From the Departments of Bone and Cartilage Biology,
¶ Medicinal Chemistry,
Protein Biochemistry, and
** Mechanistic Enzymology, SmithKline Beecham Pharmaceuticals, King of
Prussia, Pennsylvania 19406
Received for publication, November 27, 2000
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ABSTRACT |
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Cathepsins K and L are related cysteine proteases
that have been proposed to play important roles in osteoclast-mediated
bone resorption. To further examine the putative role of cathepsin L in
bone resorption, we have evaluated selective and potent inhibitors of
human cathepsin L and cathepsin K in an in vitro assay of
human osteoclastic resorption and an in situ assay of
osteoclast cathepsin activity. The potent selective cathepsin L
inhibitors (Ki = 0.0099, 0.034, and 0.27 nM) were inactive in both the in situ cytochemical assay (IC50 > 1 µM) and the
osteoclast-mediated bone resorption assay (IC50 > 300 nM). Conversely, the cathepsin K selective inhibitor was
potently active in both the cytochemical (IC50 = 63 nM) and resorption (IC50 = 71 nM)
assays. A recently reported dipeptide aldehyde with activity against
cathepsins L (Ki = 0.052 nM) and K
(Ki = 1.57 nM) was also active in both
assays (IC50 = 110 and 115 nM, respectively)
These data confirm that cathepsin K and not cathepsin L is the major protease responsible for human osteoclastic bone resorption.
Osteoclasts are multinucleated cells of hematopoietic origin that
are responsible for resorbing bone (1, 2). Following tight attachment
to the bone surface, osteoclasts begin the resorptive process by
secreting protons into the extracellular space (3), which results in
the removal of bone mineral and the exposure of the underlying matrix.
It also provides optimal conditions for the activity of
osteoclast-derived proteases that ultimately solubilize these matrix
components, which include type I collagen, osteopontin, osteonectin,
and other components. The identification of the key enzyme(s)
responsible for this protein matrix degradation is essential for
understanding the mechanisms of bone metabolism and for the design of
inhibitors of the resorptive process for the intervention of metabolic
bone diseases such as osteoporosis.
Numerous reports have suggested that cathepsin L and/or cathepsin K may
be the major proteases involved in this process (4-6). However, recent
studies have indicated that mRNA for cathepsin L is undetectable in
human osteoclasts (5), and the cathepsin L null mouse does not show a
bone-related phenotype (7). In contrast, it has been reported that the
knockout of cathepsin K results in osteopetrosis that is characterized
by osteosclerosis (8, 9). Both microcomputerized tomography and
histomorphometry indicate an increase in trabecular number and in
trabecular and cortical thickening in the knockout animals when
compared with their wild-type littermates (9). Furthermore, this
mutation leads to the elimination of all osteoclast-related cathepsin
activity and ultimately to a reduction in osteoclast-mediated matrix degradation.
The importance of cathepsin K in the resorptive process is further
supported by a rare skeletal disorder in humans called pyknodysostosis,
which occurs as a result of mutations in the cathepsin K gene,
resulting in elimination of cathepsin K activity (10). The disorder is
characterized by a reduction in the rate of bone turnover, which leads
to poor quality dense bone that is predisposed to fracture (11, 12).
Other skeletal manifestations of the disorder include a dysplasia in
the bones of the face and clavicle, abnormal tooth eruption, and a lack
of closure in the cranial sutures.
Several reports claim that potent and selective inhibitors of cathepsin
L are able to inhibit bone resorption both in vitro and
in vivo (4, 6). Similarly, several studies suggest that small molecule inhibitors of cathepsin K are also capable of inhibiting in vitro and in vivo bone resorption (13, 14). In
this study, we address this discrepancy and determine the relative
importance of cathepsins L and K in human osteoclast-mediated bone
resorption. In the course of a program directed at specific inhibition
of cathepsin K, we have discovered several inhibitors that are
selective for this enzyme, but we also have found selective inhibitors
of cathepsin L. We also have studied the inhibitor of Woo et
al. (4) that we find to be a potent inhibitor of both cathepsins L
and K. Nonselective inhibitors from our inhibitor collection have also
been studied. Our data suggest that cathepsin K and not cathepsin L is
the major protease responsible for human osteoclastic bone resorption.
