biomol libraries based on their degree of inhibition.

Figure 2.
The distribution of potential inhibitor hits from KD2 and Biomol libraries based on their degree of inhibition.

(A) Hits identified from the KD2 screen in the FP assay. A total of 62 compounds were found to disrupt CatK/C4-S* complex formation (>40% FP signal), representing a total hit rate of 1.3% and 30 compounds were highly effective (>80% FP signal; hit rate: 0.6%). (B) From the Biomol library, a total of 12 compounds were effective in disrupting CatK/C4-S* complex formation (>40% FP signal hit rate: 3.9%) and four compounds were highly effective at complex disruption (>80% FP signal hit rate: 1.3%).

Figure 2.
The distribution of potential inhibitor hits from KD2 and Biomol libraries based on their degree of inhibition.

(A) Hits identified from the KD2 screen in the FP assay. A total of 62 compounds were found to disrupt CatK/C4-S* complex formation (>40% FP signal), representing a total hit rate of 1.3% and 30 compounds were highly effective (>80% FP signal; hit rate: 0.6%). (B) From the Biomol library, a total of 12 compounds were effective in disrupting CatK/C4-S* complex formation (>40% FP signal hit rate: 3.9%) and four compounds were highly effective at complex disruption (>80% FP signal hit rate: 1.3%).

An additional 361 pharmaceutically active natural products from Biomol were screened in the FP assay and 18 compounds disrupted complex formation by greater than 40% with four of them by greater than 80% (Figure 2B). Six of these compounds displayed active site-directed inhibition and thus were not subjected to further analysis. In all, 12 compounds were further evaluated in follow-up assays. A complete list of FP-active compounds that had no inhibitory activity on the hydrolysis of Z-FR-MCA is shown in Supplementary Table S1.

Anti-collagenase and inhibitory activity on other substrates

Commercially available active compounds identified in the FP assay were then tested for their ability to inhibit collagen degradation. Out of the 18 commercially available compounds from both libraries, eight compounds displayed collagenase inhibitory activities with IC50 values between 5 and 190 µM (Table 1 and Supplementary Table S2). The most potent compounds included suramin, sclareol, and ATC with IC50 values below 10 µM. Ellipticine, abscisic acid (AA), epigallocatechin gallate (EGCG), and acetyl-strophanthidin were active as collagenase inhibitors with IC50 values between 10 and 100 µM, which translates into a 25- to 250-fold molar excess over CatK assay concentrations. SGC had the weakest IC50 value with 186 µM (Table 1).

Table 1
Active compounds identified from KD2 and Biomol libraries
Chemical name Chemical structure Collagenase IC50 (µM) Other substrate inhibition (100 µM) (in % inhibition) 
Z-FR-MCA Gelatin 
ATC  9.0 ± 3.0 1.7 ± 0.3 
Ellipticine  88 ± 28 4.0 ± 1 
SGC  186 ± 37 14 ± 2 
EGCG  75 ± 10 12 ± 1 
Suramin  5.0 ± 1.0 16 ± 4 
Acetyl-strophanthidin  14.3 ± 4.5 5.4 ± 1.2 
AA  19.5 ± 2.5 4.9 ± 1.8 
Sclareol  9.3 ± 3.8 3.7 ± 0.8 
Chemical name Chemical structure Collagenase IC50 (µM) Other substrate inhibition (100 µM) (in % inhibition) 
Z-FR-MCA Gelatin 
ATC  9.0 ± 3.0 1.7 ± 0.3 
Ellipticine  88 ± 28 4.0 ± 1 
SGC  186 ± 37 14 ± 2 
EGCG  75 ± 10 12 ± 1 
Suramin  5.0 ± 1.0 16 ± 4 
Acetyl-strophanthidin  14.3 ± 4.5 5.4 ± 1.2 
AA  19.5 ± 2.5 4.9 ± 1.8 
Sclareol  9.3 ± 3.8 3.7 ± 0.8 

To further rule out any significant active site inhibition of CatK by the anti-collagenase inhibitors, their effect on the hydrolysis of Z-FR-MCA at 100 µM and the macromolecular substrate, gelatin, was analyzed. Both of these substrates do not require ectosteric sites and rely exclusively on the active site activity of the enzyme. None of the eight compounds displayed more than 16% inhibition of Z-FR-MCA hydrolysis at a molar CatK to inhibitor ratio of 20 000 : 1. This molar ratio is markedly higher than the 465-fold molar excess characterized for the IC50 value of the weakest collagenase inhibitor, SGC. None of these compounds displayed any observable inhibition of the gelatinase activity of CatK (Table 1).

