Exploring the chemical space of ureidothiophene-2-carboxylic acids as inhibitors of the quorum sensing enzyme PqsD from Pseudomonas aeruginosa

Article abstract of DOI:10.1016/j.ejmech.2015.04.007

Pseudomonas aeruginosa employs a quorum sensing (QS) communication system that makes use of small diffusible molecules. Among other effects, the QS system coordinates the formation of biofilm which decisively contributes to difficulties in the therapy of

Full text of DOI:10.1016/j.ejmech.2015.04.007

European Journal of Medicinal Chemistry 96 (2015) 14e21
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Exploring the chemical space of ureidothiophene-2-carboxylic acids
as inhibitors of the quorum sensing enzyme PqsD from Pseudomonas
aeruginosa
J. Henning Sahner a, Martin Empting a, Ahmed Kamal a, Elisabeth Weidel a,
a
b
a, *
€
Matthias Groh , Carsten Borger , Rolf W. Hartmann
a Pharmaceutical and Medicinal Chemistry, Saarland University & Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Department of Drug
Design and Optimization, Campus C2 3, 66123 Saarbrücken, Germany
b PharmBioTec GmbH, Science Park 1, 66123 Saarbrücken, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Pseudomonas aeruginosa employs a quorum sensing (QS) communication system that makes use of small
diffusible molecules. Among other effects, the QS system coordinates the formation of biofilm which
decisively contributes to difficulties in the therapy of Pseudomonas infections. The present work deals
with the structure-activity exploration of ureidothiophene-2-carboxylic acids as inhibitors of PqsD, a key
enzyme in the biosynthetic pathway of signal molecules in the Pseudomonas QS system. We describe an
improvement of the inhibitory activity by successfully combining features from two different PqsD in-
hibitor classes. Furthermore the functional groups, which are responsible for the inhibitory potency,
were identified. Moreover, the inability of the new inhibitors, to prevent signal molecule formation in
whole cell assays, is discussed.
Received 1 October 2014
Received in revised form
1 April 2015
Accepted 2 April 2015
Available online 6 April 2015
Keywords:
Quorum sensing
Pseudomonas aeruginosa
Bacterial cell-to-cell communication
Ureidothiophene-2-carboxylic acids
Antibacterial agents
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
signaling allows the bacterial population to assess its cell density
and coordinate group behavior. The Pseudomonas QS system con-
Antibiotic therapy is characterized by the everlasting competi-
tion between the generation of novel antibacterial substances and
the development of the respective bacterial resistances [1,2]. One
outstanding example is the opportunistic pathogen Pseudomonas
aeruginosa which is responsible for severe infections and is a
leading cause of death in cystic fibrosis patients [3]. Its ability to
rapidly form resistances against the currently used antibiotics ne-
cessitates new approaches for antibacterial treatment [4,5]. Typi-
cally, antibiotics affect bacterial viability and thus cause a selection
pressure that inevitably leads to the development of resistances. In
recent years several research groups have been trying to break out
of this vicious cycle by reducing the virulence of the pathogens
instead of affecting their viability [6]. One approach is the inhibition
of the bacterial cell-to-cell communication systems like QS [7]. In
QS, bacterial cells release a variety of small diffusible molecules
which can be detected by other bacteria. This kind of molecular
sists of two N-acylhomoserine lactone (AHL) regulatory circuits (las
and rhl) linked to an 2-alkyl-4-quinolone (AQ) system [3]. Whereas
the AHL systems are widespread among Gram negative bacteria [8],
our group focuses on the so called Pseudomonas quinolone signal
quorum sensing (PQS-QS) system, an AQ system that exclusively
exists in P. aeruginosa and some Burkholderia strains [9]. It is
involved in the production of a number of virulence factors that
contribute to their pathogenicity [10]. Moreover, it takes part in
regulating the formation of biofilms, a main cause for bacterial
resistance against conventional antibiotics in clinical use [11]. The
pqsABCDE operon, whose expression is controlled by the tran-
scriptional regulator PqsR (MvfR), directs the AQ biosynthesis in
P. aeruginosa. Molecules, produced upon activity of this operon are
among others, 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas
quinolone signal, PQS) and its biosynthetic precursor 2-heptyl-4-
quinolone (HHQ) [12]. HHQ and PQS themselves activate PqsR
leading to an auto-induction of the pqsABCDE operon [13,14]. Be-
sides, they can modulate the innate immune response of
mammalian cells, affecting the first defense barrier of the host
* Corresponding author.
