Design, synthesis and biological evaluation of inhibitors of cathepsin K on dedifferentiated chondrocytes

Article abstract of DOI:10.1016/j.bmc.2019.02.003

Selective proteinase inhibitors have demonstrated utility in the investigation of cartilage degeneration mechanisms and may have clinical use in the management of osteoarthritis. The cysteine protease cathepsin K (CatK) is an attractive target for arthritis therapy. Here we report the synthesis of two cathepsin K inhibitors (CKIs): racemic azanitrile derivatives CKI-E and CKI-F, which have better inhibition properties on CatK than the commercial inhibitor odanacatib (ODN). Their IC₅₀ values and inhibition constants (K_i) have been determined in vitro. Inhibitors demonstrate differential selectivity for CatK over cathepsin B, L and S in vitro, with K_i amounting to 1.14 and 7.21 nM respectively. We analyzed the effect of these racemic inhibitors on viability in different cell types. The human osteoblast-like cell line MG63, MOVAS cells (a murine vascular smooth muscle cell line) or murine primary chondrocytes, were treated either with CKI-E or with CKI-F, which were not toxic at doses of up to 5 μM. Primary chondrocytes subjected to several passages were used as a model of phenotypic loss of articular chondrocytes, occurring in osteoarthritic cartilage. The efficiency of CKIs regarding CatK inhibition and their specificity over other proteases were validated in primary chondrocytes subjected to several passages. Racemic CKI-E and CKI-F at 0.1 and 1 μM significantly inhibited CatK activity in dedifferentiated chondrocytes, even better than the commercial CatK inhibitor ODN. The enzymatic activity of other proteases such as matrix metalloproteinases or aggrecanases were not affected. Taken together, these findings support the possibility to design CatK inhibitors for preventing cartilage degradation in different pathologies.

Full text of DOI:10.1016/j.bmc.2019.02.003

Accepted Manuscript
Design, synthesis and biological evaluation of inhibitors of cathepsin K on de-
differentiated chondrocytes
Xiao-Yu Yuan, Zhongyuan Ren, Yuqing Wu, Carole Bougault, Leyre Brizuela,
David Magne, René Buchet, Saida Mebarek
PII:
S0968-0896(18)32031-5
https://doi.org/10.1016/j.bmc.2019.02.003
BMC 14739
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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5 December 2018
27 January 2019
1 February 2019
Please cite this article as: Yuan, X-Y., Ren, Z., Wu, Y., Bougault, C., Brizuela, L., Magne, D., Buchet, R., Mebarek,
S., Design, synthesis and biological evaluation of inhibitors of cathepsin K on dedifferentiated chondrocytes,
Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.02.003
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Design, synthesis and biological evaluation of inhibitors of cathepsin K on
dedifferentiated chondrocytes
Xiao-Yu Yuan1,3, Zhongyuan Ren1, Yuqing Wu1, Carole Bougault2, Leyre
Brizuela2, David Magne2, René Buchet2 and Saida Mebarek2*
1State Key Laboratory of Supramolecular Structure and Materials, Jilin University,
Changchun, 130012, China.
2Univ. Lyon, Univ. Claude Bernard Lyon 1, CNRS UMR 5246, ICBMS, F-69622, Lyon,
France
3College of Food Science and Technology, Henan Agricultural University, Zhengzhou,
450002, China.
Corresponding author
Dr. Saida Mebarek
Université Lyon 1, ICMBS UMR CNRS5246
Batiment Raulin
43 Boulevard du 11 Novembre 1918
69622 Villeurbanne Cedex France
Email: saida.mebarek@univ-lyon1.fr
1

ABSTRACT:
Selective proteinase inhibitors have demonstrated utility in the investigation of cartilage
degeneration mechanisms and may have clinical use in the management of osteoarthritis. The
cysteine protease cathepsin K (CatK) is an attractive target for arthritis therapy. Here we
report the synthesis of two cathepsin K inhibitors (CKIs): racemic azanitrile derivatives CKI-
E and CKI-F, which have better inhibition properties on CatK than the commercial inhibitor
odanacatib (ODN). Their IC50 values and inhibition constants (Ki ) have been determined in
vitro. Inhibitors demonstrate differential selectivity for CatK over cathepsin B, L and S in
vitro, with Ki amounting to 1.14 and 7.21 nM respectively. We analyzed the effect of these
racemic inhibitors on viability in different cell types. The human osteoblast-like cell line
MG63, MOVAS cells (a murine vascular smooth muscle cell line) or murine primary
chondrocytes, were treated either with CKI-E or with CKI-F, which were not toxic at doses of
up to 5 µM. Primary chondrocytes subjected to several passages were used as a model of
phenotypic loss of articular chondrocytes, occurring in osteoarthritic cartilage. The efficiency
of CKIs regarding CatK inhibition and their specificity over other proteases were validated in
primary chondrocytes subjected to several passages. Racemic CKI-E and CKI-F at 0.1 and 1
µM significantly inhibited CatK activity in dedifferentiated chondrocytes, even better than the
commercial CatK inhibitor ODN. The enzymatic activity of other proteases such as matrix
metalloproteinases or aggrecanases were not affected. Taken together, these findings support
the possibility to design CatK inhibitors for preventing cartilage degradation in different
pathologies.
Keywords: Azanitrile • cathepsin K inhibitors • chondrocyte • dedifferentiation •
osteoarthritis
Abbreviations: CatK: cathepsin K, CKI: cathepsin K inhibitor, ODN: odanacatib, OA:
Osteoarthritis
2