Osteoclast bone resorption assays using the most potent anti-collagenase compounds

We selected six compounds (suramin, ATC, sclareol, EGCG, AA, and SGC) that showed potent to moderate anti-collagenase activity (Table 1) and tested them in an osteoclast-mediated bone degradation assay to assess their ability to inhibit bone resorption. Figure 3A,B shows the quantification of the osteoclast numbers and metabolic activity. None of the compounds tested revealed cytotoxic effects at a 5 µM concentration. The analysis of the release of CTx, a C-terminal degradation fragment of type I collagen specifically generated by CatK [27], revealed that only EGCG and ATC were effective in significantly reducing osteoclastic bone resorption (Figure 3C). Figure 3D shows toluidine-stained osteoclast-mediated resorption events on the bone surface in the absence or presence of inhibitors (each at 2 µM). In the untreated cultures, there were numerous large trenches and smaller round pits, indicating extensive bone resorption (Figure 3D). Cultures treated with EGCG and ATC (Figure 3D) showed a significant reduction in trenches and revealed mostly small round demineralized pits indicating effective inhibition of CatK-mediated bone resorption. Small resorption pits represent CatK-independent demineralization events. These results corroborate well with the reduction in CTx release. Finally, we determined the IC50 values for both compounds for the inhibitory activity on the % eroded surface and % trench surface. The IC50 values for EGCG were 2.1 ± 0.3 and 2.1 ± 0.4 µM, respectively, and for ATC 1.7 ± 0.3 and 1.8 ± 0.4 µM, respectively (Figure 4A,B).

Effect of anti-collagenase inhibitors on the viability and activity of human osteoclasts.

Figure 3.
Effect of anti-collagenase inhibitors on the viability and activity of human osteoclasts.

(A) The number of TRAP+ osteoclasts and (B) metabolic activity after treatment with the compounds compared with untreated cells show no significant differences on cell viability at 5 µM inhibitor concentrations. (C) Quantification of bone resorption using CTx levels under untreated and inhibitor-treated (2 µM) conditions show significant reduction for EGCG and ATC (P < 0.05). The values were determined from three independent experiments where five bone slices from each condition were analyzed. (D) Representative images of osteoclast-generated resorption cavities in the presence or absence of the compounds (Suramin, AA, sclareol, EGCG, ATC, SGC). Large trenches were observed and represent collagen degradation events. Small round cavities indicate demineralized pit areas with no or minimal collagen degradation (scale bar = 40 µm). Mature human osteoclasts were cultured on bovine bone slices for 72 h in the absence or presence of inhibitors (2 µM). All assays were done in three independent experiments and data represent mean ± SD. ‘ns’, not significant; **P < 0.01; n = 3.

Figure 3.
Effect of anti-collagenase inhibitors on the viability and activity of human osteoclasts.

(A) The number of TRAP+ osteoclasts and (B) metabolic activity after treatment with the compounds compared with untreated cells show no significant differences on cell viability at 5 µM inhibitor concentrations. (C) Quantification of bone resorption using CTx levels under untreated and inhibitor-treated (2 µM) conditions show significant reduction for EGCG and ATC (P < 0.05). The values were determined from three independent experiments where five bone slices from each condition were analyzed. (D) Representative images of osteoclast-generated resorption cavities in the presence or absence of the compounds (Suramin, AA, sclareol, EGCG, ATC, SGC). Large trenches were observed and represent collagen degradation events. Small round cavities indicate demineralized pit areas with no or minimal collagen degradation (scale bar = 40 µm). Mature human osteoclasts were cultured on bovine bone slices for 72 h in the absence or presence of inhibitors (2 µM). All assays were done in three independent experiments and data represent mean ± SD. ‘ns’, not significant; **P < 0.01; n = 3.