Enzyme Assays--
Inhibition of human cathepsin K activity was
measured as previously described (13). Inhibition of cathepsin L was
measured under identical experimental conditions using the substrate
Z-Phe-Arg-aminomethylcoumarin. As appropriate, linear
portions of the initial velocity data from product progress curves were
analyzed to generate steady-state constants. A standard curve with AMC
was used in the conversion of fluorescence to molar units.
Tissue Processing--
Human osteoclastoma tissue (Jefferson
Hospital, Philadelphia, PA) was obtained (with informed consent) at the
time of surgery and frozen as described previously (15). Cryostat
sections (7 µm) were cut on a Hacker cryostat (Hacker Instruments,
Inc., Fairfield, NJ) equipped with a finely polished tungsten-tipped
steel knife and flash-dried onto glass 4-well slides.
In Situ Cytochemical Cathepsin Activity Assay--
Cryostat
sections of human osteoclastoma were assayed for cathepsin activity
using a slightly modified version of the azo-coupling procedure
described by Dodds et
al.1 Each section was
incubated in the presence or absence of inhibitors (0.01-1
µM) for 10 min at 37 °C in 150 µl of the following
reaction medium: 20% polypeptide (Sigma) in 0.1 M
phosphate buffer, pH 5.5, containing 2.5 mM EDTA and
7 mM substrate
Ac-Leu-Arg-4-methoxy- In Vitro Human Osteoclast Resorption Assay--
Human
osteoclastoma-derived osteoclasts were isolated as previously described
(17) and were used in an in vitro bone resorption assay
(18). Isolated osteoclast-enriched cell preparations were seeded onto
bovine cortical bone slices in the presence of compound (0.001-3
mM) or vehicle (dimethyl sulfoxide) for 48 h at
37 °C. Compounds were not tested at concentrations higher than 3 mM, because the vehicle is inhibitory at high
concentrations. The culture supernatants were harvested, and the levels
of the C-terminal peptide of the Compounds--
The compounds were synthesized in the Department
of Medicinal Chemistry (SmithKline Beecham). Details of the design and
synthesis of these compounds will be reported elsewhere. SB 412515, a
potent cathepsin L and cathepsin K inhibitor, was originally
synthesized and described by Woo et al. (4). SB 290190, a
potent and selective cathepsin K inhibitor, is described by Veber
et al. (20).
Enzyme Inhibitory Activity--
The human cathepsin L
inhibitors (SB 468420, SB 468432, and SB 468433 in Table
I) used in this study are very potent
inhibitors of this enzyme (Ki = 0.0099 ± 0.0003, 0.033 ± 0.006, and 0.272 ± 0.026 nM,
respectively) and demonstrate impressive selectivity compared with the
closely related cysteine protease, human cathepsin K (66- to
>50,000-fold selectivity). The dipeptide aldehyde SB 412515 is also a
potent cathepsin L inhibitor (Ki = 0.052 ± 0.002 nM), but it also shows activity against cathepsin K
(Ki = 1.57 ± 0.367 nM, Table I).
This compound was originally described as a potent cathepsin L
inhibitor (4), but no data were presented showing its activity against
human cathepsin K. In contrast, SB 290190 is a potent inhibitor of
cathepsin K (Ki = 0.050 ± 0.050 nM) and shows ~72-fold selectivity over cathepsin L
(Ki = 3.58 nM, Table I). The data in Table I are presented as Ki ± S.D.
Activity of the Inhibitors in an in Situ Cytochemical Assay of
Human Cathepsin Activity and an in Vitro Human Osteoclast Resorption
Assay--
To determine whether the cathepsin L selective inhibitors
could inhibit native osteoclast cathepsins within whole tissue sections of human osteoclastoma, the compounds were evaluated in an in situ cytochemical assay using the cathepsin K/L substrate,
Ac-Leu-Arg-4M- Conflicting data suggest that cathepsin L may play a pivotal role
in human osteoclast-mediated resorption. This study was designed to
determine whether potent and selective inhibitors of this protease can
indeed inhibit the resorptive process and to contrast them with potent
and selective cathepsin K inhibitors. The data presented here show a
lack of inhibition of both human osteoclast cathepsin activity and
resorption by these selective cathepsin L inhibitors. In contrast, the
compounds that show potent inhibition of cathepsin K (with some
cathepsin L inhibitory activity) potently inhibit cathepsin activity
and resorption in human osteoclasts. Taken together, these data
indicate that cathepsin K is the single most important protease in
human osteoclast-mediated bone resorption and that cathepsin L is not
likely to play a role in this process.