1. INTRODUCTION
Osteoarthritis (OA) is a very common age-related disease, affecting approximately 15% of the
global population (1). Due to the demographic trends and ageing, OA is expected to affect
more people in the forthcoming years (2). OA is a degenerative joint disorder characterized by
irreparable destruction of the articular cartilage. Engineered cartilage has great potential in
joint regeneration and reconstruction. A common cell source is chondrocytes, which are the
sole cell type in articular cartilage. Its use is however restricted since autologous chondrocyte
availability is limited in patients. Various in vitro cell expansion methods have been adopted
to increase cell numbers before transplantation, but dedifferentiation of chondrocytes
during this process remains an important challenge (3-6). Dedifferentiation evidences include
cell shape change and decreased expression of cartilaginous matrix components such as
collagens type II and type IX, as well as aggrecan (the major proteoglycan in the extracellular
matrix) (3-6). Several experimental approaches are being developed to overcome the pitfalls
of dedifferentiation (7). For instance, culturing chondrocytes on three-dimensional scaffold
and/or adding cartilage growth factors may delay the dedifferentiation or favor the
redifferentiation (8, 9). Of note, a knockdown of cathepsin K (CatK) has been shown to
maintains the phenotype in expanded chondrocytes (10), indicating possible applications of
CatK inhibition in tissue engineering and regenerative medicine.
CatK belongs to the family of papain-like cysteine peptidases (11). It is one of the main
enzymes involved in bone resorption. Indeed, inhibition of CatK may serve as drug therapy to
prevent osteoclast-related diseases (12, 13). Whereas the activity of most other cathepsins is
restricted to the lysosomal compartment, CatK works extracellularly, preferentially under
acidic pH conditions. At the molecular level, CatK is unique due to its ability to cleave the
triple helix of collagen molecules at multiple locations. This activity is unparalleled among
human collagenases (14). CatK has been demonstrated to be secreted by chondrocytes in OA
patients and participates to the breakdown of cartilage collagen as well as proteoglycan
3

networks (15). A decrease in pH from 7 to 5.5 in the OA cartilage favors cysteine cathepsins
over metalloproteinases (MMPs) as the major matrix degrading proteases in OA, at least in
the later stages of the disease (15). The implication of CatK in OA cartilage degradation was
demonstrated in mice, guinea pig, canine and rabbit models, and further confirmed by a
chondroprotective effect of CatK inhibitors (CKIs) in OA pathology (16-18). Increased CatK
expression and activation in human OA cartilage and synovial tissues were reported (12),
indicating a possible effect of CatK on the pathogenesis of OA(12).
Many low molecular weight CKIs have been synthesized and tested for their specificity and
ability to inhibit bone resorption (12, 13). Inhibitors that have emerged in recent years were
designed to minimize unfavorable side effects, especially by synthesizing other classes of
electrophiles like ketones and nitriles, capable of trapping the nucleophilic cysteine in a
covalent yet reversible fashion (19-26). Several cathepsin inhibitors have previously fallen by
the wayside. Novartis dropped its balicatib in 2006 because of dermatological adverse events
(27, 28), and GlaxoSmithKline abandoned its relacatib in 2007, possibly because of off-target
toxicity (29). Phase III clinical trials for odanacatib (ODN) (30-32) were stopped in 2016 by
Merck, due to stroke risk (33). Among the cathepsin inhibitors that reached phase II trials,
MIV-701 (34) was dedicated to the treatment of bone and cartilage related disorders in
humans, such as OA, while ONO-5334 was evaluated for osteoporosis treatment in humans
(35). Alternate CKIs, especially to prevent cartilage degradation in OA are needed, since only
very few were tested to prevent cartilage degradation. In this report, we described the
synthesis of two racemic CKIs, CKI-E and CKI-F, whose structures derived from previous
CKIs (19), to improve inhibition properties against CatK. We compared their effects with
those of the commercial inhibitor ODN. Inhibition parameters and selectivity over other
cathepsins were assessed in vitro. Cell viability was tested in different cell types. Moreover,
primary chondrocytes were dedifferentiated during several passages to mimic the phenotype
4

