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A. Di Pizio et al.
Matrix metalloproteinases (MMPs) are zinc-dependent
ECM-degrading-endopeptidases. 23 MMPs have been
identified in humans, and, despite their high structural
similarity, no functional redundancy is shared between
them [7]. Each MMP influences ECM properties differ-
ently, and consequently, enzyme selectivity is essential to
develop MMP inhibitors, especially for anticancer therapy.
Indeed, MMP-2 and MMP-9 essentially contribute to the
invasion and dissemination of tumor cells [8], but several
MMPs showed protective effects in cancer [9], with MMP-
8 as a recognized anti-target [10, 11].
inhibition of the enzyme activity [25]. EGCG and analogs
have been previously tested on MMP-9, and the pyrogallol
hydroxyl groups were shown to be essential for MMP-9
inhibition [26]. Interestingly, it has been found that the
gallate itself inhibits MMPs and is more selective on
MMP-2 than on MMP-8 [15]. To understand the molecular
features that are responsible for the MMP-2 binding and
inactivation, we performed a structure-based analysis of
EC (1) and EGCG (2) into the MMP-2 enzyme, which then
led to the design of compound 3. EC (1) and EGCG (2)
were purchased from Aldrich Chemicals (Milan, Italy);
compound 3 was obtained via condensation of tribenzy-
loxybenzoic acid with resorcinol and debenzylation of the
resulting ester through catalytic hydrogenation (Scheme 1).
Even though EGCG is active in preventing also the
proMMP-2 activities [25], we focused our analysis on the
active form of MMP-2. Particularly, EC (1), EGCG (2),
and compound 3 were submitted to enzyme inhibition
assays for MMP-2, but also for MMP-9 and MMP-8,
which, as mentioned above, are considered target and anti-
target, respectively, for cancer therapy (Table 1).
Commonly, synthetic MMP inhibitors (MMPIs) consist
of a metal coordinating function, called zinc-binding group
(ZBG), as it binds to the catalytic zinc, and moieties that
reach into surrounding binding pockets. The binding to the
zinc ion ensures the potency, while the interaction with the
pockets modulates the selectivity of the inhibitor. The
hydroxamate function is one of the most used ZBGs for the
development of potent MMPIs [12]. However, it is charac-
terized by a poor pharmacokinetic profile and by toxic
effects in long-term treatments [13]. To overcome the
problems of selectivity and/or toxicity, two approaches have
been followed to synthesize MMPIs: the use of alternative
ZBGs [14–16] and the development of novel non-competi-
tive inhibitors not interacting with the catalytic zinc (also
classified as the third generation MMPIs) [17–19].
Binding modes of EGCG and EC into MMP-2
enzyme
Previous attempts were done to identify the binding mode
of the green tea polyphenols into the MMP-2 enzyme [25].
Docking studies by Chowdhury et al. suggested that EC
and EGCG H-bond with Glu202 side chain through the
catechol and the pyrogallol, respectively; instead, the
benzodihydropyran of the two ligands is oriented differ-
ently, forming an H-bond with the Pro221 main chain in
the case of EC and with the backbone of Leu197 and
His201 residues in the case of EGCG.
Intriguingly, several natural compounds have shown
promising inhibition of MMP-2, including curcumin from
Curcuma longa, polyphenols from Camellia sinensis,
ageladine A from the marine sponge Agelas nakamurai,
fucoidan extracts from seaweeds Claisiphon novaecaledo-
niae, and methanolic extracts from marine red algae
Cavalina pilulifera [20–23]. This opens the possibility of
using non-toxic natural compounds as starting points for a
drug design project. Here, the putative binding modes of
(–)-epicatechin (1, EC) and (–)-epigallocatechin gallate (2,
EGCG), polyphenols from Camellia sinensis, have been
investigated. This work provides insights for the opti-
mization of catechins as selective MMP-2 inhibitors and
suggests the molecular features that lead to enzyme inac-
tivation. Moreover, we generated a structure-based phar-
macophore model that was used to virtually screen food-
derived compounds (collected in the freely available
cules were selected as potential MMP-2 inhibitors.
Recently, the MMP-8 enzyme was crystallized in com-
plex with N-(3,4-dihydroxyphenyl)-4-diphenylsulfonamide
(Fig. 1), and, using the X-ray structure (PDB ID: 5H8X),
the binding modes of several catechol-containing inhibitors
into MMP-2, -8, and -9 have been investigated. This
analysis revealed a pivotal role of water molecules in
mediating the ligand–protein interaction [27]. As shown in
Fig. 1, three clusters of water molecules were observed
around the catalytic site.
The work from Tauro et al. [27] provides valuable tools
to further analyze the binding mode of polyphenolic
compounds into MMPs. Particularly, we characterized the
binding conformation of EC and EGCG into the MMP-2,
MMP-8, and MMP-9 enzymes by superimposing EC and
EGCG to the ligand co-crystallized with MMP-8 (PDB ID:
5H8X). Then, the complexes of EC and EGCG with the
MMP-2, -8, and -9 were generated and minimized.
In the MMP-2 enzyme, the catechol of EC establishes an
H-bond with Glu202 side chain (Fig. 2), as previously
Results and discussion
Green tea polyphenol (-)-epigallocatechin gallate (2) was
found to inhibit the growth of malignant cells via modu-
lation of MMP-2 [24]. While EGCG (2) inhibits markedly
MMP-2 activities, epicatechin (1) did not show noticeable
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Di Pizio, Antonella