IC50 determination of human osteoclast bone resorption parameters for EGCG and ATC.

Figure 4.
IC50 determination of human osteoclast bone resorption parameters for EGCG and ATC.

(A) For the compound EGCG, the IC50 values for the inhibitory effect on the % eroded surface and % trench surface were determined to be 2.1 ± 0.3 and 2.1 ± 0.4 µM, respectively. (B) For ATC, the IC50 values for the inhibitory effect on the % eroded surface and % trench surface were 1.7 ± 0.3 and 1.8 ± 0.4 µM, respectively. The IC50 values were determined from three independent experiments where five bone slices from each condition were analyzed.

Figure 4.
IC50 determination of human osteoclast bone resorption parameters for EGCG and ATC.

(A) For the compound EGCG, the IC50 values for the inhibitory effect on the % eroded surface and % trench surface were determined to be 2.1 ± 0.3 and 2.1 ± 0.4 µM, respectively. (B) For ATC, the IC50 values for the inhibitory effect on the % eroded surface and % trench surface were 1.7 ± 0.3 and 1.8 ± 0.4 µM, respectively. The IC50 values were determined from three independent experiments where five bone slices from each condition were analyzed.

Molecular docking of collagenase and resorption inhibitors

The most potent compounds in the collagenase and osteoclast-based assays were then investigated using a molecular docking approach to predict their interactions with the enzyme. Two potential binding sites previously implicated in complex formation were assessed: (i) ectosteric site 1 and (ii) the C4-S-binding site. Ectosteric site 1 lies on the L-domain of CatK consisting of the loop ranging from residues Ser84 to Pro100. This site has been shown to be a protein–protein interaction site required for oligomerization of collagenolytic CatK complexes [18]. The formation of collagen-degrading CatK complexes also requires the binding of C4-S at the C4-S-binding site opposite of the active site on the R-domain [19,28]. The C4-S-binding site contains several electropositive residues, which interact strongly with the negatively charged sulfate groups on C4-S. Compounds binding at either ectosteric site 1 or the C4-S-binding site on the enzyme would have an inhibitory effect on complex formation and thus prevent collagen degradation.

Molecular docking for EGCG, ATC, suramin, and sclareol was performed using the Glide suite (Maestro) with extra-precision (XP) mode docking to both ectosteric site 1 and the C4-S-binding site at the physiologically relevant pH of 5.5. The overall best-predicted binding poses for these compounds are shown in Figure 5. Table 2 summarizes the theoretical Kd values for the best poses of the compounds for both ectosteric binding sites.

Ectosteric sites required for CatK-mediated collagen degradation and the best binding poses predicted for the most potent anti-collagenase activity inhibitors.

Figure 5.
Ectosteric sites required for CatK-mediated collagen degradation and the best binding poses predicted for the most potent anti-collagenase activity inhibitors.

(A) Two ectosteric binding sites implicated in the collagenase activity of CatK. The active site Cys25 residue is indicated in yellow. Ectosteric site 1 represents the protein oligomerization site and is indicated in orange. The C4-S interaction site located on the R-domain is colored in blue. The CatK molecule on the right is a 180° rotation. (BE) Top predicted poses for the most potent anti-collagenase compounds docking at either ectosteric site 1 or the C4-S-binding site using Glide. (B) Sclareol (light blue) was predicted to effectively interact with ectosteric site 1 due to its hydrophobic character. (C) ATC, (D) suramin, and (E) EGCG (all in yellow) were predicted to interact effectively with the residues surrounding the C4-S-binding site due to their negatively charged or hydrophilic functional groups. The predicted binding affinities are listed in Table 2.

Figure 5.
Ectosteric sites required for CatK-mediated collagen degradation and the best binding poses predicted for the most potent anti-collagenase activity inhibitors.