The pivotal role for cathepsin K in osteoclast-mediated bone matrix
degradation has been described using multiple approaches. Northern blot
(16, 21) and in situ hybridization (5) studies have
demonstrated that cathepsin K mRNA is abundant in osteoclasts. Immunocytochemical studies, using an anti-cathepsin K antibody, indicate that the enzyme is present in osteoclasts opposed to the bone
surface (5). Furthermore, the enzyme is activated as the cells approach
bone and distributed in a polarized fashion to the surface of the cell
that interacts with the bone surface. In contrast, in situ
hybridization studies have shown that mRNA for cathepsins L,
B, and S is undetectable in human osteoclasts (5). Mutations in the
cathepsin K gene also indicate the importance of cathepsin K in the
resorptive process. The cathepsin K knockout mouse (8, 9) and the human
disease, pyknodysostosis (10), are characterized by marked
osteopetrosis as a result of retarded osteoclastic bone resorption. In
contrast, a null mutation in the cathepsin L gene that resulted in mice
lacking cathepsin L mRNA and biological activity did not present
with bone abnormalities (7).
We have previously described the development and characterization
of a human in vitro resorption assay that uses
osteoclastoma-derived osteoclasts (18). This assay provides a robust
and reproducible system for the evaluation of inhibitors of
osteoclast-mediated bone resorption including the inhibitors of
cathepsin K. The potency of the cathepsin K compounds correlates
strongly with the activity against recombinant human cathepsin K enzyme
activity (r2 = 0.80). In addition to the
resorption assay, we recently described the development of a
quantitative cytochemical assay that measures cathepsin activity in
osteoclasts in sections of human tissue.1 Previous studies
using this assay on bone from cathepsin K knockout mice1
indicated that cathepsin K was the enzyme activity being measured within osteoclasts in this assay. Consequently, the cathepsin cytochemical assay provides confirmation of the activity of the cathepsin K inhibitors in the resorption assay and is an essential part
of our screen for potential cathepsin K inhibitors.
It has been reported previously by Woo et al. (4) that the
dipeptidyl aldehyde SB 412515 was a potent cathepsin L inhibitor that
could inhibit both in vitro and in vivo bone
resorption. Although this report describes the compound as a more
potent inhibitor for cathepsin L than other cysteine proteases, such as
cathepsin B and calpain II, its inhibition of cathepsin K was not
reported. Similarly, Yasuma et al. (6) reports the
preparation of a series of peptide aldehyde derivatives that
demonstrated potent activity against cathepsin L; data were not
reported for cathepsin K inhibition. One of these compounds inhibited
bone resorption in vitro and prevented bone loss in the
ovariectomized mouse (6). Our enzyme activity studies revealed
that SB 412515 had potent activity against human cathepsin K, as well
as cathepsin L, and the compound from the Yasuma study (6) is a potent
inhibitor of both human and mouse cathepsin K (data not shown). The
lack of activity of the cathepsin L selective compounds confirm that it
is the potent cathepsin K activity of SB 412515 and SB 290190 that
explains their potent antiresorptive activity.
In conclusion, we demonstrate using two distinct in
vitro assays of human osteoclast function that cathepsin
L selective inhibitors are unable to inhibit human
osteoclast cathepsin activity, and this results in a lack of inhibition
of osteoclast-mediated bone resorption. In contrast, potent inhibitors
of cathepsin K reproducibly inhibited osteoclast function thus
providing unequivocal evidence that it is cathepsin K and not cathepsin
L that plays the pivotal role in osteoclast-mediated matrix degradation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-naphthylamide. The medium was removed,
and the sections were postcoupled with 0.25 mg/ml Fast Blue B Base
(Sigma) for 10 min at room temperature. The sections were then rinsed
in phosphate-buffered saline and finally incubated for 10 min at
room temperature with 100 mM CuSO4. The
reaction product was measured on a per cell basis (× 40 objective at
550 nm) in a minimum of 10 osteoclasts in duplicate sections of
osteoclastoma using a Vickers M85 scanning and integrating microdensitometer as described previously.1 Results are
presented as IC50 for individual inhibitors as calculated from the resulting dose response curves.