E-mail address: rolf.hartmann@helmholtz-hzi.de (R.W. Hartmann).
http://dx.doi.org/10.1016/j.ejmech.2015.04.007
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

J.H. Sahner et al. / European Journal of Medicinal Chemistry 96 (2015) 14e21
15
[15,16,17]. Compounds, interfering with the PQS-QS system have
proven to be promising candidates for drug development. Treat-
ment with antagonists of the PqsR receptor enabled the survival of
P. aeruginosa infected Galleria mellonella larvae [18]. Furthermore
we could show that inhibitors of the enzyme PqsD, a key player in
the AQ biosynthesis (Scheme 1), are able to reduce biofilm in
P. aeruginosa cultures [19]. It was recently reported by Dulcey et al.,
that PqsD produces 2-aminobenzoylacetate-coenzyme A (2-ABA-
CoA), a highly active intermediate in the HHQ biosynthesis, by
converting anthraniloyl-coenzyme A (ACoA) with malonyl CoA.
Firstly, ACoA is covalently transferred to C112 of PqsD, followed by
the reaction with malonyl-CoA. Further conversion of the inter-
mediate 2-ABA-CoA in several enzymatic steps finally results in
HHQ and PQS (Scheme 1) [17]. Interestingly, PqsD is also capable of
Fig. 1. Docking pose of B inside the binding channel of PqsD.
directly producing HHQ by converting ACoA with b-ketodecanoic
acid [20]. In the recent past we frequently used this enzymatic
reaction to assess the activity of PqsD inhibitors [19,21e25]. For
compounds interfering with the first enzymatic step, the formation
of the PqsD-CoA complex, this is still a valid method [23,24].
In a recent work, we reported on the class of ureidothiophene-
2-carboxylic acids as potent inhibitors of PqsD. Biophysical
methods were used to confirm a binding hypothesis derived from
molecular docking studies. This approach enabled the structure-
based optimization of a screening hit compound resulting in a se-
ries of highly active molecules (e.g. A and B in Chart 1) [21]. Ac-
cording to our docking pose and the results from SPR competition
experiments, the most active derivative B occupies an area, span-
ning from the entrance of the binding channel to the active site,
leaving a gap of about 6 Å to the bottom of the pocket where the
catalytic residues are located. Its carboxylic acid groups are sup-
posed to interact with Asn154 and Arg262 respectively anchoring
the inhibitor in the binding channel of PqsD The phenylalanine
residue perfectly fits into a narrow pocket at the channel's entrance
delimited by Arg 223 and Phe226 and (Fig. 1).
In this work we describe further exploration of the chemical
space, the structure activity relationships (SAR) and the intracel-
lular effects of this class of inhibitors.
Fig. 2. Illustration of the proposed clash between the additional methoxy group and
the protein residue Arg223.
2. Results and discussion
According to our binding hypothesis, the carboxylate group of
the amino acid part in A and B interacts with Arg262 at the
entrance of the PqsD binding channel (Fig. 1). The natural substrate
anthraniloyl CoA (ACoA) builds several ion bridges between its
phosphoric acid groups and the arginines on the surface of the
protein. Such interactions are considered very potent in literature
[26,27]. Inspired by ACoA we replaced the carboxylic acid moiety by
a phosphoric acid group (1). In comparison to the glycine derivative
A the activity did not increase. Based on these findings, the phos-
phoric acid was not considered for further optimization due to
synthetic reasons.
The so far most active inhibitor B carries a phenylalanine sub-
stituent at the ureido motif. In order to fine-tune the electronic
properties of the aromatic system we investigated electron
donating and electron withdrawing substituents in para-position
(Table 1). In case of an electron donating hydroxyl function (2), the
activity dropped to 29
mM similar to an additional methoxy group
(3; IC50: 31 M). In case of the latter this is probably due to steric
m
reasons, which is in good agreement with our binding hypothesis
(Fig. 2).
