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Using inhibitors of metalloproteinases to treat arthritis. Easier said than done

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ARTHRITIS & RHEUMATISM Volume 37
Number 8, August 1994, pp 1115-1126
8 1994, American College of Rheumatology
Arthritis & Rheumatism
Official Journal of the American College of Rheumatology
REVIEW
USING INHIBITORS OF METALLOPROTEINASES TO
TREAT ARTHRITIS
Easier Said Than Done?
MATTHEW P. VINCENTI, IAN M. CLARK, and CONSTANCE E. BRINCKERHOFF
Introduction
Irreparable degradation of the extracellular matrix is a hallmark of rheumatic disease, and it is now
well documented that much of this degradation occurs
through the enzymatic action of proteinases on the
various components of the extracellular matrix (for
review, see refs. 1 and 2). Although each of the 4
classes of proteinases found in mammalian cells
(serine, cysteine, aspartic, and metallo-) contributes to
matrix destruction, the majority of the degradation is
mediated by metalloproteinases. These enzymes are
termed matrix metalloproteinases (MMPs) and comprise a multigene family of at least 10 members (for
review, see refs. 3-6). All are active at neutral pH,
have zinc at their active sites, and require Cat+ for
activity. They are synthesized and secreted in a latent
proenzyme form, and activation is accompanied by
proteolytic cleavage of a propeptide domain at the
N-terminus of the molecule.
Dr. Brinckerhoffs work was supported by NIH grant
AR-26599, and by grants from the Council for Tobacco Research
and the RGK Foundation (Austin, TX). Dr. Vincenti’s work was
supported by an NIH postdoctoral fellowship (NSRA-AR-08216).
Dr. Clark’s work was supported by an ARC Copeman Fellowship
(UW.
Matthew P. Vincenti, PhD: Dartmouth Medical School,
Hanover, New Hampshire; Ian M. Clark, PhD: Dartmouth Medical
School (currently at Addenbrooke’s Hospital, Cambridge, UK);
Constance E. Brinckerhoff, PhD: Dartmouth Medical School.
Address reprint requests to Constance E. Brinckerhoff,
PhD, Dartmouth-Hitchcock Medical Center, Dartmouth Medical
School, Hanover, NH 03755-3833.
Submitted for publication January 6, 1994; accepted in
revised form March 2, 1994.
MMPs can be grouped into 3 main classes
(Table 1). Group 1 includes MMP-1 (interstitial) and
MMP-8 (neutrophil) collagenase, whose major substrates are collagen types I, 11, and 111. Group 2
contains the gelatinasedtype IV collagenases. These
are the 72-kd gelatinase A (MMP-2) and the 92-kd
gelatinase B (MMP-9), which degrade gelatin and type
IV collagen in basement membrane. The third group is
made up of the stromelysins: stromelysin 1 (MMP-3),
stromelysin 2 (MMP-lo), and pump-1 (MMP-7). The
stromelysins are active against a broad spectrum of
substrates, e.g., proteoglycans, laminin, fibronectin,
and some collagens.
In arthritic disease, collagenase (MMP-1) and
stromelysin I (MMP-3) play an important role (1,2).
Normal fibroblasts produce very low levels of both
enzymes (1, 2, 7, and refs. therein). In both rheumatoid arthritis (RA) and osteoarthritis (OA), however,
levels increase markedly in response to a variety of
stimuli (1,2,7). Cytokines such as interleukin-la (ILla) and IL-lp, epidermal growth factor (EGF), plateletderived growth factor, and tumor necrosis factor a are
all potent inducers of both collagenase and stromelysin, as are crystals of monosodium urate monohydrate, phagocytosis of debris, and formation of
multinucleated giant cells. In populations of stimulated
cells, such as those found in proliferating rheumatoid
synovial tissue, collagenase and stromelysin become
major gene products of the tissue and may comprise as
much as 2% of the messenger RNAs (mRNAs) produced by the synovial fibroblasts (7). The rise in
1115
1116
VINCENT1 ET AL
Table 1. Matrix metalloproteinases (MMPs)
Enzyme
Group 1
Interstitial collagenase
(MMP-I)
Neutrophil collagenase
(MMP-8)
Group 2
72-kd gelatinase A
(MMP-2)
92-kd gelatinase B
(MMP-9)
Group 3
Stromelysin 1
(MMP-3)
Stromelysin 2
(MMP-10)
Pump-1 (MMP-7)
Matrix substrate
Collagen types I, 11, 111, VII, and X;
gelatin
Collagen types I, 11, and I11
Gelatins I, 11, and 111; collagen types
IV, V, VII, and X; fibronectin;
elas tin
Gelatins I and V; collagen types IV
and V
Proteoglycan; fibronectin; laminin;
gelatins I, 111, IV, and V; collagen
types 111, IV, V, and IX;
procollagen propeptides; activates
procollagenase
Gelatins I,-III, IV, and V; collagen
types 111, IV, and V (low specific
activity); fibronectin; activates
procollagenase
Gelatins I, 111, IV, and V; proteoglycan;
fibronectin; activates procollagenase
mRNA is paralleled by a rise in secreted protein. For
example, after addition of an inducer such as urate
crystals, collagenase mRNA levels increase within 6
hours, and this is followed shortly by an increase in
collagenase protein (7). This protein is not stored
within the cells, and the time required for the synthesis
and secretion is approximately 45 minutes (8).