-1 chain of human type I collagen
were quantified with a biochemical readout of resorption using a second
generation one-step enzyme-linked immunosorbent assay (Osteometer
Biotech A/S, Herlev, Denmark). This is a modification of the
original competitive assay described by Foged et al. (19).
The results are expressed as percent inhibition of resorption. The
IC50 values were determined from the resultant dose
response curves.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Enzyme inhibitory activity of the cathepsin inhibitors
-naphthylamide. Using this assay none of the cathepsin
L selective compounds at concentrations of up to 1 µM
inhibited enzyme activity (Table II). The
selective cathepsin L inhibitors also failed to decrease bone
resorption at all concentrations tested (0.001-300 nM,
Table II), even though their potency against cathepsin L ranged from Ki = 0.097 nM to Ki
=1.7 nM (Table I). In contrast, SB 290190, a potent
and selective cathepsin K inhibitor (Ki = 0.050 ± 0.005 nM), showed potent inhibition of osteoclast
cathepsin activity in the cytochemical assay (Fig.
1, IC50 = 63 ± 4 nM), and this activity translated into potent inhibition of
bone resorptive activity in the in vitro resorption assay
(Fig. 1, IC50 = 71 ± 25 nM, Table II, and
Fig. 2). Significantly, SB 412515, a
compound that had been reported previously as a selective inhibitor of cathepsin L (4), also demonstrated potent activity in both the in
situ cytochemical assay (IC50 = 110 ± 5 nM) and the in vitro resorption assay
(IC50 = 115 ± 40 nM). Subsequent
measurement of the enzyme inhibitory activity of this compound revealed
that it is a potent inhibitor of both human cathepsins K and L. Together these data indicate that inhibition of cathepsin K is required for the inhibition of bone resorption in vitro. Potent
inhibition of cathepsin L without cathepsin K inhibition has no effect
on this process.
Potent and selective cathepsin L inhibitors do not block cathepsin
activity or in vitro resorption by human osteoclasts
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Fig. 1.
The cathepsin L selective compounds are
inactive in both the resorption assay (A) and the in
situ cytochemical assay (data not shown). SB 290190, a
potent and selective inhibitor of cathepsin K, inhibits both in
situ cathepsin activity and osteoclastic resorption with similar
potency. The compound was tested in the human osteoclast resorption
(n = 3) (B) and cathepsin cytochemical
assays (n = 4) (C). The data are presented
as percent inhibition ± S.E.
View larger version (12K):
[in a new window]
Fig. 2.
SB 412515, a potent dual cathepsin L and
cathepsin K inhibitor, restricts in vitro human
osteoclast-mediated resorption and in situ cathepsin
activity. The compound was tested in the human osteoclast
resorption (n = 4) (A) and cathepsin
cytochemical assays (n = 4) (B). The
compound was active in both assays and showed similar potency. The data
are presented as percent inhibition ± S.E.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Dr. Richard Lackman of the Jefferson Hospital (Philadelphia, PA) for supplying the osteoclastoma tissues.
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FOOTNOTES |
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* This work was funded by SmithKline Beecham Pharmaceuticals.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd., P. O. Box 1539, King of Prussia, PA 19406. Tel.: 610-270-6257; Fax: 610-270-5598; E-mail: ian_e_james@sbphrd.com.
Published, JBC Papers in Press, January 8, 2001, DOI 10.1074/jbc.M010684200
1 Dodds, R. A., James, I. E., Rieman, D., Ahern, R., Hwang, S. M., Connor, J. R., Thompson, S. D., Veber, D. F., Drake, F. H., Holmes, S., Lark, M. W., and Gowen, M. (2001) J. Bone Miner. Res. 16, 478-486.
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