Competitive Live-Cell Profiling Strategy for Discovering Inhibitors of the Quinolone Biosynthesis of Pseudomonas aeruginosa

Article abstract of DOI:10.1021/jacs.8b07629

Quinolones of the human pathogenPseudomonas aeruginosaserve as antibacterial weapons and quorum sensing signals and coordinate the production of important virulence factors. A central enzyme for the biosynthesis of these quinolones is the synthetase PqsD.

Full text of DOI:10.1021/jacs.8b07629

Communication
pubs.acs.org/JACS
Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Competitive Live-Cell Profiling Strategy for Discovering Inhibitors of
the Quinolone Biosynthesis of Pseudomonas aeruginosa
̈
Michaela Prothiwa, Felix Englmaier, and Thomas Bottcher*
Department of Chemistry, Konstanz Research School Chemical Biology, Zukunftskolleg, University of Konstanz, 78457 Konstanz,
Germany
S
* Supporting Information
ability to occupy and defend niches in the human body, quino-
ABSTRACT: Quinolones of the human pathogen Pseudo-
monas aeruginosa serve as antibacterial weapons and quorum
sensing signals and coordinate the production of important
virulence factors. A central enzyme for the biosynthesis of
these quinolones is the synthetase PqsD. We developed an
activity-based probe strategy that allows to screen for
PqsD inhibitors in a cellular model system of live cells of
Escherichia coli overexpressing PqsD. This strategy allowed
us to determine IC50 values for PqsD inhibition directly in
live cells. Our most potent inhibitors were derived from the
anthranilic acid core of the native substrate and resulted in
single-digit micromolar IC50 values. The effectiveness of our
approach was ultimately demonstrated in P. aeruginosa by
the complete shutdown of the production of quinolone
quorum sensing signals and quinolone N-oxides and a
considerable inhibition of the production of phenazine
virulence factors.
lone biosynthesis is a prime target for potential antivirulence
strategies. A common step in the biosynthesis of all quinolones is
the decarboxylative coupling reaction of coenzyme A-activated
anthranilic acid (ACoA) with malonyl-CoA, which is catalyzed
by PqsD.22,23 Hydrolysis of the resulting thioester by PqsE leads
to 2-aminobenzoylacetate (2-ABA), which is the precursor
for the subsequent reactions to 2-alky-4-quinolones (AQs),
3-hydroxy-AQs, and AQNOs (Figure 1a).24 We here report a
competitive in-cell probe strategy that allowed to detect a potent
mechanism-based PqsD inhibitor resulting in a global and
complete shutdown of quinolone biosynthesis in P. aeruginosa.
The active site of PqsD comprises a nucleophilic cysteine
residue, which is involved in the transfer of the anthraniloyl
moiety.23 We have demonstrated with purified enzyme that
simple electrophilic α-chloroacetamide probes have a remark-
able selectivity for this cysteine residue (Cys112), and its
mutation to alanine prevented probe labeling.15
We now aimed for a live-cell labeling strategy using the
α-chloroacetamide probe CA (Figure 1b, Figure S1). Hereby,
we incubated intact cells with the probe, removed excess probe
by washing steps and after cell lysis employed click chemistry
to append a fluorescent tetramethylrhodamine tag to the ter-
minal alkyne group for visualization by SDS-polyacrylamide gel
electrophoresis (PAGE). However, when we applied the probe at
100 μM to live cells of a stationary phase culture of P. aeruginosa,
no fluorescent band in the size range of PqsD was detected.