Increased amounts of collagenase and stromelysin are present in cartilage both from patients with
RA and from patients with OA, and the level of
enzyme activity correlates with the severity of the
lesion (9,lO). Similarly, synovial fluid (SF) from patients with these diseases, especially those with RA
(11,12), shows an increase in both enzymes. More
recently, the use of in situ hybridization techniques to
demonstrate the presence of mRNAs for these enzymes in arthritic tissue has provided further evidence
of their role in the pathophysiology of these diseases
(13-15).
Because collagenase and stromelysin play such
a fundamental role in the pathophysiology of rheumatic disease, and because the connective tissue destruction they cause is largely irreversible, their inhibition would seem to be of utmost importance in
designing effective therapeutic strategies. However,
only a few of the established treatments currently used
in arthritis are thought to influence levels of enzyme.
These include D-penicillamine, which, as a chelator of
divalent cations (16), has the theoretical potential to
inhibit enzyme activity, and the glucocorticoid hormones, which decrease enzyme synthesis (1,2,6,7).
Thus, there are 2 principal ways to decrease levels of
metalloproteinases: inhibition of enzyme activity or
inhibition of enzyme synthesis. While considerable
success has been achieved with both modalities in
experimental systems, the successful application of
either to the actual treatment of clinical disease has
been more difficult.
Inhibition of enzyme activity
Let us first consider the inhibition of enzyme
activity. There are several naturally occurring inhibitors of activity, the most prominent of which is a2macroglobulin (aZM), a large (750-kd) protein produced by the liver. It is found in normal serum and in
the serum and SF of patients with RA and OA and can
inhibit all 4 classes of enzymes, not just metalloproteinases (17). It functions by means of a “bait” region
that can be cleaved by the enzyme. This then leads to
a conformational change in the a2M, which “traps”
the proteinase and sterically blocks access to protein
substrates (18,19). In some destructive arthritides, the
level of inhibitors in the SF can be exceeded by the
level of active enzymes, thus permitting matrix degradation to proceed. For example, in SF from patients
with untreated septic arthritis, the level of proteinases
from invading neutrophils exceeds the level of inhibitor. This leads to the presence of active metalloproteinases in the SF, accompanied by rapid tissue destruction. Antibiotic treatment cures the sepsis and leads to
a reduction in neutrophils. This results in a return of
excess inhibitor, and tissue destruction is halted (20).
However, the large size of the a2M may exclude it
from many sites of connective tissue turnover in the
joint, e.g., deep within the cartilage, and this may
decrease its effectiveness as an inhibitor.
There are inhibitors that are specific for metalloproteinases. These are produced locally by chondrocytes and synovial fibroblasts and are called tissue
inhibitors of metalloproteinases (TIMP). TIMP-1 is a
glycoprotein with an M , of -28,000 and contains 6
disulfide bonds which constrain the molecule into 2
major domains (21,22). A recombinant protein containing only the N-terminal domain of TIMP-I still has
inhibitory activity, though the mechanism for inhibition is not yet known (23). TIMP-2 shares -40%
sequence identity with TIMP-1, with retention of the 6
disulfide bonds and 2-domain structure. TIMP-2 is not
METALLOPROTEINASE INHIBITORS IN ARTHRITIS TREATMENT
glycosylated, however, and has an M , of -22,000 (24).
Both inhibitors bind specifically with the active matrix
metalloproteinases to form 1: 1, noncovalent, but tightbinding complexes which inactivate the enzymes. In
addition, TIMP-1 can form a bimolecular complex
with the latent form of the 92-kd gelatinase (MMP-9),
while TIMP-2 can form a similar complex with latent
72-kd gelatinase (MMP-2) (25,26). Some samples of SF
from patients with RA and OA contain complexes of
TIMP-2 and the latent form of the 72-kd gelatinase,
although the role of this complex in the disease process is presently unclear (27).
A further member of the TIMP family, TIMP-3,
has recently been described in chickens, where it is
localized in the extracellular matrix. The mouse and
human equivalents of TIMP-3 have been cloned, but
their physiologic role is not yet known (28). The facts
that TIMPs are 1) produced locally by the same cells
that make collagenase and stromelysin and are 2)
specific inhibitors of metalloproteinases suggest that
they may play an important role in dampening connective tissue degradation. The question is whether this
role can be exploited for therapeutic purposes.