change of chondrocytes in OA cartilage. Dedifferentiated chondrocytes served to validate the
efficiency of CKIs regarding CatK inhibition and their specificity over other proteases. In
agreement with the literature (10), passages induced chondrocyte dedifferentiation with a loss
of chondrogenic matrix production and an increase in CatK activity. We evaluated the ability
of racemic CKI-E and racemic CKI-F to prevent collagen digestion by CatK in chondrocytes
subjected to 0, 1, 2 and 3 passages. We aimed to provide a proof-of-concept of the potential of
CKI treatment for preventing cartilage degradation during OA.
2. MATERIALS AND METHODS
2.1 Chemicals and reagents
Thin layer chromatography was performed on aluminum sheets from Branch of Qingdao
Haiyang Chemical Co., Ltd. Preparative column chromatography was performed on silica gel
1
60, 54-74 μm. H NMR (500 MHz) and 13C NMR (126 MHz) spectra were recorded on a
Bruker Avance 500 spectrometer. ESI-MS spectra were recorded on a Bruker HCT mass
spectrometer. (4-(Methylsulfonyl) phenyl)boronic acid was bought from Alfa Aesar China
(Tianjin Co., Ltd.); LDA (lithium diisopropylamide) was obtained from Shanghai Darui
Finechemical Co., Ltd.; 1-iodo-2-methylpropane and THF (tetrahydrofuran) were bought
from J&K Scientific Ltd.; 1,2-dimethylhydrazine dihydrochloride was obtained from TCI Co.,
Ltd.; cyanogen bromide was bought from ACROS Co., Ltd.
2.2 Synthesis routes for CKI-E and -F.
Compound 1Figure was obtained by using the same synthesis procedure as described
before (19, 36), therefore its synthesis will not be described in detail.
5

Figure 1. Synthesis route for compounds CKI-E and CKI-F.
2.2.1
N'-cyano-N,N',4-trimethyl-2-(4'-(methylsulfonyl)-[1,1'-biphenyl]-4-
yl)pentanehydrazide (racemic CKI-E)
100 mL round-bottomed Schlenk flask equipped with a magnetic stir bar was charged with
0.34 g compound 1(bromide as para position)1 mmol), 0.24 g (4-(methylsulfonyl) phenyl)
boronic acid (1.2 mmol), 0.058 g tetrakis(triphenylphosphine) palladium (0.05 mmol), 2 mL
of 1 M aqueous solution of Na2CO3, and 30 mL of tetrahydrofurane (37) (Figure 1). The
mixture was degassed using five cycles of vacuum/nitrogen back-fill firstly, and then it was
heated up to 70 °C and kept there for 2.5 h with vigorous stirring. In following, the mixture
6

was cooled down to room temperature, and the solvent was removed under reduced pressure.
The residue was then suspended in H2O (10 mL) and ethyl acetate (30 mL), the organic layer
was separated while the aqueous layer was extracted with ethyl acetate (3×15 mL). The
combined organic phases were washed with brine (2×15 mL), and then dried with Na2SO4 and
evaporated to obtain the crude product of N'-cyano-N,N',4-trimethyl-2-(4'-(methylsulfonyl)-
[1,1'-biphenyl]-4-yl)pentanehydrazide, which was then purified by column chromatography
on silica gel using ethyl acetate / petroleum ether (1:1) as eluent. HPLC chromatogram
indicated that CKI-E was highly pure (around 95%) as estimated by negligible intensities of
contaminant compounds (HPLC: t = 2.5min).
1H NMR (500 MHz, CDCl3): δ 8.03 (d, J = 8.2 Hz, 2H), δ 7.78 (d, J = 8.3 Hz, 2H), δ 7.61 (d,
J = 8.2 Hz, 2H), δ 7.46 (dd, J = 22.0, 9.8 Hz, 2H), δ 4.26 (t, J = 7.2 Hz, 1H), δ 3.21 (s, 4H), δ
3.12 (s, 3H), δ 2.62 (s, 3H), δ 2.09 - 1.98 (m, 1H), δ 1.66 (dt, J = 29.6, 11.4 Hz, 1H), δ 1.52
(dt, J = 13.7, 7.0 Hz, 1H), δ 1.01-0.94 (m, 6H),
13C NMR (126 MHz, CDCl3): δ 174.48 (s), δ 145.85 (s), δ 140.32 (s), δ 139.28 (s), δ 137.99
(s), δ 128.74 (s), δ 127.91 (d, J=18.6 Hz), δ 113.91 (s), δ 45.81 (s), δ 44.61 (s), δ 43.58 (s), δ
40.59 (s), δ 30.64 (s), δ 25.67 (s), δ 22.71 (s), δ 22.40 (s).
MS (ESI) m/z: 414.1841[M + H]+, 827.3567 [2M + H]+.
2.2.2
N'-cyano-N,N',4-trimethyl-2-(4'-(methylsulfonyl)-[1,1'-biphenyl]-3-
yl)pentanehydrazide (racemic CKI-F)
CKI-F (Figure 1) was produced by using the similar procedures as those for CKI-E except
compound 1(bromide as meta position). The crude product of N'-cyano-N,N',4-trimethyl-2-
(4'-(methylsulfonyl)-[1,1'-biphenyl]-4-yl)pentanehydrazide was obtained and purified
similarly by column chromatography on silica gel using tetrahydrofuran / petroleum ether
7

