Chemical probes for competitive profiling of the quorum sensing signal synthase PqsD of Pseudomonas aeruginosa

Article abstract of DOI:10.3762/bjoc.12.277

The human pathogen Pseudomonas aeruginosa uses the pqs quorum sensing system to coordinate the production of its broad spectrum of virulence factors to facilitate colonization and infection of its host. Hereby, the enzyme PqsD is a virulence related quoru

Full text of DOI:10.3762/bjoc.12.277

Chemical probes for competitive profiling of the quorum
sensing signal synthase PqsD of Pseudomonas aeruginosa
Michaela Prothiwa, Dávid Szamosvári, Sandra Glasmacher and Thomas Böttcher*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2016, 12, 2784–2792.
Department of Chemistry, Konstanz Research School Chemical
Biology, University of Konstanz, 78457 Konstanz, Germany
doi:10.3762/bjoc.12.277
Received: 16 September 2016
Accepted: 08 December 2016
Published: 20 December 2016
Email:
Thomas Böttcher* - thomas.boettcher@uni-konstanz.de
* Corresponding author
This article is part of the Thematic Series "Chemical biology".
Guest Editor: H. B. Bode
Keywords:
activity-based probes; PqsD; protein labelling; Pseudomonas
aeruginosa; quinolones
© 2016 Prothiwa et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
The human pathogen Pseudomonas aeruginosa uses the pqs quorum sensing system to coordinate the production of its broad spec-
trum of virulence factors to facilitate colonization and infection of its host. Hereby, the enzyme PqsD is a virulence related quorum
sensing signal synthase that catalyzes the central step in the biosynthesis of the Pseudomonas quinolone signals HHQ and PQS. We
developed a library of cysteine reactive chemical probes with an alkyne handle for fluorescence tagging and report the selective and
highly sensitive in vitro labelling of the active site cysteine of this important enzyme. Interestingly, only one type of probe, with a
reactive α-chloroacetamide was capable of covalently reacting with the active site. We demonstrated the potential of our probes in a
competitive labelling platform where we screened a library of synthetic HHQ and PQS analogues with heteroatom replacements
and found several inhibitors of probe binding that may represent promising scaffolds for the development of customized PqsD in-
hibitors as well as a chemical toolbox to investigate the activity and active site specificity of the enzyme.
Introduction
The emergence of multi-drug resistant bacterial strains urges the virulence factors is coordinated on population level by several
rapid discovery of new antibiotics and the development of novel layers of hierarchically interconnected quorum sensing systems
antiinfective strategies [1]. One of the leading causes for noso- [6]. Quorum sensing signals are released from the cells and
comial infections is the opportunistic human pathogen Pseudo- accumulate in a growing bacterial population to a certain
monas aeruginosa, which, by chronic infections, also poses a threshold by which they start inducing the production of viru-
major threat for cystic fibrosis patients [2,3]. P. aeruginosa lence factors. This simple signaling strategy thus regulates bac-
deploys numerous virulence factors such as toxins, extracel- terial behaviour in dependence of population density. One of
lular enzymes, and small molecule factors that are responsible these quorum sensing systems, the pqs system, uses 2-alkyl-4-
for the bacterium’s ability to invade the host and cause a broad quinolones (AQs) as signals of which the Pseudomonas
spectrum of different diseases [4,5]. The production of these quinolone signal (PQS) and its biosynthetic precursor 2-heptyl-
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4-quinolone (HHQ) are the two best studied AQs (Figure 1A) 2-aminobenzoylacetate (2-ABA) [13]. The PqsBC complex
[7]. A variety of virulence factors are under control of the pqs finally generates HHQ or other AQs in a decarboxylative con-
quorum sensing system, including the production of elastase, densation reaction of 2-ABA with fatty acids loaded on PqsC
pyocyanin, PA-IL lectin, and rhamnolipids, as well as popula- (Figure 1B) [14].
tions dynamic behaviours such as biofilm formation. However,
the exact roles of the different AQs are still not completely For the condensation step of an anthraniloyl residue with
understood [6,8].