It is thought that much of the connective tissue
destruction seen in OA and RA may be due to a local
imbalance between activated matrix metalloproteinases and TIMPs. Extracts of cartilage from both OA
patients and RA patients show an increase in metalloproteinase activity over TIMP activity in macroscopic
lesions compared with the surrounding “normal” tissue (10). Two recent studies have examined the level
of TIMP mRNA in the synovium of RA and OA
patients by in situ hybridization (14,15). The results
suggested that the overall level of TIMP gene expression was similar in the 2 diseases, but there was a
relative decrease in TIMP compared with proteinase
levels in highly inflammatory RA. This is probably due
to increased production of metalloproteinases in conjunction with a constant level of TIMP production.
Experiments in which exogenous TIMPs were
administered in order to ablate connective tissue destruction have had mixed results. Interestingly, daily
intraperitoneal administration of TIMP-1 at a dose of
2 mg in a mouse model of type I1 collagen-induced
arthritis significantly reduced the seventy of disease
relative to untreated controls (29). However, with
such a route of administration, it is uncertain whether
there was any increase in TIMP concentration in the
joints of the treated animals, and hence whether this
was responsible for the improvements noted. In other
experiments, the addition of exogenous TIMP-1 or
1117
TIMP-2 to resorbing cartilage in vitro inhibited the
release of collagen but did not prevent glycosaminoglycan release (Cawston TE: personal communication). This may reflect diaculties in the ability of the
TIMP to penetrate into the cartilage prior to the
removal of proteoglycan, since synthetic, low molecular weight inhibitors of matrix metalloproteinases can
decrease the release of both glycosaminoglycan and
collagen (see below). Perhaps the breakdown of collagen represents the irreversible step in cartilage degradation. If a network of collagen exists, glycosaminoglycan can be replaced. However, once the collagen is
compromised, destruction of the cartilage is inevitable. Thus, it is uncertain whether administration of
exogenous TIMPs, per se, will be useful therapeutically. Nonetheless, a thorough understanding of their
mechanism of action may well enable us to design new
synthetic inhibitors with such potential.
An alternative approach is the reduction of
connective tissue destruction by increasing local production of TIMPs. Indeed, there are a number of
agents that increase TIMP-1 mRNA and protein, and
these agents either decrease or do not affect expression of metalloproteinase genes. They include alltrans-retinoic acid and synthetic vitamin A analogs
(retinoids) and several cytokines, including transforming growth factor P (TGFP), IL-6, IL-11, leukemia
inhibitory factor (LIF), and oncostatin M. All of these
compounds have multiple effects on cells. All-transretinoic acid increases TIMP-1 gene expression in
fibroblasts derived from a variety of sources (30-32).
TGFP can generally be considered an anabolic factor
in the joint. It increases the level of TIMP-1 in monolayer cell culture, either alone (31,33) or in combination with other growth factors (34). It can also prevent
release of glycosaminoglycan from explants of articular cartilage stimulated with IL-1 (35).
IL-6, IL-11, LIF, and oncostatin M share many
activities. All affect hematopoietic cells and all induce
the release of acute-phase proteins from hepatocytes.
In addition, IL-6 increases TIMP production from
synovial fibroblasts and chondrocytes in vitro. It is
present in high levels in inflammatory conditions
(36,37) and is increased in SF of patients with RA
(38,39). IL-11 has also been found in SF from RA
patients (40), and it, too, can be induced in articular
chondrocytes and synovial fibroblasts. Many of its
biologic effects are similar to those of IL-6, including
induction of TIMP production. Recently, LIF and
oncostatin M have been added to the list of cytokines
that increase TIMP, and since all of these molecules
.
VINCENT1 ET AL
1118
CMT-3
OH
Figure 1. The tetracycline molecule and its chemical derivatives.
The tetracycline molecule, with the indicated chemical changes for
the various chemically modified tetracycline (CMT) molecules, is
depicted. Hatched area depicts addition; shaded areas depict deletions. (Modified, with permission, from Critical Reviews in Oral
Biology and Medicine [a].)
utilize the signaling molecule gp130, a common pathway may be involved (41). They also share significant
similarities in amino acid sequence, predicted secondary structure, and exon organization of their genes,
suggesting a biologic relationship among them (42).
Thus, numerous cytokines increase expression
of TIMP-1 mRNA, and although this increase is at
least partially transcriptional, details on the exact
mechanism(s) are lacking. The issue then becomes
how to use any or all of these cytokines to specifically
up-regulate TIMP expression in arthritic joints and still
avoid some of their other, more generalized effects.
This problem has been successfully addressed with the
specific localized application of TGFP to repair retinal
lesions (43), and the similar localized nature of synovial lesions may mean that targeted, local induction of
TIMPs is a viable option for the future.