Introduction of an electron withdrawing and lipophilic chlorine
substituent (4) also resulted in decreased inhibitory potency
(14
mM). As both kinds of substituents with inverse electronic
properties were detrimental to the activity, we considered the
unsubstituted ring as most favorable. In the next step we shortened
(5) and prolonged (6) the linker between the a-position and the
phenyl ring. In the crystal structure, the entrance to the sub-pocket,
delimited by Arg223 and Phe226 and Glu227 is narrow and
therefore requires a special conformation. This conformation is
obviously only provided by the compound with the methylene
linker as both, 5 and 6 displayed significantly weaker inhibitory
potency. As a last trial to explore the SAR at this part of the
Scheme 1. Biosynthetic pathway of the signal molecules HHQ and PQS according to
Dulcey et al. [15].

16
J.H. Sahner et al. / European Journal of Medicinal Chemistry 96 (2015) 14e21
Fig. 3. Energy minimized conformations showing the orientation between the phenyl ring and the thiophene core of B, 15 and 16.
Fig. 4. a) Docking pose of B (green) and C turquoise). b) Docking pose of B (green) and D (yellow). (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
focused on the opposite side of the molecule. Firstly parts of the
methoxy equipped ring of B were removed to determine the
essential functional groups (Table 2). Demethylation, resulting in
hydroxyl compound 12 decreased the activity. Further omitting this
OH-group (13) however partially restored it. This leads to the
assumption that the oxygen of the methoxy group contributes to
the activity to a certain extent, presumably as a hydrogen bond
acceptor. In absence of an appropriate interaction partner the
hydrogen bond donor of 13 is surrounded by highly ordered water
molecules, leading to an entropic loss and, therefore, a lowered
activity. Removal of the entire methoxyphenyl ring (14) results in a
total loss of inhibitory potency. To reveal the bioactive conforma-
tion and the orientation of the methoxyphenyl ring towards the
thiophene, 15 and 16 were investigated. The methyl group of 15
increased the IC50 to 8 mM suggesting an ortho-effect and thus an
unfavorable perpendicular orientation of the two rings or a steric
clash of the additional substituent. Rigidification by an ethylene
linker (16), which directly connects the two aromatic systems
causing a planar structure (Fig. 3), did not improve the activity as
well. The tight shape of the PqsD binding channel that demands
certain flexibility from entering inhibitors can once more be an
explanation for these findings.
Fig. 5. Docking pose of B (green) extended with the NAC moiety (blue) present in 24,
illustrating how the additional residue is directed outside the binding channel of PqsD.
(For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
The proposed interaction between the carboxylic acid at the
thiophene core with Asn154 was corroborated by compound 17.
Removal of the carboxylic group decreased the activity by about
twentyfold.
By the application of SPR competition experiments in the above
mentioned earlier work [21], we were able to narrow down the
position of the ureidothiophene-2-carboxylic acids within the
binding channel compared to known PqsD inhibitors from the 2-
(nitrophenyl)-methanol class (C and D in Chart 2). Whereas the
shorter compound C (Fig. 4a turquoise) did not affect the binding
affinity of B, the longer derivative D (Fig. 4b yellow) prevented
binding of B (data not shown), fitting to our binding hypothesis.
According to the docking pose, a linkage of the two inhibitor
classes in order to combine their interactions with PqsD should be
Chart 1. Structures of the PqsD inhibitors A and B.
molecule, further readily available (S)-amino acids were intro-
duced. None of the resulting compounds (7e11) outperformed the
potency of B.
We proceeded, retaining the phenylalanine residue at the
ureido-motif, as the most promising moiety and subsequently

J.H. Sahner et al. / European Journal of Medicinal Chemistry 96 (2015) 14e21
17
Table 1
Inhibitory activities of 1ꢀ11 against P. aeruginosa PqsD in vitro.
Table 2
Inhibitory activities of 12ꢀ17 against P. aeruginosa PqsD in vitro.
