Antagonism of a zinc metalloprotease using a unique metal-chelating scaffold: Tropolones as inhibitors of P. aeruginosa elastase

Article abstract of DOI:10.1039/c3cc41191e

Tropolone emerged from the screening of a chelator fragment library (CFL) as an inhibitor of the Zn²⁺-dependent virulence factor, Pseudomonas aeruginosa elastase (LasB). Based on this initial hit, a series of substituted tropolone-based LasB inhibitors was prepared, and a compound displaying potent activity in vitro and in a bacterial swarming assay was identified. Importantly, this inhibitor was found to be specific for LasB over other metalloenzymes, validating the usage of tropolone as a viable scaffold for identifying first-in-class LasB inhibitors.

Full text of DOI:10.1039/c3cc41191e

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Antagonism of a zinc metalloprotease using a unique
metal-chelating scaffold: tropolones as inhibitors of
P. aeruginosa elastase†
Cite this: Chem. Commun., 2013,
49, 3197
Received 13th February 2013,
Accepted 5th March 2013
a
b
Jessica L. Fullagar,z Amanda L. Garner,z Anjali K. Struss,b Joshua A. Day,a
David P. Martin,a Jing Yu,b Xiaoqing Cai,b Kim D. Janda*b and Seth M. Cohen*a
DOI: 10.1039/c3cc41191e
www.rsc.org/chemcomm
Tropolone emerged from the screening of a chelator fragment
library (CFL) as an inhibitor of the Zn2+-dependent virulence factor,
Pseudomonas aeruginosa elastase (LasB). Based on this initial hit,
a
series of substituted tropolone-based LasB inhibitors was
prepared, and a compound displaying potent activity in vitro and
in a bacterial swarming assay was identified. Importantly, this
inhibitor was found to be specific for LasB over other metallo-
enzymes, validating the usage of tropolone as a viable scaffold for
identifying first-in-class LasB inhibitors.
Fig. 1 Metal-binding groups (MBGs) and derived inhibitors with IC50 values
listed for LasB inhibition.
Tropone, and its hydroxylated derivative tropolone, are planar,
7-membered, unsaturated rings that are found in natural products of chelator fragment libraries (CFLs). CFLs are specifically
with diverse biological activities, including compounds displaying designed with fragments that can coordinate metal ions in
antibacterial, antiinflammatory, antitumor, and antiviral activity.1–3 the active site of metalloproteins. This approach has revealed
The metal-binding capacity of tropolone is well known, using its novel scaffolds such as hydroxypyrones, hydroxypyridiones,
exocyclic oxygen donor atoms to bind metal ions (i.e. O,O donor hydroxyquinolines, and quinolone sulfonamides to be effective
ligand, Fig. 1),4 and the employment of tropolone-based units in MBGs against a variety of metalloproteins, including MMPs, LF,
the design of metalloprotein inhibitors has been explored.5,6 and several others.4,7,10
Tropolone has been identified as an inhibitor of several
LasB11,12 is one of several virulence factors produced by
Zn2+-dependent metalloenzymes including carboxypeptidase A, P. aeruginosa to promote infection within a host.13,14 Previous
thermolysin, matrix metalloproteases (MMP-2 and -3), and mutation-15 and vaccine-based16 studies have revealed that LasB
anthrax lethal factor (LF) with IC50 values ranging from plays a critical role in promoting virulence through targeted proteo-
0.003ꢀ1.4 mM.1,4,7 Tropolone has also been found to be a potent lysis of host tissue proteins and immune system components.11
inhibitor of the dinuclear copper-dependent enzyme tyrosinase Moreover, LasB has also been linked to the establishment of
(IC50 value of B400 nM);8 however, a recent crystal structure of antibiotic-resistant biofilm17 and swarm colonies.18,19 Because
tropolone bound to tyrosinase revealed that the natural product evidence exists supporting the investigation of virulence factors
does not act by coordinating to the metal ion.9
as promising new antibiotic targets,20–22 the pursuit of non-
In an effort to identify suitable metal-binding groups (MBGs) peptidic, small molecule inhibitors of LasB is of interest.