Several chemotherapeutic agents, antibiotics,
and synthetic peptides can also inhibit the activity of
MMPs. Of the antibiotics, tetracycline and its related
compounds show some promise for clinical efficacy.
Although several synthetic peptides are potent inhibitors of MMPs in vitro, their biostability currently
limits their usefulness in treating arthritis.
Tetracycline (Figure l), and its semisynthetic
forms doxycycline and minocycline, have potent MMP
inhibitory properties which are independent of their
antimicrobial activity (44). Several studies have demonstrated that tetracyclines can inhibit interstitial collagenase (MMP-1) both in vitro and in vivo, as well as
the 72-kd gelatinase (MMP-2) and macrophage elastase
(MMP-12) in vitro. The 50% inhibition concentrations
(1C5,) of doxycycline, minocycline, and tetracycline for
the inhibition of purified interstitial collagenase are
15 p M , 190 p M , and 350 pM, respectively (45). Given
the greater inhibitory activity of the semisynthetic
tetracyclines compared with the parent compound,
these compounds will probably be more useful in
disease intervention.
In addition to doxycycline and minocycline,
other derivatives which have no antimicrobial activity,
but which still inhibit collagenase, have been synthesized (Figure 1). There are at least 5 chemically modified
tetracycline (CMT) compounds that have side chain
additions andor deletions from the 4-ring structure of the
parent molecule (44). CMT-5 is missing the carbonyl
oxygen from carbon 11 of the C ring and the hydroxyl
from carbon 12 of the B ring and does not inhibit
collagenase, suggesting that these side chains are necessary for chelation of Zn++ ions. However, the actual
interaction of these groups with zinc ions of collagenase
has not been directly demonstrated.
The anthracycline antibiotics, which are structurally similar to tetracyclines, inhibit type 1V collagenase activity purified from Walker 256 carcinoma (46). In
addition, the antineoplastic anthracycline drugs daunorubicin, doxorubicin, and epirubicin inhibit basement
membrane collagedegrading activity in a reversible
and noncompetitive manner (47). The IC,, for these
compounds ranges from 90 pM to 37 pM, which is
equivalent to concentrations of these drugs used clinically, and comparable with the IC,, values reported for
the tetracycline analogs. Interestingly, the 3 anthracyclines mentioned above contain 4 rings like the tetracycline molecule, and possess the carbon 11 carbonyl
oxygen and carbon 12 hydroxyl groups, which are required for activity in the tetracycline derivatives. With
their structural similarity to tetracycline and their noncompetitive mode of action, these compounds probably
function like tetracycline, by chelating metal ions.
Given the efficacy of tetracyclines in reducing
collagenolysis in vitro and in vivo (48) and in preventing degradation of type XI collagen from osteoarthritic
collagen in vitro (49), it has been suggested that these
drugs may be useful in the treatment of arthritis.
Unfortunately, recent data from animal models and
clinical trials have not been encouraging. Greenwald
and colleagues (50) tested the ability of tetracyclines to
inhibit joint destruction in the rat model of adjuvantinduced arthritis. Treatment of rats with minocycline,
doxycycline, or CMT did not significantly decrease the
amount of joint swelling or radiologically detectable
bone and joint damage. This was surprising since
collagenase and gelatinase activity was reduced in
extracts from arthritic paws of treated animals. The
1119
METALLOPROTEINASE INHIBITORS IN ARTHRITIS TREATMENT
authors did find that the combination of tetracyclines
with the nonsteroidal antiinflammatory drug (NSAID)
flurbiprofen was more effective at suppressing metalloproteinase activity and radiographic joint damage
than was either drug alone.
The use of tetracyclines to treat patients with
rheumatic disease has resulted in mixed findings. In an
unblinded and uncontrolled study (5l), minocycline
given in conjunction with existing NSAID regimens
improved a variety of clinical parameters during treatment. However, an earlier double-blind study indicated that, compared with placebo, tetracycline did
not significantly improve standard RA evaluation parameters (52). Perhaps the ineffectiveness of tetracycline
in that study was due to its low inhibitory activity
relative to the semisynthetic forms. It seems that
additional, well-controlled studies using the tetracycline derivatives are needed to determine whether
these drugs have therapeutic value in arthritis.
Considerable effort is also being devoted to the
development of synthetic peptides that are designed to
specifically inhibit metalloproteinases. Although this is
a laudable goal, the task is formidable since developing
an enzyme inhibitor to be used as a drug is a complex
process. Issues of bioavailability , circulation through
the body and successful delivery to the target tissue(s),
and effective concentration are all of paramount importance (53). Furthermore, the inhibitor should not
be immunogenic, and since many if not all of the
inhibitors designed to date are small peptides, antigenicity is a possibility. On the other hand, the relatively
small size of many synthetic peptides means that they
are subject to rapid hydrolysis in the gut, in the blood,
and in tissues. Many are often poorly absorbed from
the gastrointestinal tract, and when they do enter the
circulation, they are rapidly cleared by the liver and
kidneys (53). Some of the problems associated with
proteolytic breakdown may be overcome by protecting the amino and carboxy termini and/or by cyclization, which protects against exo- and endopeptidase
attack.