malonyl-CoA by PqsD, a cysteine residue (Cys112) is involved
in the formation of a covalent thioester intermediate. We were
Besides HHQ and PQS, in total more than 50 structurally speculating that activity-based electrophilic probes may be
related AQs have been detected in P. aeruginosa [9]. Key to applicable to target this enzyme in vitro which could allow to
this large diversity of natural AQs are their common biosynthe- study its active site reactivity in greater detail and apply a
sis steps by enzymes encoded in the pqsABCDE operon [10]. competitive labelling platform to discover potential PqsD inhib-
The biosynthesis of AQs has been matter of a long-standing itors.
debate that could only recently be resolved. Although HHQ
Results and Discussion
could be produced in vitro by a PqsD catalyzed “head-to-head”
decarboxylative Claisen condensation of activated anthranilic Electrophilic activity-based probes
acid with β-keto fatty acid derivatives [10,11], isotope labelled The primary structure of PqsD comprises in total six cysteines.
feeding experiments indicated an entirely different mechanism However, only one of them, Cys112, is engaging in the cataly-
for its biosynthesis [12]. This mechanism has been elucidated tic process forming a covalent reaction intermediate. We thus
step by step in recent efforts by the work of various research aimed at exploring the possibility to selectively label the active
groups. Hereby, PqsA activates anthranilic acid to anthraniloyl- site cysteine residue using chemical probes.
CoA which is transferred to PqsD which catalyzes the conden-
sation with malonyl-CoA to form 2-aminobenzoylacetyl-CoA. Activity-based protein profiling (ABPP) has become a power-
The thioesterase PqsE hydrolyses the thioester to produce ful tool to study protein function and elucidate targets of pro-
Figure 1: Quinolone signals of Pseudomonas aeruginosa. A) Structures of HHQ and PQS. B) Proposed mechanism for the synthesis of 2-alkyl-4-
quinolones [12-14].
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tein-reactive natural products in complex proteomes [15-18]. alkyne handle were introduced to investigate potential differ-
Various types of probes with an electrophilic core have been ences in selectivity. Different alkyne amines were used to
applied as tools for in vitro and in situ experiments of activity- generate α,β-unsaturated amide probes UA1–3 by reaction with
based protein profiling [19-21]. ABPP uses probes with a reac- acrylic acid chloride (Figure 2A) and α-chloroacetamide probes
tive chemical group selectively targeting the active site of an CA1–3 by reaction with chloroacetyl chloride (Figure 2B). The
enzyme and a reporter group that allows in-gel imaging and/or α,β-unsaturated ketone probe UK1 was synthesized via the
affinity enrichment of target enzymes [22].
Weinreb–Nahm amide in a Grignard reaction with vinyl-
magnesium bromide (Figure 2C). An overview of the small
We thus synthesized a small library of chemical probes with ABPP probe library is given in Figure 2D.
electrophilic α-chloroacetamide, α,β-unsaturated amide, and
α,β-unsaturated ketone moieties as protein reactive groups, Active site specific labelling of PqsD
which have been reported to exhibit selectivity for active site Next, we were interested to investigate if any of the ABPP
cysteines [19] (Supporting Information File 1, Figure S1). Each probes was capable of labelling PqsD. Therefore, we cloned the
probe was equipped with a terminal alkyne handle for in-gel pqsD gene of Pseudomonas aeruginosa PAO1 into an expres-
analysis by fluorescence tagging via click chemistry with a cor- sion vector encoding an N-terminal strep-tag. The protein was
responding rhodamine azide. Variations of linker length and heterologously expressed in Escherichia coli BL21 followed by
side group decorations between the reactive group and the affinity purification by an ÄKTA chromatography system
Figure 2: Synthesis of electrophilic ABPP probes. A) Synthesis of α,β-unsaturated amide probes UA1–3. B) Synthesis of α-chloroacetamide probes
CA1–3, and C) synthesis of α,β-unsaturated ketone UK1. D) Structures of the ABPP probe library.