for targeting metalloprotein active sites, a fragment-based drug Recently, the screening of CFL-1.1 against P. aeruginosa elastase
discovery (FBDD) approach has been applied via the development (LasB) was shown to produce several hits.19 Among the initial hits
was 3-hydroxy-1,2-dimethylpyridine-4(1H)-thione (3,4-HOPTO, 1),
a chelator that is also a potent inhibitor of MMPs, LF, and others
metalloproteins.10 Because 3,4-HOPTO displayed activity against
LasB (IC50 value 81 mM, Fig. 1), an existing sublibrary of
3,4-HOPTO derivatives10 was examined, which revealed com-
pound 2 (Fig. 1) as a potent LasB antagonist and the first
mechanism-based compound to exhibit swarming antagonism.19
Despite its exceptional activity, the 3,4-HOPTO MBG (1) is known
to inhibit a broad set of metalloproteins and it is likely that 2
a Department of Chemistry and Biochemistry, University of California, La Jolla,
San Diego, CA, USA 92093. E-mail: scohen@ucsd.edu
b Departments of Chemistry and Immunology and Microbial Science, The Skaggs
Institute for Chemical Biology, The Worm Institute for Research and Medicine,
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, USA.
E-mail: kdjanda@scripps.edu
† Electronic supplementary information (ESI) available: Detailed synthesis, char-
acterization, and assay procedures. See DOI: 10.1039/c3cc41191e
‡ These authors contributed equally to the manuscript.
c
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Fig. 2 Tropolone fragments with IC50 values listed for LasB inhibition.
and related compounds will not be highly selective for LasB
(indeed compound 2 is known to inhibit MMPs and LF at
concentrations of 1–4 mM).10
Screening of CFL-1.1 against LasB had produced other,
albeit less active, MBG hits. It was hypothesized that these hits,
which still showed good ligand efficiencies,23,24 could be used
to develop potent, and more selective LasB inhibitors. Herein, we
report the discovery of tropolone-based inhibitors of LasB. Impor-
Fig. 3 Structures and IC50 values for tropolone sublibrary compounds.
tantly, the identified antagonists are not only more potent than MBG (analogous to compound 4 above). Compound 10 also
the previously reported 3,4-HOPTO-derived compound 2, but are showed a B50-fold reduction in activity (IC50 value of 58.4 ꢁ
also highly selective for LasB over other metalloproteins.
0.1 mM, Fig. S3†), providing strong evidence that metal coordi-
From our prior screening of CFL-1.1 against LasB,7,10 tropolone nation is essential for LasB inhibition by tropolone compounds.
(3, Fig. 2) emerged as a ‘‘hit’’, with an IC50 value of 387 ꢁ 1 mM.19 As This was further verified by acetylation of another potent
mentioned earlier, tropolone is known to inhibit Zn2+-dependent derivative 7e (Fig. S1, Table S1†), which resulted in an even
thermolysin via metal coordination.25,26 Importantly, acetylation of greater loss of potency (>100-fold, data not shown).
3 to produce acetyltropolone (4, Fig. 2)1 resulted in a B3-fold
Because the 3,4-HOPTO chelating motif found in 2 (Fig. 1)
reduction in potency (IC50 value of 1.29 ꢁ 0.24 mM) suggesting that was known to inhibit other Zn2+ metalloproteins, including
Zn2+ chelation is required for LasB inhibitory activity. Again, this MMPs,7,10 the specificity of 2 versus tropolone 7a was examined
parallels findings with thermolysin, where acetylation of tropolone against a panel of metalloenzymes. Three Zn2+-dependent metallo-
resulted in a loss of activity.1 Also contained in CFL-1.1 was proteins, MMP-2, MMP-9, and human carbonic anhydrase II
b-thujaplicin (5, Fig. 2), a tropolone natural product containing (hCAII), and one Cu2+-dependent metalloenzyme, tyrosinase, were
an isopropyl group in the 4-position of the ring.2,3 The IC50 examined as a gauge of selectivity. Specificity was determined by
value of 5 was 1.34 ꢁ 0.37 mM, which is B3-fold less potent than treating each enzyme with 50 mM of either 2 or 7a and monitoring
3, perhaps suggesting that steric repulsion at the 4-position is percent enzymatic activity. Both 2 and 7a had a modest effect on
prohibiting proper Zn2+ chelation within the active site. However, hCAII, each reducing activity by B25% at 50 mM (Fig. S5†). In
5-aminotropolone (6, Fig. 2) was found to be a more potent hit, contrast, 2 was found to completely suppress the enzymatic activity
with an IC50 value of 180 ꢁ 1 mM, suggesting that selected of MMP-2 and MMP-9 at 50 mM, while 7a showed only weak
substitutions may be tolerated on the ring system.
inhibition (o40% at 50 mM, Fig. 4). Finally, neither 2 nor 7a
Encouraged by these findings, a sublibrary of compounds based showed any inhibition against tyrosinase. The loss in activity of
on fragment 6 was constructed via a one-step condensation of 6 7a against tyrosinase is a key observation, as unsubstituted
with isocyanates under microwave irradiation conditions (Fig. 3, tropolone (3) is a very potent inhibitor of this enzyme (IC50
Fig. S1†). This sublibrary was screened using a fluorescence-based value B400 nM, Fig. S5†). Similar results were obtained with
assay (Fig. S2†),19,25 and several compounds were found to have several other tropolone-derived inhibitors (data not shown).