Coomassie staining indicated, that PqsD was of low abundance
in the native proteome of P. aeruginosa, which was likely too
low for in situ labeling by the CA probe (Figure 1b). We thus
speculated that Escherichia coli cells overexpressing PqsD may
provide a useful in situ model for in-cell labeling. Indeed,
incubation of cells with 100 μM of CA probe revealed a band
only for E. coli cells expressing PqsD but not for the parent
E. coli strain (Figure 1c). The labeling hereby achieved remark-
able sensitivity down to submicromolar probe concentrations
(Figure 1d). The band of the corresponding protein size was
cut out and after tryptic digest subjected to proteomic analysis
by mass spectrometry. The results confirmed the sequence of
PqsD and in addition the active site cysteine as the site of
covalent attachment of the probe (Figure S2). Since our probe
selectively and specifically labeled the active site of PqsD in the
background of the native proteome of live E. coli cells, we now
aimed to apply this probe as tool for live-cell compatible
creening approaches with purified proteins have yielded
Snumerous potent enzyme inhibitors. Yet, in vitro activity
does not always go along with biological efficacy in an organ-
ism. Reasons are often permeability barriers, active detox-
ification mechanisms, or promiscuous binding to off-targets.1,2
Thus, strategies for probing enzyme inhibition in a living cell
are highly desirable. Activity-based probes have become
powerful tools for the discovery of target proteins of small
molecules,3−5 for diagnostic purposes6,7 and for dissecting
enzyme functions and biochemical pathways.8−11 Competitive
ABPP strategies have been successfully applied to discover
selective enzyme inhibitors in the background of complex
proteomes.12−14 We have recently shown that simple electro-
philic chemical probes can selectively target the active site
cysteine of the Pseudomonas aeruginosa quinolone biosynthesis
enzyme PqsD in vitro.15 Since this approach failed to generate
in situ inhibitors of quinolone biosynthesis we now aimed for a
live-cell inhibitor screening strategy. The human pathogen
P. aeruginosa produces more than 50 different quinolones.16
For instance, 2-heptyl-4-quinolone (HHQ) and the Pseudomonas
quinolone signal (PQS) serve as quorum sensing signals
controlling the production of virulence factors and modulate
the host immune response.17−19 2-Alkylquinolone N-oxides
(AQNOs) inhibit growth of competing bacterial species such as
Staphylococcus aureus and contribute to autolysis of aged
P. aeruginosa cultures (Figure 1a).20,21 Since quinolones have
many important roles for the virulence of P. aeruginosa and its
Received: July 19, 2018
© XXXX American Chemical Society
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Figure 1. In situ labeling of PqsD. (a) Biosynthesis of the different quinolone classes of Pseudomonas aeruginosa and their biological effects.
(b) In situ labeling of P. aeruginosa PAO1 by probe CA showed no fluorescent band for PqsD. (c) Live E. coli BL21 cells overexpressing PqsD
labeled by probe CA (100 μM) resulted in a strong band for PqsD compared to the parent BL21 strain. (d) Dose-down of probe CA with live
E. coli overexpressing PqsD. (e) Scheme of the competitive labeling platform. 2-HABA: 2′-hydoxyaminobenzoylacetate.
Figure 2. In situ screening of potential PqsD inhibitors. (a) Inhibitor design based on the anthraniloyl-CoA core. (b) Synthesis of
α-chloroacetophenone 1. (c) Focused compound library of PqsD substrate analogs. (d) Initial screening of the compound library in PqsD
overexpressing E. coli cells. (e) Concentration-dependent labeling experiments with active inhibitors.
inhibitor discovery. Effective PqsD inhibitors would block the
active site and thereby inhibit intracellular labeling by the
probe (Figure 1e). To this aim, we compiled a small focused
library of 14 compounds comprising an α-halocarbonyl-moiety,
some of which mimicked the core of the native PqsD substrate
anthraniloyl-CoA (Figure 2a). We hypothesized that fusing
of the core structure of anthranilate and the electrophilic
α-chloroacetyl group of the probe CA could lead to covalently
binding inhibitors exhibiting high affinity toward PqsD.