Entry
Structure
Inhibition of PqsDa
12
21.6 3.9
mM
Entry
R
Inhibition of PqsDa
7.0 0.7
13
3.8 1.4 mM
1
mM
2
3
4
5
29.0 0.01 mM
14
15
no inhibition @ 50 mM
31.3 3.7
13.8 1.1
~40% @ 50
m
M
7.5 1.8 mM
mM
m
M
M
M
16
17
a
23.7 0.02 mM
6
7
33.7 1.5
31.9 3.0
m
m
11.5 2.0 mM
8
9
19.0 2.6
13.2 0.9
m
M
M
IC50 values of PqsD.
m
sum of interactions of the respective single compounds B and D. To
confirm, that the nitro-substituted ring of 22 reaches deep into the
binding channel, SPR experiments were performed. Firstly, the
binding responses of B, 21 and 22 towards PqsD were recorded in
the absence of ACoA. In a second experiment, the PqsD loaded
sensor chip was treated with ACoA. According to the catalytic
mechanism of PqsD this results in the anthranilate-PqsD complex,
in which the anthranilic acid is covalently bound to Cys112. Sub-
sequently the binding responses of B, 21 and 22 to this complex
were determined. Only the signals of 22 showed significant dif-
ferences between treated and untreated PqsD (Figure S1 in
supporting information). Since this behavior is typical for the 2-
nitrophenylmethanol derivatives [22], occupation of the same
binding site can be assumed.
10
5.0 0.9 mM
11
30.3 0.3 mM
a
IC50 values of PqsD.
possible. We decided to enlarge the unsubstituted compound 13
step by step to achieve a full combination with D. Due to the
absence of strong interactions with the protein, docking studies
never delivered an unambiguous orientation of the aryl ring at the
thiophene core within the pocket. Even though an attachment in 3-
position (18,19) should be favorable according to the dockings with
compound B (compare Fig. 4), expansion in 4-position (20ꢀ22)
delivers better results (Table 3). We hypothesized that this is once
again due to the tight shape of the binding channel which hampers
the entrance of the stronger tilted compounds 18 and 19. Whereas
the introduction of the first phenyl ring (20) did not result in better
inhibition, elongation with a hydroxy-methyl function (21), which
is supposed to mimic the one of C and D increased the activity by
twentyfold compared to 13 (factor three compared to B). Attach-
ment of the second, nitro-substituted ring (22) forfeits parts of the
activity gain. A possible explanation could be that the final com-
pound is large and inflexible and, is therefore, incapable of adapting
to conformational changes which would be necessary to retain the
Although the inhibitors displayed high potency in the cell free
enzyme assay, none of them was able to reduce the HHQ levels in a
whole cell P. aeruginosa assay. These findings can be attributed to
different reasons like permeation- or efflux problems. Several steps
were taken to achieve an intracellular activity.
Chart 2. Structures of the PqsD inhibitors C and D and their in vitro IC50 values against
P. aeruginosa PsqD.

18
J.H. Sahner et al. / European Journal of Medicinal Chemistry 96 (2015) 14e21
Table 3
3. Conclusions
Inhibitory activities of 18ꢀ22 against P. aeruginosa PqsD in vitro.
In conclusion, we further explored the chemical space of the
ureidothiophene-2-carboxylic acids as inhibitors of PqsD. The
pharmacophore of the inhibitor class was determined and the es-
sentiality of several functional groups was clarified. Moreover, two
inhibitor classes could be successfully merged without having ac-
cess to structural information of protein-ligand x-ray structures.
The resulting compounds display higher inhibitory activity by
profiting from the combined interactions with the protein.
Following this approach, the most potent PqsD inhibitors described
so far were obtained. Although the potency in cell free assay was
high, an intracellular activity could not be achieved even by
attachment of a cell penetrating peptide. We assume that the class
of inhibitors is subject to efflux causing natural resistance of
P. aeruginosa towards the newly developed antibacterial agents.
Therefore, we consider the ureidothiophene-2-carboxylic acids to
be not eligible for further development in the field of Pseudomonas
quorum sensing inhibitors. The problem could eventually be solved
by a combined application with efflux pump inhibitors, or by using
pharmaceutical technological methods, but this is beyond the
frame of this work. Nevertheless, important interactions of func-
tional groups with the protein were revealed that can be used to
improve the inhibitory activity of other PqsD inhibitors with better
intracellular effects.