(1:2) as eluent . HPLC chromatogram indicated that CKI-F was highly pure (about 95%) as
estimated by negligible intensities of contaminant compounds (HPLC: t = 2.7min).
1H NMR (500 MHz, CDCl3): δ 8.04 (d, J = 8.1 Hz, 2H), δ 7.80 (dd, J = 18.9, 8.1 Hz, 1H), δ
7.58 - 7.51(m, 2H), δ 7.47 (dd, J = 15.1, 7.4 Hz, 1H), δ 4.30 (dt, J = 14.8, 7.2 Hz, 1H), δ 3.20
(s, 3H), δ 3.12 (s, 4H), δ 2.59 (s, 2H), δ 2.15 - 1.81 (m, 2H), δ 1.50 (ddd, J = 22.6, 14.0, 7.2
Hz, 1H), δ 0.97 (dd, J = 10.9, 4.3 Hz, 6H)
13C NMR (126 MHz, CDCl3): δ 174.50 (s), δ 146.02 (s), δ 140.75 (s), δ 139.87 (s), δ 139.49
(s), δ 129.69 (s), δ 128.54 – 127.67 (m), 126.61 (d, J = 8.7 Hz), δ 126.29 (s), δ 46.19 (s), δ
44.64 (s), δ 43.73 (s), δ 40.57 (s), δ 30.67 (s), δ 25.74 (s), δ 22.64 (s), δ 22.47 (s).
MS (ESI) m/z: 414.1839 [M + H]+, 827.3571 [2M + H]+.
2.3 In vitro performed inhibition assays
Inhibition assays were performed using commercial recombinant cathepsins to determine the
concentrations of half maximal inhibition (IC50) of CKI-E and CKI-F. The experimental
conditions for the inhibition assays of human CatK (Merck 219461, His-Tag, Human,
recombinant, E.coli. Germany), cathepsin L (CatL) (Merck 219402, Human Liver, Germany),
cathepsin S (CatS) (Merck 219343, Human, recombinant, E. coli, Germany) and cathepsin B
(CatB) (Merck 219362, human liver, Germany) were described before (21, 36). We have
previously determined the Michaelis constant (Km) for CatK, L, S and B to be 18.06 ± 0.22
μM, 3.525 ± 0.405 μM, 102.2 ± 1.52 μM and 157.5 ± 2.5 μM, respectively (21). IC50 values
determined in this work, Km and the concentration of substrate [S] were used to calculate the
inhibition constant of the inhibitors (Ki), by using the following equation:
(1)
8

2.4 Cell culture
2.4.1 Ethics statements
All experiments were carried out according to the guidelines laid down by the French
Ministère de l’Agriculture (n° 87-848) and the E.U. Council Directive for the Care and Use of
Laboratory Animals of November 24th, 1986 (86/609/EEC). Animal experiments were
performed under the authorization n°69-266-0501 (INSA-Lyon, DDPP-SV, Direction
Départementale de la Protection des Populations - Services Vétérinaires du Rhône). MLC
(n°692661241), AG (n°69266332) and COS (n°69266257) hold special licenses to experiment
on living vertebrates issued by the French Ministry of Agriculture and Veterinary Service
Department. The experiments were realized on euthanized animals by dislocation of cervical
vertebra, which did not require surgery and were not painful.
2.4.2 Cell models
9