The largest group of synthetic peptide inhibitors
are the collagen substrate analogs (for review, see ref.
53). All are generally <6 amino acids long. Up to 3 of
the amino acid residues are found on either side of the
collagen scissile bond, and the peptide is linked to a
Zn++-binding moiety (Figure 2). The left-hand side
inhibitors contain amino acid residues N-terminal to
the collagen cleavage site with the Zn++-binding molecule at the C-terminus of the peptide. Right-hand side
inhibitors contain amino acids C-terminal to the colla-
Scissile
Bond
I
Giyclne-
-Giutamlne-Glyclne
Prollne
P3
P2
I
-Alanine -Giyclne
%oleucine
P1
P1
P2
P3
Left Hand Side Inhibitors:
P3
Zinc
- -P1-Blndlng
Group
p2
Right Hand Side inhibitors:
- P2
P’3
- -
P3
Zlnc
BlndlngGroup
P’1
Zlnc
BlndlngGroup
P’1
Left and Right Hand Side Inhibitors:
P3
- P2
PI-
~ ‘ 2
Figure 2. The a1 type I collagen cleavage site. The amino acid
sequence around the scissile bond of the bovine and human a1 type
I collagen molecule is shown. The “P” sites indicate homologous
amino acids used in the design of metalloproteinase inhibitors. The
relative positions of homologous amino acid residues and zincbinding moieties are shown for the left-hand side, right-hand side,
and left- and right-hand side inhibitors. (Modified, with permission,
from Osteoarthritis: Current Research and Perspectives fur Pharmacological Intervention [53].)
gen cleavage site, with the Zn++-binding molecule at
the N-terminus of the peptide. Inhibitors that are both
left- and right-handed also exist. In these compounds,
the zinc-binding group is centered between peptides
corresponding to sequences on either side of the
collagen cleavage site. A variety of Zn++-chelating
moieties have been used, among them hydroxamic
acid, thiols, and carboxyalkyl groups (Figure 3). Current theory suggests that these compounds act by
binding to the active site of the collagenase molecule,
interacting with the necessary zinc molecule and
thereby inactivating the enzyme.
When tested in vitro, both in cell-free and in cell
culture systems, the hydroxamate-based compounds
are generally considered to be the most potent inhibitors (Figure 4). The carboxyalkyl and hydroxamate
derivatives have a significantly lower IC,, for strome-
VINCENT1 ET AL
1120
NCH
-cH3
CH2
0
I
GlY
II
tors must enter the cartilage. This means they must
penetrate the dense network of collagen and proteoglycan aggregates. These aggregates are highly negatively charged and this, in turn, may further impede
entry of an inhibitor. Indeed, testing of a hydroxamic
acid peptide collagenase inhibitor on explants of rabbit
articular cartilage revealed significant inhibition of
matrix degradation only in the presence of 10-1,000
times more enzyme than what was needed to inhibit
purified collagenase in a cell-free in vitro system.
Since the ICs0 for many inhibitors ranges from 10 nM
to -400 nM (53), this becomes a serious problem.
Despite these difficulties, some success has
been achieved in delivering inhibitors of activity to
localized sites. DiMartino et a1 showed that one such
inhibitor (BB16; ICso 5 nM against collagenase, 40 nM
against stromelysin), when administered intraperitoneally, moderately decreased the degree of soft tissue
swelling, the extent of the inflammatory process, and
the severity of bone and cartilage loss in the rat
adjuvant-induced arthritis model (58). Davies et al (59)
established ovarian carcinoma ascites in nude mice
and then decreased the tumor burden in these mice
with intraperitoneal injections of a hydroxamate-based
inhibitor. The inhibitor used in this study, BB94, has
an ICs0 of 3 nM for interstitial collagenase, 20 nM for
stromelysin, 4 nMfor the 72-kd gelatinase A (MMP-2),
and 1-10 nMfor the 92-kd gelatinase B (MMP-9). Even
though the inhibitor displayed inhibitory activity
against several metalloproteinases, the authors proposed that inhibition of the 92-kd gelatinase was
responsible for the increased survival seen in treated
mice. However, no direct evidence of decreased MMP
-
Zinc Binding Group (R)
-SH (THIOL)
-CH2CONHOH (HYDROXAMIC ACID)
-CH2COOH
(CARBOXYALKYL)
Figure 3. Structure of the right-hand side inhibitors. The structure
of a representative right-hand side peptide analog inhibitor is shown.