(A) Two ectosteric binding sites implicated in the collagenase activity of CatK. The active site Cys25 residue is indicated in yellow. Ectosteric site 1 represents the protein oligomerization site and is indicated in orange. The C4-S interaction site located on the R-domain is colored in blue. The CatK molecule on the right is a 180° rotation. (BE) Top predicted poses for the most potent anti-collagenase compounds docking at either ectosteric site 1 or the C4-S-binding site using Glide. (B) Sclareol (light blue) was predicted to effectively interact with ectosteric site 1 due to its hydrophobic character. (C) ATC, (D) suramin, and (E) EGCG (all in yellow) were predicted to interact effectively with the residues surrounding the C4-S-binding site due to their negatively charged or hydrophilic functional groups. The predicted binding affinities are listed in Table 2.

Table 2
Predicted binding affinities for the most potent collagenase and osteoclast resorption inhibitors
Compound Predicted binding affinities (Kd) for each binding site (µM) 
Ectosteric site 1 C4-S-binding site 
ATC Not predicted to bind 3.2 
EGCG 350 54 
Suramin Not predicted to bind 1.9 
Sclareol 28.9 575 
Compound Predicted binding affinities (Kd) for each binding site (µM) 
Ectosteric site 1 C4-S-binding site 
ATC Not predicted to bind 3.2 
EGCG 350 54 
Suramin Not predicted to bind 1.9 
Sclareol 28.9 575 

Sclareol was well accommodated in ectosteric site 1 and had a predicted Kd of 28.9 µM (Figure 5B). The best pose predicted hydrogen bond interactions between the aliphatic region of the compound and residues Ala86 and Tyr89 and hydrophobic interactions with Tyr87 (Supplementary Figure S1A).

ATC, EGCG, and suramin were all predicted to effectively bind in the C4-S-binding site (Figure 5) of the enzyme with predicted Kd values correlating well to their determined IC50 values in the collagenase and osteoclast assays (Table 2). The best-calculated pose for ATC in the C4-S-binding site (Kd of 3.17 µM) showed extensive hydrogen bond interactions between the three carboxylic acid groups and the positively charged residues Arg8, Lys9, and Lys191 found in the C4-S-binding site (Figure 5C and Supplementary Figure S1C). Additional hydrophobic interactions were seen for Tyr145, Gly148, Asn190, and Leu195. Similarly, EGCG was predicted to bind at the C4-S-binding site (Kd of 54 µM) with hydrogen bond interactions between the alcohol groups on the molecule and the residues surrounding the C4-S-binding site (Figure 5E and Supplementary Figure S1B). Owing to its high molecular mass, suramin was not expected to fit into ectosteric site 1 and was predicted to only bind at the C4-S-binding site (Kd of 1.9 µM). One of the sulfates on the compound occupies a position in close proximity to the one occupied by a sulfate residue from C4-S in the CatK/C4-S complex structure (PDBID: 3C9E) (Supplementary Figure S2B). Hydrogen-bonding interactions were observed between the suramin and Arg8, Lys9, Gln172, Asn 190, and Tyr193 residues, largely mediated through the sulfate groups on the compound. The aromatic regions of the compound also form hydrophobic interactions with Lys191, Tyr193, and Leu195, which partially encompass the C4-S-binding site (Supplementary Figure S2A). An overlay of the best-predicted suramin binding pose and the CatK/C4-S structure (PDBID: 3C9E) showed a similar geometry in the positioning of the sulfate groups and part of the aromatic sections of the compound (Supplementary Figure S2B).

Comparison of the IC50 values of FP inhibition for EGCG, ATC, sclareol, and suramin showed that they corresponded well with their experimental anti-collagenase IC50 values and the calculated Kd values in molecular docking (Supplementary Table S3). As expected, an active site-directed inhibitor such as E-64 had no effect on FP inhibition and supports the notion that the anti-collagenase activity is a result of the disruption of complex formation.