2′-Amino-α-chloroacetophenone 1 was synthesized using
Houben−Hoesch acylation (Figure 2b) and other compounds
(2−14) were obtained commercially. For an initial screening,
PqsD expressing E. coli cells were preincubated with the library
compounds at 50 μM for 15 min followed by incubation with
100 μM probe CA. Successful inhibitors that bind to the active
site of PqsD would be detected by reduction in fluorescence
signal intensity in the gel. All compounds with an anthranilic
acid-derived core were active and completely abolished
labeling. Compounds that did not considerably reduce probe
labeling (10−14) had a rather bulky nonflat architecture. For
compound 7, we considered electronic reasons of the pyridine
core being responsible for its poor activity. The remaining
compounds were tested in concentration series.
Quantifying the intensity of competitive probe labeling over
a broad range of concentrations ranging from 0.05 to 100 μM
provided the unique opportunity to determine IC50 values for
PqsD inhibition in living cells (Figure 3a, Figures S3−S5). Com-
pounds resembling the anthranilic acid core of the native sub-
strate were among the most potent inhibitors, such as 1−6 with
an α-chloroketone motif exhibiting IC50 values of 1−3 μM
(Figure 3a). Also compound 9 with an α-chloroacetamide
motif was highly active, while closely related compound 8 was
significantly less active, indicating that already small changes in
substitution pattern had major impacts. Using proteomic ana-
lysis of PqsD inhibited by compound 1 in live cells, confirmed
that the compound only bound to the active site cysteine
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(Cys112) although the protein structure comprises in total
six cysteines, five of which are not involved in catalysis (Figure 3b).
This indicates a highly selective mechanism-based mode of action.
Off-target protein bands labeled by simple electrophilic probes
in P. aeruginosa were not competed by compound 1, suggesting
the inhibitor was more specific than these probes (Figure S6).
Consequently, we were interested to investigate if also quino-
lone production of P. aeruginosa could be inhibited. Since
primary amines facilitate the accumulation of compounds in
Gram-negative bacteria25 and due to the close structural resem-
blance with the anthranilic substrate, we selected compound 1
for in situ assays with P. aeruginosa. Growth remained largely
unaffected with compound 1 and 2 at concentrations up to
100 μM, ruling out that potential effects on virulence would be
an artifact of growth inhibition (Figure S7). We thus could
investigate the effects on the biosynthesis of quinolones. PqsD
is responsible for the biosynthesis of more than 50 quinolones,
including AQs such as HHQ and its 3-hydroxy-derivative PQS,
as well as different AQNOs.16 We established a LC-MS/MS
method using characteristic mass transitions for quantitative
analysis of metabolites in the supernatants of P. aeruginosa
(Table S1, Figures S8−S10). A library of phenazines and
synthetic AQs and AQNOs that we reported recently served as
standards.26 We also analyzed phenazines, including phena-
zine-1-carboxamide (PHZ-CA), 1-hydroxyphenazine (1-OH-
PHZ), and pyocyanin (PYO), which are produced as virulence
factors partially under control of the PQS quorum sensing
system (Figure 4a).
Quantifying quinolone production over time revealed that
AQs and AQNOs are produced between 5 and 10 h and then
remain constant to 24 h (Figure S11). We thus incubated
cultures of P. aeruginosa strains PAO1 and PA14 with different
concentrations of 1 for 24 h and used extracted culture
Figure 3. Inhibition of PqsD in situ. (a) Dose−response curves
of compounds 1 and 8 and IC50 values of all active compounds.
(b) Tryptic peptide fragment with compound 1 covalently bound to
the active site cysteine of PqsD after in situ inhibition.
Figure 4. Quinolone and phenazine inhibition in P. aeruginosa PAO1 cultures treated with compound 1. (a) Structures of quinolones and
phenazines analyzed by LC-MS/MS. (b) Chromatograms of mass transitions from extracted supernatants after incubation with 1 at different
concentrations for 24 h. (c) Percentage of inhibition of extracellular quinolones and phenazines.