The “P” sites indicate homologous amino acids used in the design of
metalloproteinase inhibitors; the circled “R” indicates the position
of zinc-binding groups, such as thiols, hydroxamates, and carboxyalkyls. (Modified, with permission, from Osteoarthritis: Currenf
Research and Perspectives for Pharmacological Intervention [53].)
lysin than for collagenase (53). This is surprising since
the inhibitors were designed to mimic the type I
collagen scissile bond. However, this fact may explain
the ability of these compounds to inhibit collagen as
well as proteoglycan breakdown in in vitro cartilage
degradation assays (54-56). Since activation of latent
collagenase is potentiated by stromelysin ( 3 4 the net
inhibition of collagenase activity may have an even
better therapeutic effect. The thiol-based inhibitors are
less potent peptide analogs than the carboxyalkyls and
hydroxamates and work equally well for stromelysin
and collagenase (53). For these compounds, sequence
around the cleavage site does not appear to play as
large a role in function. In fact, thiol-based tripeptide
inhibitors are considerably more potent if the second
amino acid on the carboxy-terminal side of the cleavage site has an aromatic side chain and the third amino
acid is an alanine (57). It may be that these compounds
become better Zn++ chelators by taking advantage of
hydrophobic pockets near the enzyme’s active site.
Perhaps the greatest hurdle is that of achieving
therapeutic concentrations in vivo. To effectively decrease cartilage breakdown, metalloproteinase inhibi-
Leu (P’l)
CH3
Hydroxamate
I
A18 (P3)
Leu
CH
-
CH3
CH3
Figure 4. Complete structure of a representative hydroxamate-base
peptide analog inhibitor. The “P’sites indicate homologous amino
acids used in the design of metalloproteinase inhibitors. (Modified,
with permission, from Osteoarthritis: Current Research and Perspectives for Pharmacological Intervention [531.)
METALLOPROTEINASE INHIBITORS IN ARTHRITIS TREATMENT
activity in situ was presented. It is not clear if intravenous or oral administration of these drugs would be
as effective.
An alternative approach to the design of MMP
inhibitors has been to use peptides that mimic sequences in the conserved region of the propeptide.
Metalloproteinases are secreted as latent proenzyme
forms which must be proteolytically cleaved before
they are fully active. This can take place by means of
autoproteolysis, as in the case of the 72-kd gelatinase
(26), or proteolysis mediated by another enzyme, as in
the activation of interstitial collagenase by kallikrein
or stromelysin (3,5,6). The region of the proenzyme
that is removed, or prosegment, contains a cysteinyl
residue, which can complex with the Zn++atom of the
active site. This association inhibits cleavage by the
activating enzyme and helps maintain the latent form.
Recently, synthetic peptides that are homologous to
the cysteinyl-containing region of the prosegment have
been constructed. These peptides can inhibit the 72-kd
gelatinase (60) and can prevent tumor cell invasion in
an in vitro assay (61). However, concentrations as
high as 30 pM were needed to achieve an 80% maximal
level of inhibition of invasion. Given the biologic
instability of small peptides, it is likely that the therapeutic potential of such inhibitors will be limited
unless they can be chemically modified.
Inhibition of enzyme synthesis
Now let us focus on the other means of decreasing MMP levels: decreasing the actual synthesis of
these proteins. As with inhibitors of activity, there are
both naturally occurring and synthetic inhibitors of
synthesis. Naturally occurring compounds are TGFp
and all-trans-retinoic acid (33, 62, and references
therein). Synthetic compounds also include the synthetic derivatives of retinoic acid (the retinoids) (7,62)
and glucocorticoid hormones such as dexamethasone
(1,6,62). All 3 classes of compounds appear to suppress MMP synthesis by suppressing transcription, although posttranscriptional effects cannot be ruled out.
We are just beginning to understand the transcriptional mechanisms that control MMP gene expression. In general, transcription is activated by the
presence of a core of proteins (transcription initiation
factors) that bind to defined sequences known as the
TATA box in the promoter region of genes (63-65).
Transcription is further influenced by “enhancers,”
sequences of DNA that can be located anywhere
throughout the gene and that augment transcription
1121
(64-66). One enhancer element, the %basepair sequence 5‘-TGAGTCAC-3’,is located approximately at
position -77 in the promoters of collagenase and
stromelysin (67-71). This sequence is called the activator protein-1 or AP-1 site, and it binds the transcription factors fos and jun (69). Although the AP-1 site is
important in the transcriptional activation of the collagenase and stromelysin genes, additional sequences
located upstream in the promoters are also important,
and may even be necessary (70,71). Some of these
upstream sequences may function independently of
the AP-1 site, while others may act in cooperation with
it (70,72,73). Thus, interaction of multiple elements
within the promoter appears to be essential for transcriptional control, and may provide a mechanism for
regulation of metalloproteinase gene expression in response to a variety of stimuli under a variety of normal
and pathologic conditions.
