Enzymatic activation of a matrix metalloproteinase inhibitor

Article abstract of DOI:10.1039/b923302d

Matrix metalloproteinase inhibitors (MMPi) possessing a glucose protecting group on the zinc-binding group (ZBG) show a dramatic increase in inhibitory activity upon cleavage by β-glucosidase.

Full text of DOI:10.1039/b923302d

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Enzymatic activation of a matrix metalloproteinase inhibitorw
Jody L. Major Jourden and Seth M. Cohen*
Received (in Austin, TX, USA) 5th November 2009, Accepted 20th November 2009
First published as an Advance Article on the web 18th January 2010
DOI: 10.1039/b923302d
Matrix metalloproteinase inhibitors (MMPi) possessing a
glucose protecting group on the zinc-binding group (ZBG)
show a dramatic increase in inhibitory activity upon cleavage
by b-glucosidase.
designed for MMPs.22–29 Only one example of a glycoside
MMP proinhibitor has been prepared; however, efforts to
release the active MMPi through enzymatic cleavage proved
unsuccessful.27 In this report, we show that an MMPi can be
formulated and inactivated as a proinhibitor, deprotected and
released by an enzymatic signal, and hence exhibit triggered
control over inhibitory activity. The compounds described
here allow for spatial and temporal control of MMP inhibition,
making them potentially valuable as therapeutics or as
chemical tools for studying the roles of MMPs in disease
models and developmental biology.30,31
Matrix metalloproteinases (MMPs) are a ubiquitous class of
zinc(II)-dependent hydrolytic enzymes that have been associated
with a wide range of pathologies including cancer, arthritis,
heart disease, and stroke.1–3 Clinical trials of matrix metallo-
proteinase inhibitors (MMPi) have frequently been hampered
by the onset of musculoskeletal syndrome (MSS), which
manifests as severe joint pain, and has been attributed
to non-specific, systemic inhibition of MMPs and other
metalloenzymes.4,5 One approach to eliminate or minimize
MSS and other complications would be to develop prodrugs
or ‘proinhibitors’ that are triggered in a localized fashion,
hence restricting inhibitory activity both spatially and temporally.
The prodrug concept has been applied to a variety of
targets, triggered by stimuli such as light, changes in pH,
and enzymatic activity.6,7
Building on the strategy of glycosidic protecting groups, we
first focused our attention on the development of glucose-
protected non-hydroxamate zinc-binding groups (ZBGs)32,33
that can be activated by enzymatic cleavage of the protecting
group with b-glucosidase to release the ZBG and glucose
(Fig. 1). The synthesis of the protected ZBGs (2, 4, and 6)
was accomplished following a literature procedure used by
Orvig and coworkers for enzyme-activated metal-binding
chelators (Scheme S1).9,34 The ZBG was protected with
acetobromo-a-D-glucose in a 1 : 1 solution of 1.0 M NaOH
and CH2Cl2 in the presence of (nBu)4NBr. The desired
products were obtained by cleavage of the glucose acetate
groups using NaOMe in MeOH.
A common strategy in the development of prodrugs and
proinhibitors is through the use of carbohydrate protecting
groups. The development of glycoconjugate prodrugs offers
the ability to increase water solubility, enhance targeting, and
minimize the side effects associated with systemic delivery of
cytotoxic drugs.8–11 The use of glycoside protecting groups,
such as glucoside moieties, have been reported for use with
targeted treatments such as ADEPT (antibody directed enzyme
prodrug therapy) and PMT (prodrug monotherapy).12–14 The
ADEPT approach, developed over 20 years ago, has been
widely investigated as a means to deliver drugs specifically to
cancerous tissue and has shown promise in both animal studies
and clinical trials.15–17 Glucose-derivatized compounds can
also enhance uptake in cancer cells18–20 and the brain.9,10,21
Metalloenzyme inhibitors such as MMPi are particularly
suitable to the proinhibitor approach because such compounds
generally employ a metal-binding moiety, which if blocked
with a protecting group, abolishes inhibitory activity. Specifi-
cally, using glucose as a protecting group, a proinhibitor
MMPi can be developed to release the active MMPi in the
presence of b-glucosidase to restore MMP inhibitory activity.