Two commercial cell lines were used: the mouse vascular smooth muscle cell line MOVAS
(ATCC® CRL-2797™) and the human osteoblast-like cell line MG-63 (ATCC® CRL-1427™). In
addition, primary chondrocytes were isolated as described previously (38) from the
epiphyseal cartilage of the humerus and femur of 5-6 day-old mice (SWISS strain). All three
cell models were cultured in growing medium containing DMEM (Dulbecco's Modified Eagle
Medium) with 10% (v:v) Fetal Bovine Serum (FBS), 100 U mL-1 penicillin and 100 μg mL-1
streptomycin. When primary cells reached confluency, they were harvested using trypsin-
EDTA (Sigma Aldrich, Lyon, FR) and split in two sets. One set was sub-cultured, that is to
say passaged, and the other set was collected for further analysis. This procedure was repeated
so that primary chondrocytes were analyzed at 4 time points: passages 0, 1, 2 and 3 (P0, P1,
P2, P3). All cells were incubated in a humidified atmosphere consisting of 95 % air and 5 %
CO2 at 37 °C.
2.5 Cell viability assay
For cell viability assay, the cells were plated in 12-well plates (Corning inc, 60000 cells/well
for chondrocytes, 100 000 cells/well for cell lines) and were cultured in DMEM, containing
10% (v:v) FBS, 100 U.mL-1 penicillin and 100 μg.mL-1 streptomycin without (control) or
with CKIs at different concentrations until confluence. Their viability was measured using the
MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay
methods (Roche Diagnostics, Meylan, France) as described previously (39). MTT labeling
reagent (0.125 mg mL-1 final concentration) was added to each well and the cells were further
incubated for 4 h. Culture supernatant was replaced by 1 mL of solubilization solution
(dimethyl sulfoxide). Cell viability was directly related to absorbance measured at 570 nm
using a Tecan Infinite M200 (Salzburg, Austria) micro-titre plate reader. Results were
normalized relative to their respective controls (0.1% DMSO (v:v) without CKI), taken as
10

100% of viability. For each inhibitor/cell combination, three distinct sample pools were
analyzed in a triplicate manner (n = 9).
2.6 Zymography of matrix metalloproteinases and CatK in culture medium and cell
lysates
Zymography of matrix metalloproteinases (MMPs) and CatK were performed accordingly to
literature (40, 41). Briefly, extracellular medium (containing secreted MMPs) and primary
chondrocyte lysate (containing intracellular CatK) were collected at 4 time points (P0, P1, P2,
P3), in extraction buffer (pH 7.5) containing 20 mM Tris-HCl, 5 mM ethylene glycol
tetraacetic acid (EGTA), 150 mM NaCl, 10 mM NaF, 20 mM ß-glycerol phosphate (ß-GP), 1
mM sodium orthovanadate, 1% (v:v) Triton X-100 and 0.1% (v:v) Tween 20. Equal amounts
of protein were resolved either by 12% SDS-polyacrylamide gels containing 2 mg.mL-1
gelatin for revealing enzymatic activity of CatK, or by 8% (v:v) SDS-polyacrylamide gels
containing 1.2 mg.mL-1 gelatin, for revealing enzymatic activity of (MMPs). Gels were
washed in renaturing buffer containing 65 mM Tris, (pH=7.38), 20% (v:v) glycerol for 45
min, twice. An acidic buffer (pH 6.0) containing 100 mM sodium phosphate buffer, 1 mM
EDTA and 2 mM dithiothreitol, was used to reveal the activity of CatK selectively. To reveal
the activity of MMPs, gels were incubated in buffer (pH 7.4) containing 50 mM Tris, 20 mM
NaCl and 5 mM CaCl2. After 30 min, these activity buffers were exchanged to fresh ones
containing 0.1% DMSO (v:v) without (control) or with CKI for 24 h of incubation at 37 °C.
Finally, gels were rinsed twice with deionized water before incubation for 1 h in a Coomassie
staining solution (2.5 mg mL-1 Coomassie blue), 45% (v:v) methanol, 10% (v:v) acetic acid)
for labeling the gelatin. This step was followed by a bath in distaining solution (30% (v:v)
methanol, 10% (v:v) acetic acid). The gels were scanned using a Canon scanner.
Disappearance of gelatin from gels, as evidenced by lack of coloration, indicated activity of
CatK or MMPs.
11

2.7 Fluorescence measurement for quantitative determination of CatK activity
Chondrocyte lysates were obtained by adding the same extraction buffer that was used for
zymography. CatK activity was detected at the cellular level according to the method of
Dolbeare and Smith (42) by means of the substrate Z-Gly-Pro-Arg-4-methoxy-β-
naphthylamide (Z-GPR-4MβNA, 1 mg mL-1). The hydrolysis of Z-GPR-4-MβNA by CatK
liberated 4-methoxy-β-naphthylamide (4MβNA). To quantify the CatK activity, 4MβNA was
precipitated and detected by 5-nitro-salicylaldehyde at 10 µM final concentration (Sigma-
Aldrich), which is an accumulating fluorescent product. The CatK activity was normalized to
the protein concentration [C] according to the equation:
(2)
where Fsc was the fluorescence of the control (containing the substrate and DMSO 0.1%
(v:v)), while Fsi was the fluorescence of the sample containing the substrate with or without
CKIs. Fluorescence was determined with a F-4500 fluorescence spectrophotometer (Thermo
Scientific, Germany) at least three times for each condition, with excitation and emission
wavelength of 488 and 525 nm (fluorescein setting), respectively. Protein concentration was
measured using Braford assay (43).
2.8 Alcian Blue staining
Chondrocyte phenotype was evaluated by measuring the extracellular sulfated
glycosaminoglycans (sGAG) deposition using Alcian blue staining. Briefly, the culture
medium was removed from culture plates, cells were gently washed once with phosphate-
buffered saline (PBS, 137 mM sodium chloride, 10 mM phosphate, 2.7 mM potassium
chloride; pH 7.4) and then fixed in 4% (v:v) of formaldehyde solution for 30 min at room
temperature (42). After fixation, cells were rinsed with PBS and stained for 30 min with 1%
Alcian blue (m:v) solution prepared in 0.1 N HCl. Finally, staining was dissolved with 33%
12