Differential inhibition of the collagenase and elastase activity of CatK

Based on the molecular docking results and our previous studies elucidating the structural requirements for the collagenase and elastase activity of CatK [16], we investigated whether the identified ectosteric inhibitors were selective for the collagenase or elastase activity. In addition to being required for the oligomerization of CatK in its collagenase activity, ectosteric site 1 is also required for elastin degradation [15,16]. Therefore, compounds binding at this site would block both collagenase and elastase activity. But, since the C4-S binding site is only required for the collagenase activity, compounds binding here should not interfere with CatK elastase activity.

To demonstrate this, we used dihydrotanshinone I (DHT1) as a previously characterized and selective inhibitor binding at ectosteric site 1 [29]. As expected, DHT1 significantly blocked both collagenase (at 10 and 50 µM) and elastase (at 10 µM) activities (Figure 6 and Supplementary Figure S3). In contrast, EGCG and ATC, which were predicted to bind preferentially or exclusively at the C4-S-binding site (Table 2), predominantly inhibited only the collagenase activity at their tested concentrations (100 and 500 µM for EGCG; 10 and 50 µM for ATC; Figure 6 and Supplementary Figure S3). The inhibitory effect on the elastase activity was only weak at the highest respective inhibitor concentration tested. To the best of our knowledge, this is the first time that differential inhibition of the activity of a protease was achieved by targeting different ectosteric sites.

Enzymatic inhibition of the collagenase and elastase activity of CatK by DHT1, ATC, and EGCG at high and low inhibitor concentrations.

Figure 6.
Enzymatic inhibition of the collagenase and elastase activity of CatK by DHT1, ATC, and EGCG at high and low inhibitor concentrations.

DHT1 (10 and 50 µM) was observed to inhibit both collagenase and elastase activities. ATC (10 and 50 µM) and EGCG (100 and 500 µM) only inhibited the collagenase activity of the enzyme without disrupting its elastase activity. All assays were done in three independent experiments and data represent mean ± SD.

Figure 6.
Enzymatic inhibition of the collagenase and elastase activity of CatK by DHT1, ATC, and EGCG at high and low inhibitor concentrations.

DHT1 (10 and 50 µM) was observed to inhibit both collagenase and elastase activities. ATC (10 and 50 µM) and EGCG (100 and 500 µM) only inhibited the collagenase activity of the enzyme without disrupting its elastase activity. All assays were done in three independent experiments and data represent mean ± SD.

Discussion

CatK has previously been characterized as a promising target for the treatment of osteoporosis and osteoarthritis and multiple CatK inhibitors have been evaluated in clinical trials [3032]. However, these drugs have all been active site-directed inhibitors, which have either failed due to adverse side effects, or are on hold or discontinued for marketing reasons. The exact mechanisms of the side effects remain unknown but may be caused by the complete inhibition of the CatK activity including its regulatory functions [8]. Inhibiting the collagenase activity of CatK by targeting an ectosteric site could potentially save the enzyme as a drug target. Complex formation of CatK monomers in the presence of GAGs has been shown to be a prerequisite for its collagenase activity. Previous site-directed mutagenesis experiments demonstrated that mutation at these sites led to the reduction or complete loss of the collagenase/elastase activity of CatK without affecting the hydrolysis of other substrates [16,28,33]. Using an in silico computational approach, we recently identified collagenase inhibitors targeting ectosteric site 1 [21]. In the present study, we used an in vitro high-throughput FP-screening method to directly find complex formation inhibitors of the protease.

From two drug libraries containing a total of 4761 compounds, we identified 38 compounds (FP hit rate: 0.8%) that were active in disrupting the complex formation of CatK and C4-S without affecting the active site of the protease. Eight of these compounds were able to prevent collagen degradation, with IC50 values as low as 5 µM (Table 1). When tested for potential active site-directed inhibition, none of the compounds displayed significant inhibition in the cleavage of Z-FR-MCA or gelatin at a 20 000-fold molar excess of inhibitor over protease (Table 1). These results suggest that the inhibitory activity observed is not due to the blocking of the active site but is selective for the collagenase activity of the enzyme through the inhibitor binding at an ectosteric site.