Surprisingly, metalloenzyme proinhibitors have not been
widely investigated and very few proinhibitors have been
To evaluate the ability of these compounds to be enzymati-
cally activated, cleavage of the protected ZBGs in the presence
of b-glucosidase (from almond extract, Fluka) was followed
using electronic spectroscopy. To a solution of the protected
ZBG in HEPES buffer was added b-glucosidase, and the
change in absorbance was monitored over time. As can be
Department of Chemistry and Biochemistry, University of California,
San Diego, La Jolla, California, 92093-0358, USA.
E-mail: scohen@ucsd.edu; Fax: +1 858-822-5598;
Fig. 1 Structures of inhibitors and proinhibitors tested in this
study. Compounds include the ZBGs 1-hydroxy-pyridin-2(1H)-one (1),
3-hydroxy-2-methyl-4-pyrone (3) and 3-hydroxy-2-methyl-4-pyrothione
(5) as well as the full-length MMPi 1,2-HOPO-2 (7).
Tel: +1 858-822-5596
w Electronic supplementary information (ESI) available: Synthetic
details, characterization of all compounds, and details of cleavage
assays. Scheme S1, Fig. S1–S10. See DOI: 10.1039/b923302d
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Scheme 1 Synthesis of the glucose-protected MMPi 8.
Fig. 2 Absorption spectra of the glucose-protected ZBG 2 (0.05 mM,
HEPES buffer, pH = 7.5) in the presence of b-glucosidase (16 U)
monitored over time. The heavy lines are the initial (dashed) and final
(solid) spectra and arrows indicate the change in spectra over time.
The spectrum of an authentic sample of ZBG 1 (dotted) is also shown.
The ability of the protected compounds to inhibit MMP-9
(gelatinase-B) in the presence of b-glucosidase was evaluated
using a fluorescence-based assay.36 Compounds 1–8 were
evaluated at a concentration close to their reported IC50 values
in the presence and absence of b-glucosidase (Fig. 3). The
percent inhibition of MMP-9 with the ZBGs (1, 3, and 5) is
close to 50% when tested with and without b-glucosidase indi-
cating that the presence of low concentrations of b-glucosidase
in the assay has little effect on MMP inhibition. The protected
ZBGs (2, 4, and 6) show attenuated inhibition when evaluated
without the activating enzyme and complete restoration of
inhibition when exposed to b-glucosidase. Compounds 2, 4,
and 6 do show some inhibition of MMP-9 (Table 1), which is
likely due to non-specific binding at the high concentrations of
the ZBGs (0.125–4 mM) used in these experiments.
seen in Fig. 2 for compound 2, the absorbance over time shows
a decrease at 292 nm while a band at 312 nm emerges,
indicative of the deprotected ZBG 1-hydroxy-2-pyridin-
2(1H)-one (1). Similar spectra were observed for the hydroxy-
pyrone derivatives 4 and 6 (Fig. S1, S2). In addition, cleavage
of the protected ZBGs was confirmed by HPLC analysis
(Fig. S3–S5). These studies demonstrate that in aqueous buffer
at room temperature, the glucose-protected ZBGs can be
readily activated in the presence of b-glucosidase providing
compelling evidence that hydroxypyridinone and hydroxy-
pyrone ZBGs are well suited for the development of enzyme-
activated MMP proinhibitors.
In the absence of b-glucosidase, the complete MMPi 8
(16 mM) displays very little inhibition of MMP-9, but upon
activation, MMP-9 activity is inhibited by 73%, which is
essentially identical to an authentic sample of inhibitor 7.