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equipped with a StrepTrap HP column. The individual probes peptides for CA2 labelled Cys112 underlining the selectivity of
were incubated with purified PqsD for 30 min and a rhodamine our probes (Figure 3C). These results indicate that probes
fluorescent reporter tag was appended by click chemistry. The CA1–3 are specific and covalently bind to Cys112 of PqsD and
remaining non-covalently bound probe and excess reporter tag are thus, to the best of our knowledge, the first account of activ-
were removed by SDS-polyacrylamide gel electrophoreses ity-based probes targeting and selectively labelling the active
(SDS-PAGE), and labelling of the protein was visualized by site of PqsD.
fluorescence imaging. Consistency of protein levels was
checked by coomassie staining (Supporting Information File 1, Interestingly, variations in the probe structure had little impact
Figures S2 and S3). While the α,β-unsaturated amide probes on labeling intensity and specificity. Although each α-chloro-
UA1–3 and the α,β-unsaturated ketone UK1 only resulted in acetamide probe had one closely related α,β-unsaturated acet-
very weak or no labeling, all three α-chloroacetamide probes amide counterpart, only the reactive group but not the structure
CA1–3 gave a strong fluorescent signal in the gel (Figure 3A). of the probe or its side groups determined active-site labeling.
These findings are surprising, as all three reactive groups are
In order to investigate the selectivity of the probes, we con- known to bind to cysteines which indicate a fine-tuned nucleo-
structed a PqsD C112A mutant, where the active site cysteine philic reactivity of the active site cysteine Cys112. The fine-
was replaced by alanine. The purified mutant PqsD C112A tuned nucleophilicity towards our probes is supported by calcu-
exhibited only low background labeling for some probes but not lations of a mechanistic model where Cys112 is activated by de-
comparable to labelling of the wild type protein by CA1–3, in- protonation by His257 [23]. Our results may also partially
dicating that the probes were selectively targeting the active site explain the potent inhibition of a PqsD inhibitor described in the
(Figure 3A). Concentration series with a dose-down of the three literature which was discovered in silico and had been equipped
CA probes showed that labelling of PqsD was concentration de- with an α-chloroacetyl group [24].
pendent and the two most potent probes CA1 and CA2 resulted
in significant labelling at concentrations as low as 200 nM. Inhibition of PqsD has been proposed as promising antiviru-
Mass spectrometric analysis of a tryptic digest of CA2 labelled lence strategy leading to disruption of AQ signaling and thereby
wild type PqsD resulted in an additional mass corresponding to to global down-regulation of virulence factor production [11].
a probe modified cysteine residue Cys112 confirming that the Consequently, PqsD has become a highly attractive target and a
CA probes indeed covalently labelled the active site cysteine. great amount of work pioneered by the Hartmann group has
Only one peptide was detected with another cysteine residue resulted in inhibitor discovery using a combination of in vitro
(Cys138) modified by the probe compared to 64 detected assay, in silico modelling and chemical lead optimization.
Figure 3: In vitro labeling of PqsD by chemical probes. A) ABPP probe library with wild-type PqsD and PqsD C112A mutant (PqsDm). B) Concentra-
tion dependence of labeling by the three active site directed probes. C) Mass spectrometric discovery of tryptic peptide fragments with probe CA2 at-
tached to the active site Cys112 (No.: number of detected peptides). D) Competitive experiment with N-ethylmaleimide and probe CA2.
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Examples of successful inhibitors are represented by the scaf- reaction for the synthesis of 2-heptyl-chromen-4-one (1-O-
folds of various 2-benzamidobenzoic acids [11,25,26], 2-nitro- HHQ, 4) which includes esterification, Baker–Venkataraman
phenyl derivatives [27-29], ureidothiophene-2-carboxylic acids rearrangement and subsequent acid-catalyzed ring closure to
[24,30], and catechol-based compounds [31].
affort the 1-O-HHQ in 60% yield [36] (Scheme 1).
Many promising in vitro inhibitors based on these leads have The 2-heptyl-3-hydroxychromen-4-one (1-O-PQS, 13) was pre-
been described and importantly, some of them also displayed in viously synthesized from the chroman-4-one 8 which was pro-
situ activity by reducing signal production and biofilm forma- duced by base-catalyzed Knoevenagel reaction from 2-hydroxy-
tion in live cultures of P. aeruginosa [28,31]. Recently, a syner- acetophenone with octanal. Although the starting material was
gistic dual PqsD and PqsR inhibitor was developed which also readily available, the reaction gave the product only in low
led to a marked decrease in the production of the virulence yield (20–30%) and separation of the starting material from the
factors pyocyanine and pyoverdine [32].
product could be difficult especially for multigram scale ap-
proaches. Since 1-O-HHQ (4) was now easily available, we
So far only laborious enzyme-based assays, docking studies or used 4 as starting point for the 1-O-PQS (13) synthesis by two
modelling resulted in new scaffolds. We were thus interested, if different approaches. First, we tried to synthesize chroman-4-
our probes could be applied as a simple tool to discover novel one 8 by hydrogenation of 4. We found that ammonium formate
scaffolds or chemical PqsD-binding motifs.