Subsequently, we investigated the potential binding mode of the four most potent compounds with CatK using a computational approach. Sclareol showed the best binding at ectosteric site 1, whereas ATC, EGCG, and suramin were predicted to bind preferentially at the C4-S-binding site. Both ectosteric site 1, which is located at the protein–protein interaction site, and the GAG-binding site are required for the formation of collagenolytically active CatK oligomers (Figure 5A).

Sclareol displayed the highest calculated affinity for ectosteric site 1 (Figure 5B). Previous characterization of this binding site showed a hydrophobic core with hydrogen bond-donating regions around the pocket [21]. Sclareol contains a hydrophobic aromatic ring system and functional groups that can engage in hydrogen-bonding interactions with residues surrounding the binding site (Figure 5B and Supplementary Figure S1A). EGCG showed a markedly lower affinity for ectosteric site 1 when compared with its predicted binding at the C4-S-binding site.

Suramin was the most potent collagenase inhibitor identified through the library screen, with an IC50 value of 5 µM. As expected, molecular docking of the compound to ectosteric site 1 did not yield any binding poses, probably due to steric incompatibility between the large compound and the binding site. However, the compound docked highly efficiently to the C4-S-binding site with a predicted Kd of 1.9 µM (Figure 5D). The enzyme contains numerous positively charged residues near the C4-S-binding site, which allows for interactions with the negatively charged sulfate groups of suramin (Supplementary Figure S2A). Binding at this site would probably disrupt interactions with C4-S and perturb the complex formation required for collagen degradation. Despite its potent anti-collagenase activity and its listing as an essential medicine by the WHO for the treatment of African Sleeping Disease [34] and River Blindness [35], suramin is unlikely to be a suitable drug candidate for the treatment of osteoporosis. The compound has moderate to severe side effects that will not allow for a long-term treatment regime as required for various musculoskeletal diseases including osteoporosis. Its lack of usefulness as an osteoporosis drug is also compounded by its weak efficacy in the osteoclast resorption assay.

Of more interest are EGCG and ATC, as both compounds showed potency in the osteoclast resorption assay (Figure 4). Their IC50 values for the prevention of osteoclastic bone resorption were ∼2 µM and only 7–8 times less potent than for tanshinone IIA sulfonate (240 nM), an ectosteric inhibitor that was highly potent as an anti-resorptive in ovariectomized mice [36]. Both compounds were predicted to bind preferentially with the C4-S-binding site. EGCG is the principal catechin in green tea [37] and has been shown to mitigate bone loss in ovariectomized rats [38,39]. The mechanism is thought to involve the inhibition of osteoclast differentiation via the RANKL pathway over a concentration range of 10–100 µM. Our current data suggest that at lower concentrations (<10 µM) it may directly block the collagenase activity of CatK, the main bone resorbing protease. Interestingly, consumption of green tea correlates with a reduced risk of osteoporosis [40].

There is no available literature information describing the anti-osteoporotic activity of ATC. However, the compound has been described as a powerful inhibitor of protein–nucleic acid interactions and thus inhibits enzymes such as topoisomerase [41] and ribonuclease [42]. Based on our docking experiments, the binding mode of ATC may be comparable to those described for various nucleases. It should be noted that ATC has been identified in multiple assays, which indicates that it may represent a Pan-Assay Interference compound (PAIN), and likely be a ‘cul-de-sac’ regarding future drug development [43,44].

We noted that among the six tested anti-collagenase inhibitors, only ATC and EGCG were effective in the osteoclast-mediated bone resorption assays. This difference could be due to the pharmacological properties of the compounds that prevented them from entering the osteoclast or the resorption lacuna where collagenolytic CatK is active. The membrane permeabilities calculated from physicochemical properties of ATC and EGCG were 2.0 and 2.1 nm/s, respectively. Suramin, the most potent compound in the collagenase assay, had a predicted permeability of only 0.03 nm/s and it was ineffective in the cell-based assay. Other compounds that were active in the collagenase assays (sclareol, AA, SGC), but ineffective in the cell-based assays, also had rather poor membrane permeabilities as well ranging from 0.5 to 1.2 nm/s. It should be mentioned that the overall clinically relevant anti-resorptive activity of these compounds, in particular that of EGCG, might have other mechanisms in addition to the direct inhibition of the collagenolytic activity of CatK. EGCG has been described to suppress Runx2, or to modulate osteoclast formation and osteoblast activity via signal transduction pathways affecting the expression of RANKL/OPG and mitogen-activiated protein kinases (MAPK) and bone morphogenic protein (BMP) [45].