Entry
3-Position
4-Position
H
Inhibition of PqsDa
6.2 0.7
18
mM
19
H
19.4 0.02
m
M
20
21
H
H
2.7 0.5
m
M
0.14 0.03
m
m
M
22
H
0.36 0.06
M
a
IC50 values of PqsD.
Fluoroquinolone and
b-lactam antibiotics are mostly zwitter-
4. Experimental procedures
ionic. According to several reports in literature, this feature signif-
icantly contributes to their transport into the cell, which was shown
4.1. Chemistry
especially for the b-lactams [28,29]. Inspired by that we introduced
S-histidine instead of S-phenylalanine, using the imidazole ring as a
bioisostere of the phenyl ring while gaining a basic function and
therefore a potentially positive charge at the same time. The
resulting compound 23 displayed weak activity against PqsD (40%
4.1.1. Materials and methods
Starting materials were purchased from commercial suppliers
and used without further purification. Column flash chromatog-
raphy was performed on silica gel (40e63
mM), and reaction
inhibition at 50
very moderate reduction of HHQ levels in the whole cell assay
(Reduction of HHQ at 250 M: 16 5%). In a second attempt, we
mM) but showed for the first time significant but
progress was monitored by TLC on TLC Silica Gel 60 F254 (Merck). All
moisture-sensitive reactions were performed under nitrogen at-
mosphere using oven-dried glassware and anhydrous solvents.
Preparative RP-HPLC was carried out on a Waters Corporation setup
containing a 2767 sample manager, a 2545 binary gradient module,
a 2998 PDA detector and a 3100 electron spray mass spectrometer.
Purification was performed using a Waters XBridge column (C18,
m
made use of an N-acetyl thioester (NAC-ester) which is frequently
used in mutasynthesis programs. The NAC adducts thereby serve as
mimics of coenzyme A esters which improve their acceptance as
precursors in biosynthesis and might also facilitate the entrance
into bacterial cells in comparison to the free acids [30]. The car-
boxylic acid moiety of the phenylalanine was considered more
suitable for the attachment of an NAC unit than the one at the
thiophene core. It is presumably positioned at the entrance of the
pocket (Fig. 5) directing the additional substituent outside the
binding channel of PqsD, and therefore avoiding steric hindrance.
Thus an intracellular cleavage of the thioester to set the active form
free might not be mandatory. The resulting compound 24 (Chart 3)
150 ꢁ 19 mm, 5 m), a binary solvent system A and B (A ¼ water
m
with 0.1% formic acid; B ¼ MeCN with 0.1% formic acid) as eluent, a
flow rate of 20 mL/min and a gradient of 60%e95% B in 8 min were
applied. 1H and 13C NMR spectra were recorded on a Bruker Fourier
spectrometer (500/300 or 125/75 MHz) at ambient temperature
with the chemical shifts recorded as
d values in ppm units by
reference to the hydrogenated residues of deuterated solvent as
internal standard. Coupling constants (J) are given in Hz and signal
patterns are indicated as follows: s, singlet; d, doublet; dd, doublet
of doublets; t, triplet; m, multiplet, br., broad signal. Purity of the
final compounds was determined by HPLC. The Surveyor LC system
consisted of a pump, an autosampler, and a PDA detector. Mass
spectrometry was performed on a MSQ electrospray mass spec-
trometer (ThermoFisher, Dreieich, Germany). The system was
still displayed reasonable activity (IC50: 32 mM) but turned out to be
inactive in the whole-cell assay. We further examined the intro-
duction of a cell penetrating peptide (25) at the same position.
Again, the inhibitory activity on the cell free level could be retained,
but no inhibitory effect in the whole cell assay was observed at the
test concentration.