isopropanol in GnCl (4.0 mol/L) and the absorbance was determined by a microplate reader
(Thermo Scientific) at 595 nm. The blue staining indicated the presence of acidic sGAG,
markers of chondrocyte phenotype.
2.9 Data analyses and statistical analyses
All graphs and statistical analyses were performed using GraphPad Prism software. For each
graphic analysis, at least three independent experiments were performed. Non-parametric
Mann-Whitney test was used to analyze data. Results were expressed as mean  standard
error of the mean (SEM) or standard error (SD), as indicated. Results were considered
significant when p < 0.05 (*), highly significant when p < 0.01 (**), and extremely significant
when p < 0.001 (***).
3. RESULTS
3.1 Synthesis routes for compounds CKI-E and CKI-F.
We have previously reported the synthesis of several CKIs (19, 36). We reasoned that the
removal of amide linkage in CKI-8 and CKI-13 (Figure 2) would improve the selectivity due
to elimination of hydrogen bonds involving the amide group.
Figure 2: Structures of CKI-8 and CKI-13 (36).
13

The compound 1’ was produced by using the same procedures as formerly reported（19, 36).
Racemic compounds CKI-E and CKI-F were synthesized as shown in Figure 1. Both
13
products’ structures were validated by C/1H NMR and MS. In addition, our HPLC results
indicated that CKI-E and CKI-F compounds were highly pure (about 95% of purity).
3.2 In vitro affinity and selectivity of racemic CKI- E and racemic CKI-Ffor CatK
The concentrations of half maximal inhibition (IC50) were determined in vitro determining
CatK activity in the presence of increasing doses of CKI-E (Figure 3A) or CKI-F (Figure 4A).
In parallel, IC50 were assessed for CatL, CatS and CatB to evaluate the selectivity of CKI-E
(Figure 3B, 3C, 3D) and CKI-F (Figure 4B, 4C, 4D).
14

Figure 3. Determination of IC50 values of racemic compound CKI-E against CatK (A),
Cat L (B), Cat S (C) and Cat B (D). Experiences were realized at least 4 times
independently. Results are represented as mean ± SD.
Figure 4. Determination of IC50 values of racemic compound CKI-F against CatK (A),
Cat L (B), Cat S (C) and Cat B (D). Experiences were realized at least 4 times
independently. Results are represented as mean ± SD.
The low IC50 of 2.4 nM for CKI-E and of 15 nM for CKI-F indicated that both compounds
were strong inhibitors of human CatK. These values, by using equation (1), allowed to
calculate inhibition constants (Ki), which amounted to 1.1 nM for CKI-E and to 7.2 nM for
CKI-F, respectively (Table 1). Moreover, both CKI-E and CKI-F showed a substantial
15

selectivity for CatK over CatL, S and B. In the case of CKI-E, the Ki for other cathepsins were
at least 170-fold higher than for CatK, and 50-fold higher in the case of CKI-F (Table 1).
According to this enzymatic in vitro analysis, CKI-E seemed superior to CKI-F due to its
higher affinity for CatK and due to its better selectivity for CatK over the other three
enzymes, especially over CatL. Both CatK and L are endopeptidases with high sequence
homology (60% identity, 76% similarity), which made it very difficult to be inhibited
selectively. We suggest that the isobutyl group of CKI-E and CKI-F were not adequately
packed into the S2 pocket formed by Leu69, Met70, Ala135, Met161, Asp162, Gly164 and
Ala214 in CatL, explaining that CKI-E and CKI-F had higher affinity for CatK as compared
to that for CatL. Indeed, the bad orientation of CKI-E and CKI-F in the S2 pocket might be
attributed to the fact that cathepsin L preferred large aromatic group as the bi(hetero)aryl
group.
Table 1. Ki constants of two racemic aza-peptide nitrile inhibitors CKI-E and CKI-F for
different cathepsins: CatK, CatL, CatS and CatB.
Ki (nM) of enzymes
CatK
CatL
CatS
CatB
Compounds
1.14±0.03
7.21±0.16
195±25
406±54
239±12
735±37
909±102
CKI-E
3345±374
CKI-F
The only difference between the isomeric inhibitors CKI-E and CKI-F is the occurrence of the
meta- and para- biphenyl substructure, respectively, which changed the orientation of the P3
substituent relative to entire molecule. This dissimilarity can distinctly affect the orientation
of the compounds within the active site of the target protease.
16