Our study also demonstrated that it is possible to exploit different ectosteric sites to selectively inhibit the distinct ECM-degrading activities of CatK. For example, EGCG and ATC are potent collagenase inhibitors but display only weak or no anti-elastase activity. So far, we have not identified an inhibitor capable of exclusively blocking the elastase activity of CatK. Theoretically, this would be possible if an identified compound specifically interacts with ectosteric site 2 in CatK that is needed for the elastase activity but not for the collagenase activity [16]. However, in the case of CatK, the dissection of its collagenase and elastase activities may only be of theoretical interest; for diseases where the enzyme is a pharmaceutical target, the inhibition of both activities is likely to be more beneficial. In bone and joint diseases, the inhibition of collagen degradation is most relevant, while in cardiovascular diseases the prevention of collagen and elastin is preferred. As osteoporosis patients are normally older in age and thus more prone to cardiovascular problems, the inhibition of both activities would likely be more beneficial.

In conclusion, we have demonstrated that the FP library screening method can be successfully exploited for the identification of compounds that disrupt the complex formation of CatK and it thus allows for the selective inhibition of the enzyme's collagenase or elastase activity. At least two of the identified ectosteric inhibitors were effective at blocking CatK-mediated bone resorption by osteoclasts without affecting the viability of the cells. This is considered highly beneficial for anti-resorptive drugs, as it will not interfere with the cross-talk between osteoclasts and osteoblasts, which is required for the maintenance of healthy bones [46]. Future work may include the screening of larger compound libraries allowing the discovery of further scaffolds for ectosteric CatK inhibitors. We also believe that a similar method can be applied to other pharmaceutical targets that require complex formation for their activities and will be effective at identifying novel ectosteric inhibitors for these targets.

Abbreviations

     
  • α-MEM

    α-minimal essential media

  •  
  • AA

    abscisic acid

  •  
  • ATC

    aurintricarboxylic acid

  •  
  • BMP

    bone morphogenic protein

  •  
  • C4-S

    chondroitin 4-sulfate

  •  
  • CatK

    cathepsin K

  •  
  • DHT1

    dihydrotanshinone I

  •  
  • DTT

    dithiothreitol

  •  
  • E-64

    L-3-carboxy-trans-2–3-epoxypropionyl-leucylamido-(4-guanidino)-butane

  •  
  • ECM

    extracellular matrix

  •  
  • EDTA

    ethylenediametetraacetate

  •  
  • EGCG

    epigallocatechin gallate

  •  
  • FP

    fluorescence polarization

  •  
  • GAG

    glycosaminoglycan

  •  
  • KD2

    Known Drugs 2

  •  
  • MAPK

    mitogen-activiated protein kinases

  •  
  • MEM

    minimal essential media

  •  
  • SGC

    sanguinarine chloride

  •  
  • TRAP

    tartrate-resistant acid phosphatase

  •  
  • Z-FR-MCA

    benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin

Author Contribution

D.B., X.D., S.L., P.P., and M.R. designed the study and its experiments. S.L., X.D., N.H., and T.P. carried out the screening of the libraries and characterization of the compounds. P.P. performed the cell-based assays. S.L. and D.B. wrote and edited the manuscript.

Funding

This work was supported by the Canadian Institutes of Health Research [grant numbers MOP-89974 (D.B.), MOP-125866 (D.B.)], the Natural Sciences & Engineering Research Council of Canada [grant number RGPIN-326803-13], and a Canada Research Chair award (to D.B.).

Acknowledgements

We are grateful to Dr Angela Tether for the editorial assistance in the preparation of the manuscript.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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Author notes

*

These authors contributed equally to this work.