Chart 3. Structures of compound 23ꢀ25, carrying moieties (highlighted in red) which should facilitate the entrance into Gram-negative cells. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)

J.H. Sahner et al. / European Journal of Medicinal Chemistry 96 (2015) 14e21
19
operated by the standard software Xcalibur. A RP C18 NUCLEODUR
100e5 (125 mm ꢁ 3 mm) column (MachereyeNagel GmbH, Düh-
ren, Germany) was used as the stationary phase. All solvents were
HPLC grade. In a gradient run the percentage of acetonitrile (con-
taining 0.1% trifluoroacetic acid) was increased from an initial
concentration of 0% at 0 min to 100% at 15 min and kept at 100% for
125 mM Na2HPO4, 50 mM KH2PO4 pH 7.6, 50 mM NaCl, 10% glycerol
(v/v), using a PD10 column and judged pure by SDS-PAGE analysis.
Then protein was stored in aliquots at ꢀ80 ꢂC.
4.2.2. Screening assay procedure for in vitro PqsD inhibition [20].
The assay was performed monitoring enzyme activity by
measuring HHQ formed by condensation of anthraniloyl-CoA and
5 min. The injection volume was 10
800
mL, and flow rate was set to
m
L/min. MS analysis was carried out at a spray voltage of
b-ketodecanoic acid. The reaction mixture contained MOPS buffer
3800 V and a capillary temperature of 350 ꢂC and a source CID of
10 V. Spectra were acquired in positive mode from 100 to 1000 m/z
at 254 nm for the UV trace.
(0.05 M, pH 7.0) with 0.005% (w/v) Triton X-100, 0.1 M of the
m
purified enzyme and inhibitor. The test compounds were dissolved
in DMSO and diluted with buffer. The final DMSO concentration
was 0.5%. After 10 min preincubation at 37 ꢂC, the reaction was
started by the addition anthraniloyl-CoA to a final concentration of
4.1.2. Synthesis and spectroscopic details
The synthesis of most of the 5-aryl-3-ureidothiophene-2-
carboxylic acids (Scheme 2) started from readily available aceto-
phenones (I) which were converted to the 5-aryl thiophene an-
thranilic acid methylesters (II) via an Arnold-Vilsmaier-Haack
reaction followed by a cyclization using methylmercaptoacetate
[31]. The esters (II) were then hydrolyzed under basic conditions to
afford the thiophene anthranilic acids (III) which were converted
into the thiaisatoic anhydrides (IV) [32,33]. The anhydrides (IV)
were reacted with various amines giving rise to the 5-aryl-3-
ureidothiophene-2-carboxylic acids (V) [34]. Further substituents
at the 5-aryl ring were introduced using boronic acids or esters,
respectively, via Suzuki coupling yielding VI [35].
5
m
M and
b-ketodecanoic acid to a final concentration of 70 mM.
Reactions were stopped by addition of MeOH containing 1
mM
amitriptyline as internal standard for LC/MSeMS analysis. HHQ was
quantified using a HPLC-MS/MS mass spectrometer (ThermoFisher,
Dreieich, Germany) in ESI mode. Ionization of HHQ and the internal
standard amitriptyline was optimized in each case. The solvent
system consisted of 10 mM ammonium acetate (A) and acetonitrile
(B), both containing 0.1% trifluoroacetic acid. The initial concen-
tration of B in A was 45%, increasing the percentage of B to 100% in
2.8 min and keeping it at 98% for 0.7 min with a flow of 500 mL/min.
The column used was a NUCLEODUR-C18, 100e3/125e3 (Macherey
Nagel, Dühren, Germany). Control reactions without the inhibitor,
but including identical amounts of DMSO, were performed in par-
allel and the amount of HHQ produced was set to 100%.
Further details on the synthesis and spectroscopic data of final
compounds and intermediates can be found in the Supporting
information.
4.2. Biology
4.2.3. Determination of extracellular HHQ and PQS levels
For determination of extracellular levels of HHQ produced by
PA14, cultivation was performed in the following way: cultures
(initial OD600 ¼ 0.02) were incubated with or without inhibitor
(final DMSO concentration 1%, v/v) at 37 ꢂC, 200 rpm and a hu-
midity of 75% for 16 h in 24-well Greiner BioOne (Frickenhausen,
Germany) Cellstar plates containing 1.5 mL of LB medium per well.