3.3 Effects of inhibitors on the viability of MG63 and MOVAS cell lines and primary
chondrocytes
The cytotoxicity of the two racemic compounds CKI-E and CKI-F was tested on three distinct
cell types: human osteoblast-like cell line MG63 (Figure 5A), murine vascular smooth muscle
cell line MOVAS (Figure 5B) and murine primary chondrocytes (Figure 5C). MTT viability
assays were performed after 3 days incubation with or without inhibitors (control). The
addition of DMSO up to 0.1% (v:v), which was the solvent used to solubilize the inhibitors,
did not affect cell viability. Racemic CKI-E and CKI-F were not toxic in the concentration
range from 1 nM to 5 µM on the different type of cells tested (Figure 5).
Figure 5. Effects of inhibitors on the viability of MG63, MOVAS line cells and primary
chondrocytes. MG-63 (A) and MOVAS cell line (B) or murine primary chondrocytes (C)
were treated with increasing concentrations of CKI-E and CKI-F. Three independent
experiments were performed in triplicate Results are represented as mean ± SEM.
3.4 Up-regulated CatK activity in dedifferentiated chondrocytes
As expected, we observed that the morphology of chondrocytes changed with increasing
number of passages (Figure 6A). After P2 and P3, cells exhibited a fibroblastic elongated
morphology, as opposed to the characteristic paving pattern of well-differentiated
chondrocytes (P0). In addition, we observed by Alcian blue staining that, as soon as P1, the
17

deposition of glycosaminoglycans in the extracellular matrix dropped drastically (Figure 6B).
Because a high content in proteoglycans is a feature of cartilaginous matrix, this analysis
confirmed chondrocyte dedifferentiation in our model. Using fluorogenic substrate assays and
zymography, we measured CatK activity from chondrocytes at different passages (Figure 6C,
6D). We observed that relative activity of CatK rose from P0 to P1 and P2 and then slightly
decreased after P3. Fluorometric assays showed an average increase in relative CatK activity
of 5%, 40% and 20% after P1, P2 and P3 respectively, by comparison to P0 (Figure 6C). This
finding was further confirmed by zymography as the activity follows the same profile, with a
particularly sustained band at P2 (Figure 6D, asterisk). This result confirmed a gradual up-
regulation of CatK activity in passaged chondrocytes, especially at P2.
Figure 6. Characterization of isolated articular chondrocytes using monolayer
passaging. Morphology of chondrocytes with increasing number of passages (A). Alcian blue
18

staining in primary chondrocytes after P0, P1, P2 or P3 (B). Effect of primary chondrocyte
passages on CatK activity measured by fluorescence assay (C) and by zymography (D). Three
independent experiments were performed in triplicate. Results are represented as mean ±
SEM. Non-parametric Mann-Whitney test was performed to evaluate significant differences.
* p <0.05, **<0.01.
3.5 Effect of racemic CKI-E and racemic CKI-F inhibitors on CatK activity compared
to ODN
We compared the effects of ODN, racemic CKI-E and racemic CKI-F on the activity of CatK
in dedifferentiated primary murine chondrocytes at P2 as probed by fluorescence with Z-
GPR-4MβNA as substrate (Figure 7A) and by zymography (Figure 7B). CKI-E, CKI-F and
ODN at 0.1 and 1 µM inhibited significantly (p value <0.001) CatK activity in chondrocytes
(Figure 7A). We detected an average reduction of 25-35% for CKI-E and 30-40% for CKI-F,
while ODN induced a decrease of 19-30%. CKI-E and CKI-F inhibited CatK significantly
better than ODN treatment. Furthermore, CKI-F was significantly more efficient than CKI-E
to block CatK activity. This inhibition was confirmed by zymography, using gels pre-
incubated with ODN, CKI-E and CKI-F at 1 µM (Figure 7B). Both CKI-E and CKI-F
inhibitors were able to block the gelatinolytic activity of CatK from P2 chondrocytes, even
better than ODN. The addition of 0.1% (v:v) DMSO used to solubilize the inhibitors did not
affect the inhibition. At equal concentration, the efficiency of both CKI-E and CKI-F to block
CatK activity seemed superior to that of ODN.
19

Figure 7. Effect of inhibitors on CatK activity in dedifferentiated chondrocytes (passage
2). (A) Effect of 0.1 and 1 μM ODN, CKI-E, CKI-F on CatK activity in chondrocytes after P2
probed by fluorescence assay. (B) Effect of ODN, CKI-E and CKI-F at 1 µM on CatK activity
in primary chondrocytes after P0, P1, P2 and P3 as revealed by zymography. Three
independent experiments were performed in triplicate. Control corresponded to CatK activity
in chondrocytes without the presence of any inhibitors. Results are represented as mean ±
SEM. Non-parametric Mann-Whitney test was performed to evaluate significant differences.
* p<0.05, *** p <0.001.
20