(NH4HCO2) as mild hydrogen source with Pd/C gave chroman-
4-one 8 in a clean reaction with a good yield of 76% whereas
the direct use of H2 with Pd/C gave mainly the fully reduced
Competitive screening approach
In order to test if our probes could be used as a competitive 2-heptylchromane. Compound 8 can be applied for the synthe-
labelling platform, we used the well-known unspecific cysteine sis of 1-O-PQS (13) as described in [33]. A new, direct way in
reactive agent N-ethylmaleimide (NEM) and dosed NEM in in- which 1-O-HHQ (4) can be used for the synthesis of 1-O-PQS
creasing concentrations to wild-type PqsD before applying the (13) was explored by epoxidation with subsequent ring opening
probe CA2 followed by click chemistry with the fluorophore. A in 41%. Thus, 1-O-PQS (13) could be produced in just two
decreasing labelling intensity at increasing NEM concentra- steps with an overall yield of 25% (Scheme 1). The synthesis of
tions indicates that NEM also blocks the active site cysteine 1-S-PQS (15) was previously accomplished by a two-step syn-
Cys112 at concentrations above 5 µM and thereby prevents thesis of thiochroman-4-one 9 and following oxidation to give
covalent attachment of the probe to PqsD (Figure 3D).
1-S-PQS (15) in 12% overall yield [33]. In our attempt to
synthesize 9 more efficiently, we used a method described by
We were thus interested to investigate if we could demonstrate Olah et al. [37] starting from commercially available (E)-dec-2-
the value of our activity-based probes in a competitive enoic acid and thiophenol which gave 9 in 53% yield without
screening approach by identifying potentially new scaffolds for the use of microwave assistance (Scheme 1). Thionation of the
PqsD inhibitors. We have recently reported the discovery of in- 4-position of 1, 4, 6, 10, 13 and 15 using P4S10 in pyridine
hibitors of the virulence factor elastase of P. aeruginosa by a under reflux conditions gave the 4-thiones 2, 5, 7, 11, 14 and 16
library of synthetic HHQ and PQS derivatives with systematic in yields between 60–80%, respectively (Scheme 1). The HHQ-
heteroatom replacements [33]. Because the interactions of PqsD oxime (3) was synthesized from HHQ (1) by conversion in the
with AQs is still not entirely understood, we reasoned that HHQ benzyl-protected chinolinol form (3a) and oximation with
or PQS analogues may be promising scaffolds for inhibitor de- hydroxylamine hydrochloride similar to the described method
velopment and we thus aimed to screen this library competi- used for the synthesis of the PQS-oxime 12 [33]. The entire
tively against the active site specific probe CA2. Therefore, we compound library of HHQ and PQS analogues is presented in
further refined the library by the synthesis of two additional Figure 4. Further details on the syntheses are given in the Sup-
HHQ analogues and implemented improved synthetic strate- porting Information File 1.
gies.
All compounds were screened at an initial concentration of
In detail, we synthesized 2-heptylquinolin-4(1H)-one (HHQ, 1) 240 µM in a competitive experiment against probe CA2. With
and 2-heptyl-3-hydroxyquinolin-4-one (PQS, 10) according to the compounds added as DMSO stocks, solubility of the com-
the described procedures by McGlacken et al. [34] and Hradil et pounds was not an issue at these concentrations. PqsD was
al. [35], respectively. We previously described the synthesis hereby pre-incubated with the compounds for 30 min, followed
of HHQ and PQS derivatives with nitrogen in position 1 by the addition of the probe. A compound interacting tightly
exchanged by oxygen and sulfur [33]. In our efforts to optimize with the active site would hinder the probe from binding to the
the synthesis of these heteroatom derivatives we used a one-pot active site. Thus, a reduced labelling intensity in a competitive
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Scheme 1: Synthesis of various HHQ and PQS analogues.