Even greater potency was observed against MMP-8, where
MMPi 8 could be activated with b-glucosidase to obtain 33%
inhibition at only 150 nM (Fig. S10), representing a 4 1000-fold
increase in activity upon enzymatic activation (Table 1). The
factor of 1000 difference in the quotient IC50 (IC50 value of
proinhibitor in the presence and absence of enzyme) has been
reported as an optimal value for the ADEPT approach to
targeted therapy,11 and hence MMPi 8 exceeds this threshold
with MMP-8. The higher activity against MMP-8 is consistent
with the potency of the cleavage product (7) against this
Having demonstrated the use of glucose as an effective
protecting group for the aforementioned ZBGs, we aimed to
incorporate a glucose protecting group into a full-length
MMPi to develop an MMP proinhibitor. We selected the
full-length inhibitor 1,2-HOPO-2 (7), a potent non-hydroxamate
inhibitor of MMPs that uses a 1-hydroxy-2-pyridin-2(1H)-one
(1) ZBG.35 Synthesis of the MMP proinhibitor was achieved
by addition of acetobromo-a-D-glucose and Cs2CO3 to 7 in
DMF at room temperature to give 8a in high yields (480%,
Scheme 1). These reaction conditions were a vast improve-
ment in yield over the aqueous reaction conditions used to
protect the ZBGs. Surprisingly, these high-yield reaction
conditions did not produce the desired products with the
ZBGs (1, 3, and 5). The final proinhibitor (8) was obtained
by deprotection of the glucose acetate groups with NaOMe in
MeOH at 0 1C for one hour.
The MMP proinhibitor 8 was first evaluated for activation
by b-glucosidase using electronic spectroscopy and HPLC
analysis (Fig. S6, S7). Results from these studies indicate that
while cleavage of 8 to produce the active MMPi 1,2-HOPO-2 (7)
goes to completion, the kinetics of the reaction are noticeably
slower than that observed for the protected ZBGs. Complete
conversion of 8 to 7 required B4 h at 37 1C; a Km value of
210 mM was determined (Fig. S8). Notably, compound 8 was
not cleaved under acidic conditions (0.1 M HCl) over 24 h
(Fig. S9). Overall, this is the first example of a glucosidase
proinhibitor that can be enzymatically cleaved to yield the
active MMPi.
Fig. 3 Percent inhibition of MMP-9 with compounds 1–8 tested at
1 mM for 1 and 2, 4 mM for 3 and 4, 125 mM for 5 and 6, and 16 mM for
7 and 8, in the absence (white) and presence (black) of b-glucosidase.
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Table 1 IC50 values of inhibitors and protected inhibitors against MMP-9 and MMP-8 as measured using fluorescence based assay
a
Inhibitor
IC50
Proinhibitor
IC50
QIC50
Enzyme
1
3
5
7
7
1.02 mM (Æ 0.03)
4.03 mM (Æ 0.05)
138 mM (Æ 5)
2
4
6
8
8
3.99 mM (Æ 0.04)
5.7 mM (Æ 0.3)
302 mM (Æ 5)
587 mM (Æ 7)
84 mM (Æ 1)
3.9
1.4
2.2
202
1120
MMP-9
MMP-9
MMP-9
MMP-9
MMP-8
2.7 mM (Æ 0.2)
0.075 mM (Æ 0.005)
QIC50 = quotient IC50 = IC50 proinhibitor/IC50 inhibitor.
a
metalloenzyme.35 The results for 8 show that incorporation
of a ZBG protection strategy into an MMPi gives near
complete abolition and recovery of inhibitory activity with
these enzyme inhibitors.
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In summary, we have demonstrated that it is possible to
passivate an MMPi as a proinhibitor, activate it through an
enzymatic reaction, and inhibit MMPs in a controlled manner.
We have shown that protection of the metal-binding moiety of
MMPi can be achieved and enzymatically removed to release
an active, intact MMPi. This general strategy of protecting the
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