Repression of metalloproteinase synthesis by
TGFP, glucocorticoids, and retinoids also occurs transcriptionally and also may require cooperation among
various elements in the promoter. TGFP mediates
repression through a 10-bp element, 5’-GAGTTGGTGA-3’, which is located at position -709 in the rat
stromelysin gene and which binds c-fos along with
some additional, still-uncharacterized, proteins (74).
For glucocorticoids and retinoids, some of the same
DNA sequences that participate in the induction of
transcription are involved in repression (70,75,76).
This conclusion is based on the finding that specific
mutations within the collagenase promoter that abolish
transcriptional activation also abolish repression by
glucocorticoids or retinoids (70,77). While several
regions within the collagenase promoter have been
implicated in both retinoid- and glucocorticoidmediated repression, the best understood is the AP-1
site (70,75430).
Glucocorticoid receptors (GR) reside in the
cytoplasm, and upon introduction of the glucocorticoid hormone (h) into the cell, the ligand-receptor
complex is translocated to the nucleus, where it binds
directly to specific nucleotide sequences within the
promoter (called glucocorticoid response elements, or
GREs) to influence gene expression (81,82). However,
when glucocorticoids down-regulate metalloproteinase production, they appear to do so by interacting
indirectly with the promoter in what has been called a
“composite” GRE because it binds both the GR-h
complex and additional proteins (81$2). As noted
above, in the absence of glucocorticoid, fos and jun
heterodimerize, bind to the AP-1 site in the promoter,
1122
VINCENT1 ET AL
GRh
..........
-3e==+-
JUd
FOS
JUN
FOS
and activate transcription. In the presence of hormone, the GR-h complex interacts with jun, and
induces a conformational change in it (82) (Figure 5 ) .
This change interferes with the ability of the fos-jun
heterodimer to bind to DNA, thereby suppressing
transcription (75,76,81,82).
Retinoids also exert their effects by means of
specific receptors. Retinoids interact with 2 classes of
nuclear receptors, the retinoic acid receptors (RARs)
(83,84) and the retinoid X receptors (RXRs) (85,86),
each with a, p, and y subtypes. RARs and RXRs
belong to the superfamily of steroid-thyroid hormone
receptors and are considered to be ligand-dependent
transcription factors (87). These 2 classes of receptors
are divergent, sharing only 29% homology in their
ligand-binding domains, with RARs binding with high
affinity (kd -0.5 nM) to both all-trans and 9 4 s retinoic acid and RXRs binding with a lower affinity
(kd -18 nM)only to 9-cis-retinoic acid (88). Furthermore, RARs often heterodimerize with the promiscuous RXRs (88-90), while RXRs can function as homodimers (88-90). Thus, combinations of ligand
specificity and homo- and heterodimerization among
the various RAR/RXR subtypes may help explain the
pleiotropic effects of retinoids on a wide variety of
cellular processes (88,89,91).
As with the glucocorticoids, retinoids act on the
collagenase gene via the AP-1 site (77-80). Proteins
from retinoic acid-treated cells bind specifically to an
oligonucleotide containing the AP- 1 site (77-80,92).
Furthermore, these proteins react with antibodies to
the RARs and to the RXRs (92). These results suggest
that both RARs and RXRs are involved in complex
promoter and that RARiRXR heterodimers mediate
retinoic acid suppression of collagenase gene expression. There is no evidence that either RARs or RXRs
bind directly to the DNA (7&80,93), and this suggests
that, like the GR-h complex, the RARs and RXRs
repress transcription by protein-protein interactions
will need to know which sequences of DNA are
involved in transcriptional activation and repression,
what transcription factors bind to these sequences,
and how specific receptors participate in these reactions. For example, we know that repression of collagenase occurs in an RAR type-specific manner (77)
and that certain retinoids have affinities for certain
RARsRXRs (88-90). This information may eventually
lead to the development of specific retinoids that are
targeted in their ability to interact with certain RAW
RXR subtypes.
Alternatively, we may be able to block transcriptional activation by other mechanisms. By using
receptor antagonists (95) or mutant agonists (96), it is
already possible to block or interrupt the signal transduction pathway that activates transcription of the
metalloproteinase genes by means of second messengers. Another possibility is the introduction of mutant
glucocorticoid receptor-like molecules into cells so as
to sequester fos and jun and subdue transcription
indirectly. Currently, these “gene therapies” are still
experimental, but as we understand more about the
specific molecular mechanisms that increase and decrease transcription, novel therapies that apply this
understanding become a real possibility.
However, recent studies suggest that transcriptional mechanisms alone are insufficient to account for
the regulation of metalloproteinase gene expression:
posttranscriptional mechanisms also appear to be important. Both EGF (97) and IL-1 (96,98,99) increase
the levels of collagenase and stromelysin mRNA substantially, but cause only a modest increase in transcription of these genes (97-99). Since the transcriptional response is too small to account for the net
METALLOPROTEINASE INHIBITORS IN ARTHRITIS TREATMENT
increase in mRNA, additional mechanisms must contribute. One of these is increased mRNA stability.