screening experiment indicates a potential hit compound To assess the stability of the probe, we incubated CA2 with two
(Figure 5A).
of the most active compounds, 11 and 14. NMR and MS data
indicate that the probe was not chemically modified even after
Strikingly, half of the derivatives were significantly active in 18 and 24 h so that the potential inactivation of the probe by the
the competitive screening and abolished probe labelling at compound scaffold during protein labelling could be ruled out
240 µM, while the other compounds had no such effect at this (Supporting Information File 1, Figures S4 and S5).
initial concentration (Figure 5B). Active compounds were found
in pairs of HHQ and their corresponding PQS derivatives and Our 4S-PQS analogues thus represent a promising novel scaf-
either comprised a 4-thionated HHQ (2, 5, 7) or PQS (11, 14, fold that inhibits the labelling of PqsD by an active-site-directed
16) scaffold or an oxime group in position 4 (3 and 12).
probe in the lower micromolar range. We have previously de-
scribed these compounds (11, 14, and 16) as potent inhibitors of
The eight active compounds were tested in a concentration-de- the virulence factor elastase (LasB) of Pseudomonas aerugi-
pendent experiment in order to assess their potency in inhibit- nosa [33]. However, the mechanism of inhibition was by direct
ing probe binding. Interestingly, the HHQ derivative 3 with an binding to the active site of elastase. While elastolytic activity
oxime group was significantly more active than its PQS coun- was completely inhibited with 11 even in situ we could not
terpart 12, with the lowest activity. In contrast, the 4-thionated confirm any significantly large inhibition of rhamnolipid or
PQS derivatives 11, 14 and 16 were always more active than pyocyanin production. Nevertheless, our new compounds may
their corresponding HHQ analogues (2, 5, and 7), and effi- be useful scaffolds for the future development of a novel gener-
ciently blocked probe labelling already around 24 µM. These ation of PqsD inhibitors.
results indicate that the 3-OH group was important for the activ-
ity of the 4-thionated compounds. In order to exclude any Conclusion
adverse effects of the compounds on the click chemistry, we Electrophilic probes represent powerful tools for investigating
performed control experiments where the most active deriva- protein reactivity and discovering customized enzyme inhibi-
tives were added directly before the last step of the click tors. We discovered α-chloroacetamide probes selectively
protocol. Intense labelling in this control group indicated that labelling the active site cysteine residue of the Pseudomonas
click chemistry was not affected by the compounds (Figure 5D). aeruginosa quorum sensing signal synthase PqsD. While these
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Figure 4: Library of HHQ and PQS analogues.
findings may guide the future development of covalent PqsD in- of 4S-PQS analogues as potent in vitro inhibitors of the active-
hibitors, we could also demonstrate the value of the probes as site labelling of PqsD. In combination, our probes and their in-
tools for investigating the reactivity of PqsD and apply them in hibitors represent a valuable toolkit for investigating this impor-
a competitive screening approach. These led to the novel class tant virulence-related enzyme.
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Figure 5: Competitive profiling platform. A) Schematic representation of the competitive labelling strategy with an alkyne probe (green) and potential
inhibitors (blue). Rh = rhodamine. B) Initial screening of our small library of HHQ (1–9) and PQS (10–16) analogues against the active site specific
chemical probe CA2. C) Concentration dependent competition experiment where the probe concentration is held constant and PqsD is pre-treated
with varying inhibitor concentrations. D) Click chemistry control where the compounds were added shortly before CuII-salt addition in the click
protocol. Cont.: DMSO control.
Chemical Biology (KoRS-CB), and CRC969 (DFG). MP was
Supporting Information
supported by a Carl Zeiss Ph.D. fellowship and DS received a
Ph.D. fellowship from KoRS-CB. We thank Jing Qiao for
Supporting Information File 1
helping with protein purification and gel-based labelling experi-
ments and Martin Mex and Daniel Hammler for help with mass
spectrometry. We also acknowledge the help of the Proteomics-
facility and NMR core facility of the University of Konstanz.
Syntheses, and full compound characterization,
experimental methods, and probe labelling.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-12-277-S1.pdf]
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