3.6 Effect of inhibitors on other cartilage matrix proteases, such as aggrecanases and
MMPs
Cultured chondrocyte dedifferentiation as well as OA chondrocyte degeneration are
accompanied by an increase in matrix-degrading enzymes (44) such as aggrecanases or
MMPs. We wondered if our inhibitors could affect these proteases (Figure 8). We previously
observed by Alcian blue staining that the deposition of glycosaminoglycans in the
extracellular matrix dropped drastically after passages (Figure 6B). Here, we demonstrated
that addition of any of our CKIs at 1 µM did not affect glycosaminoglycans content in
chondrocytes at P0, P1 and P2 (Figure 8A), indicating that these inhibitors did not prevent the
aggregan loss, whether due to a decreased production or an increased degradation by
aggrecanase activity. We also checked if racemic CKI-E and racemic CKI-F can affect MMP-
2 and -9 activity by zymography. As expected, we observed that P2 induced the activity of
MMP-2 and -9 in dedifferentiated chondrocytes, in comparison with P0 (Figure 8B, control).
Pre-incubation of the gels with ODN, CKI-E and CKI-F at 1 µM, did not affect the MMP
gelatinolytic activity of P0 or P2 chondrocytes (Figure 8B, ODN, CKI-E, CKI-F). Of note, the
addition of 0.1% (v:v) DMSO used to solubilize the inhibitors did not affect the inhibition
either. Therefore, neither aggrecanases nor MMP-2 and -9 protease activities were affected by
ODN, CKI-E and CKI-F.
21

Figure 8. Effect of inhibitors on other cartilage matrix proteases, such as aggrecanases
and MMPs. (A) Alcian blue staining in primary chondrocytes after P0, P1 and P2 in the
presence or in the absence of CatK inhibitors at 1µM. Three independent experiments were
performed in triplicate. Results are represented as mean ± SEM. Non-parametric Mann-
Whitney test was performed to evaluate significant differences. * p<0.05, *** p <0.001. (B)
Gelatinase activity (MMP-2 and 9) with or without CatK inhibitors at 1 µM was determined
by zymography. Each experiment was realized in triplicate.
4. Discussion
22

From the first generation of inhibitors (19, 21, 22, 45), we synthetized two racemic azanitrile
compounds CKI-E and CKI-F, which were found to be highly selective for CatK over B, L
and S in vitro, with Ki amounting to 1.14 and 7.21 nM respectively. CKI-E and CKI-F
showed no toxicity on three distinct cell types, up to 5 µM, which is three orders of magnitude
greater than Ki. CKI-E and CKI-F at 0.1 and 1 µM significantly inhibited CatK activity in
chondrocytes, even better than the commercial CatK inhibitor ODN. It was earlier suggested
that the prevention of cartilage degradation by CatK inhibition may represent a valid strategy
for pharmacological intervention in OA (18). The experimental evidence accumulated in this
work supports the possibility to use CKIs for preventing cartilage degradation and the interest
of the CKIs for engineered cartilage therapy and OA treatment.
A large network of proteases can orchestrate cartilage degradation in OA. This network
includes not only CatK but also aggrecanases (46), responsible for aggregan degradation, and
MMPs (47), affecting collagen degradation in degenerative cartilage diseases as OA.
Targeting enzymes degrading cartilage has already been considered as a possible strategy to
treat OA. Inhibitors of aggrecanases (47, 48), ADAMTS (49) and MMPs (50) indicated strong
promises in preventing cartilage degradation. Among the gene-based methods already
developed, only a few addressed cartilage matrix proteases. The optimization of the knock-
down of MMP-3 and -13 by nanoparticles encoding shRNA has given promising results (51).
Moreover, the inhibition of aggrecanase-1 and -2 expression by a lentiviral delivery system
(52) or the knock-down of CatK in expanded chondrocytes (51) had similar valuable effects
on phenotype maintenance. The expression of chondrocyte markers collagen II and aggrecan
was increased and the modified cells formed a more homogenous matrix enriched in collagens
and proteoglycans (51, 53). Combined therapy with CKIs and other inhibitor of proteases
could be effective to treat degenerative cartilage diseases, especially OA.
23

5. Funding Statement
Yuqing Wu appreciates the financial support from the projects of NSFC (No. 21373101);
Xiaoyu Yuan and Zhongyuan Ren appreciate the supports from National Student Fund
Committee. The China-France cooperation project was funded by PHC Cai Yuanpei (Project
N° 32065TD).
24

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