IC50 values of o10 mM (Table S1†). The best compounds identified Therefore, by derivatizing tropolone into a more advanced lead
from this sublibrary were more potent than the previously reported compound (7a), a highly selective LasB inhibitor has been
3,4-HOPTO-based inhibitors by B2-fold.19 In particular, compound obtained that, unlike HOPTO-derived inhibitors, is selective
7a was found to be the most potent non-peptidic small molecule for LasB over other Zn2+-dependent metalloproteins, and is also
inhibitor of LasB identified to date with an IC50 value of 1.16 ꢁ inactive against the Cu-dependent tyrosinase, for which the
0.06 mM. Moreover, compound 7a was found to be a competi- tropolone MBG has an inherently high affinity. This illustrates
tive inhibitor of the enzyme with a Ki of 336 nM (Fig. S4†). To that by judicious selection of both MBG and backbone target-
determine the structural relevance of the urea linker used in specific metalloprotein inhibitors can be designed.
these compounds, an analogue of 7a, containing an amide
Based on the enhanced potency and specificity of compound
linker was synthesized (8, Fig. 3) resulting in a >8-fold loss in 7a, its activity was further characterized in a cellular assay. As stated
activity. Coupling to a shorter amide linker (9, Fig. 3) produced above, LasB activity is required for the induction of swarming in
a nearly 50-fold loss in potency. These compounds demonstrate P. aeruginosa.18,19 Importantly, the ability of P. aeruginosa to form
that both the urea linkage and the linker length contribute to swarm colonies has been linked to the development of antibiotic
the activity of 7a against LasB. Finally, acetylation of 7a was resistance,27,28 indicating that small molecule inhibitors of
performed to produce compound 10 (Fig. 3) with a blocked LasB could be used as adjuvants with traditional antibiotics
c
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The present findings clearly suggest that identification of
privileged chelating scaffolds for a given metalloenzyme can
lead to the realization of both potent and selective metallo-
protein inhibitors.
We thank Dr Yongxuan Su (UCSD) and the Molecular Mass
Spectrometry Facility for obtaining mass spectrometry data,
´
Professor Eric Deziel (INRS-Institut Armand-Frappier) for kind
donation of P. aeruginosa strain PA14, and Dr David Puerta
(UCSD) for careful reading and editing of this manuscript.
This work was funded by the NIH (Grants R01 AI077644 to
K.D.J. and R01 GM098435 to S.M.C.).
Fig. 4 Inhibition of Zn2+-dependent MMP-2 (solid) and MMP-9 (lines) by a
broad-spectrum MMP inhibitor (+ control†), 2, and 7a.
Notes and references
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Fig. 5 Swarming of P. aeruginosa strain PA14 in the absence (left, DMSO
control) or presence of 7a (right, 25 mM).
to enhance the susceptibility of antibiotic-resistant P. aeruginosa
to these drugs.29 To examine the anti-swarming activity of
compound 7a, P. aeruginosa strain PA14 was grown on swarm
agar plates containing either DMSO (control) or 25 mM of 7a. As
shown in Fig. 5, this tropolone-based inhibitor was able to
completely inhibit the swarming phenotype at this concentration,
exhibiting swarming inhibitory properties comparable to 2.19
Importantly, 7a was found to be non-cytotoxic to PA14 at a
concentration of 25 mM (Fig. S6†). Finally, compound 10, which
has an acetylated tropolone MBG, was found to be much less
effective at inhibiting swarming (Fig. S7†). Thus, these results
demonstrate the potential of this natural product-based chelating
moiety for the design of antimicrobial metalloprotease inhibitors.
In conclusion, tropolone-based metalloprotein inhibitors have
been developed by a chelator-focused FBDD approach. These
compounds are the most potent non-peptidic small-molecule
inhibitors of LasB reported to date and show excellent activity
in a cell-based swarming assay. Importantly, the tropolone
MBG-derived inhibitors are more active and more selective
than the previously identified HOPTO-based compounds. The
work presented here is consistent with earlier studies on
tropolone-based metalloprotein inhibitors. While the majority
of the previous tropolone-based inhibitors were identified
by screening of natural products, this study demonstrates
how use of chelator fragment libraries and sublibraries can
rapidly identify leads for the development of such inhibitors.
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c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 3197--3199 3199