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Communication
supernatants for LC-MS/MS analysis. Increasing concentra-
tions of the PqsD inhibitor considerably decreased the inten-
sity of quinolones and phenazines in both strains (Figure 4b,
Figure S12a). Quantification of individual quinolones revealed
a global down-regulation of AQs and AQNOs. The production
of all major quorum sensing signals, PQS and the chain length
congeners of HHQ was completely inhibited in P. aeruginosa
PAO1 between 50 and 100 μM with an average IC50 < 10 μM
and to a slightly lesser degree in P. aeruginosa PA14 (Figure 4c,
Figures S12b and S13). Inhibition of quinolone signal produc-
tion consequently caused the down-regulation of phenazines,
which was strongest for 1-OH-PHZ and weakest for pyocyanin.
Finally, also production of the different quinolone N-oxide
congeners was inhibited by compound 1 in dose response
manner including the three major N-oxides HQNO, NQNO
and trans-Δ1-NQNO. In comparison, compound 2 inhibited
quinolone production less effectively (Figure S14). External
addition of synthetic 2-ABA27 to a culture of P. aeruginosa
PAO1 grown with 50 μM of compound 1 partially restored
HHQ production (Figure S15). Our results with P. aeruginosa
confirm compound 1 as highly active inhibitor with unpre-
cedented efficacy in the global inhibition of quinolone biosyn-
thesis and down-regulation of phenazine production.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.8b07629.
■
S
Additional data and methods (PDF)
AUTHOR INFORMATION
Corresponding Author
*thomas.boettcher@uni-konstanz.de
ORCID
■
̈
Thomas Bottcher: 0000-0003-0235-4825
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Prof. Dr. Andreas Marx, Malin Bein, Prof. Dr. Tanja
́
́
Gaich, David Szamosvari, and and Kevin Ru
̈
tschlin for their
generous support. We gratefully acknowledge funding by the
Emmy Noether program (DFG), EU FP7Marie Curie ZIF,
SFB969 project C06, Fonds der Chemischen Industrie, and
KoRS-CB. M.P. was supported by a Zeiss Ph.D. fellowship.
P. aeruginosa is listed by the World Health Organization on
the highest priority level requiring urgently new treatment
options.28 Quinolone-based quorum sensing has attracted
much attention due to its decisive impact on the virulence of
P. aeruginosa. Over the past years, the biosynthesis of quino-
lones in P. aeruginosa has been investigated in detail and
revealed possible intervention points.22,27,29 For instance,
inhibitors of PqsA, PqsBC as well as PqsE have been developed
by rational design and fragment-based screening, respec-
tively.27,30,31 PqsD has been in the focus of inhibitor develop-
ment with pioneering work led by the Hartmann group.32−36
Although potent in vitro inhibitors have been reported, in situ
efficacy remained rather moderate.37 More potent effects on
virulence could only be achieved by dual inhibition of PqsD
and the transcriptional regulator PqsR.38 To the best of our
knowledge, our anthranilic acid-derived covalent inhibitor 1 is
the most potent in situ PqsD inhibitor reported so far that
causes global inhibition of quinolone biosynthesis. While
compound 1 exhibits moderate toxicity to eukaryotic cells
(Figure S16), substituted α-chloroacetylindoles such as 2 were
well tolerated in a mouse model.39 Thus, our inhibitors may be
promising candidates for the development of an anti-infective
drug against P. aeruginosa. While many quinolones are signals
controlling virulence, quinolone N-oxides serve as weapons
against competing bacterial species and likely support the
colonization of various niches.20 Our group recently
demonstrated major activity of unsaturated trans-Δ1-NQNO
against Staphylococcus aureus while the saturated congeners are
much less potent26 and we speculated about functional spe-
cialization of different AQNOs.40 Our potent PqsD inhibitors
may hereby serve as valuable tools to dissect the roles of the
diverse quinolones produced by P. aeruginosa.
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