The stability of many mRNAs can be influenced
by the presence of the sequence AUUUA in the 3‘
untranslated region (100). These AU-rich regions target mRNAs for rapid degradation and were first documented in the 3‘ end of several mRNAs that encode
inflammatory cytokines (100). These transcripts have
short half-lives, and if the AUUUA sequences were
deleted, the stability of these mRNAs was enhanced.
Two possible mechanisms have been proposed
to explain the function of these AU-rich regions.
Treatment of cells with the inflammatory compound
phorbol myristate acetate stabilizes the mRNAs containing these regions, perhaps by inducing specific
proteins that complex with the AUUUA sequences in
the mRNAs (100,101). Alternatively, instability may
be associated with the presence of a specific mRNAbinding protein which accelerates the decay of certain
mRNAs, such as those recently described for the
c-myc transcript (102,103). These proteins are not,
however, generic RNases since they do not affect the
turnover of globin or histone mRNAs, nor do they
destabilize a major portion of polysomal polyadenylated mRNA.
Thus, these AU-rich regions in the 3‘ end of the
mRNA may represent significant physiologic mechanisms for regulating gene expression. The rabbit and
human collagenase mRNA contains 3 AUUUA sequences in its 3’ untranslated region, while the stromelysin mRNAs contain 1 (104-106). Given that IL-1 and
EGF both increase the levels of collagenase and
stromelysin mRNAs but have relatively little effect on
transcription of these genes, we can postulate a role
for these AUUUA sequences in regulating this increase. If this hypothesis is correct and the mechanisms behind it become understood, it may be appropriate to develop novel therapies directed at these
RNA-binding proteins. Alternatively, antisense oligonucleotide or RNA enzyme-directed gene therapies
are more general approaches that would specifically
target collagenase and stromelysin mRNAs for degradation, thereby subverting destruction of connective
tissue. Such approaches are already showing considerable promise in a variety of systems (107).
Summary and conclusion
Collagenase and stromelysin have a premier
role in the irreversible degradation of the extracellular
matrix seen in rheumatic disease. It is therefore no
1123
surprise that considerable attention has been devoted
to developing strategies to reduce their levels in diseased joints. Most efforts have focused on inhibiting
the activity of the enzymes, either by increasing the
concentration of natural inhibitors such as the TIMPs
or by introducing into the joint synthetic compounds
that will complex with the enzymes and inactivate
them. There have also been studies directed at inhibiting enzyme synthesis. These preclinical studies have
been carried out in cell-free and/or cell culture systems
and in animal models.
Despite promising preclinical data, there have
been no stunning successes in the clinical arena. The
reasons for this are several. In part, they are rooted in
the technical difficulties associated with designing inhibitors of enzyme activity that are of high affinity, and
then delivering them to the affected joints while still
maintaining specificity and efficacy. The complicated
structure of the proteoglycan and collagen that comprise articular cartilage, along with the biochemistry of
inflamed synovial tissue, only compound the difficulties.
In addition to these technical problems, the lack
of fundamental knowledge about the biochemistry and
molecular biology of the enzymes has handicapped our
efforts. We are just resolving the crystal structure of
the metalloproteinases (108) and beginning to understand the mechanisms controlling gene expression
(67,68,70-72). These advances represent significant
achievements in metalloproteinase enzymology and
biology and should form the scientific basis for a new
generation of effective therapies. For example, knowledge of the active site as derived from the crystal
structure of the enzymes may facilitate the development of tightly-binding specific inhibitors which function well in vivo. Similarly, based on our current
understanding of mechanisms controlling the regulation of both the TIMP genes and the MMP genes, we
are beginning to elucidate how to turn these genes on
or off, and hopefully, to modulate disease accordingly. Indeed, although some studies are still at a preclinical level, these possible approaches are becoming
a reality (109).
Arthritic diseases in general, and rheumatoid
arthritis in particular, represent a complicated multifaceted set of clinical disorders. The clinical symptoms
and pathologic features result from a cascade of biologic pathways that involve acute and chronic inflammation, the immune response, and metalloproteinase
biochemistry. Current therapies are often based on the
concept of “polypharmacy” in which several drugs
are administered simultaneously, with the hope that
1124
VINCENT1 ET AL
each will be targeted at interrupting one or another of
these pathways. However, these therapies are not
always specific and they often fail. As knowledge of
the basic mechanisms underlying these pathways continues to increase, we hope to formulate “designer
therapies” that are targeted at specific molecular
mechanisms and are effective in vivo. Successful
inhibition of metalloproteinases is sure to be among
them.
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