For HHQ analysis, according to the method of Lepine et al. [36],
4.2.1. General procedure for expression and purification of
recombinant PqsD WT and R223A mutant in Escherichia coli
His6-tagged PqsD (H6-PqsD) and mutants were expressed in
E. coli and purified using a single affinity chromatography step.
Briefly, E. coli BL21 (
(kindly provided by Prof. Rolf Müller, Helmholtz Institute for
Pharmaceutical Research Saarland (HIPS), Saarbrücken, Germany)
l
DE3) cells containing the pET28b(þ)/pqsD
500
mL of the cultures supplemented with 50 mL of a 10 mM
were grown in LB medium containing 50
m
g/mL kanamycin at 37 ꢂC
methanolic solution of the internal standard (IS) 5,6,7,8-
tetradeutero-2-heptyl-4(1H)-quinolone (HHQ-d4) were extracted
with 1 mL of ethyl acetate. After centrifugation (18,620 g, 12 min),
to an OD600 of approximately 0.8 units and induced with 0.2 mM
IPTG for 16 h at 16 ꢂC. The cells were harvested by centrifugation
(5000 rpm, 10 min, 4 ꢂC) and the cell pellet was resuspended in
100 mL binding buffer (10 mM Na2HPO4, 2 mM KH2PO4 pH 7.4,
3 mM KCl, 137 mM NaCl, 20 mM imidazole, 10% glycerol (v/v)) and
lysed by sonication for a total process time of 2.5 min. Cell debris
were removed by centrifugation (18500 rpm, 40 min, 4 ꢂC) and the
400
mL of the organic phase were evaporated to dryness and
redissolved in methanol. UHPLC-MS/MS analysis was carried out as
described in detail by Storz et al. [19] The monitored ions were
(mother ion [m/z], product ion [m/z], scan time [s], scan width [m/
z], collision energy [V], tube lens offset [V]): HHQ: 244, 159, 0.5,
0.01, 30, 106; HHQ-d4(IS): 248, 163, 0.1, 0.01, 32, 113. For each
sample, cultivation and sample work-up were performed in tripli-
cates. Inhibition values of HHQ formation were normalized to
supernatant was filtered through a syringe filter (0.20
mm). The
clarified lysate was immediately applied to a Ni-NTA column,
washed with binding buffer and eluted with 500 mM imidazole.
The protein containing fractions were buffer-exchanged into
OD600
.
Scheme 2. Synthesis of 5-aryl-3-ureidothiophene-2-carboxylic acids V or VI, respectively. Reagents and conditions: (a) POCl3, DMF, 50 ꢂC to rt, then NH2OH$HCl, up to 150 ꢂC. (b)
Methylthioglycolate, NaOMe, MeOH, reflux. (c) KOH, MeOH, THF, H2O, reflux. (d) COCl2, THF. (e) Amine, H2O, 100 ꢂC then at 0 ꢂC conc. HCl. (f) Na2CO3, Pd(PPh3)4, boronic acid or
ester, THF, H2O, toluene, 80 ꢂC.

20
J.H. Sahner et al. / European Journal of Medicinal Chemistry 96 (2015) 14e21
[2] C. Walsh, Molecular mechanisms that confer antibacterial drug resistance,
4.2.4. Surface plasmon resonance
Nature 406 (2000) 775e781.
4.2.4.1. General. SPR binding studies were performed using a
Reichert SR7500DC instrument optical biosensor (Reichert Tech-
nologies, Depew, NY, USA) and CMD500M sensor chips obtained
from XanTec (XanTec Bioanalytics, Düsseldorf, Germany). Scrubber
2 software (Version 2.0c, 2008, BioLogic Software) was used for
proceeding and analyzing the data. Changes in refractive index due
to DMSO dependent solvent effects were corrected by use of a
calibration curve (seven solutions, 4.25%e5.75% DMSO in buffer).
[3] J.-F. Dubern, S.P. Diggle, Quorum sensing by 2-alkyl-4-quinolones in Pseudo-
monas aeruginosa and other bacterial species, Mol. Biosyst. 4 (2008) 882e888.
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4.2.4.2. Immobilization of His6-PqsD. PqsD (38 kDA, >90% pure
based on SDS-PAGE) was immobilized at an level of 5919 RU on a
CMD500M (carboxymethyldextran-coated) sensor chip at 18 ꢂC
analogous to the method described by Henn et al. [37].
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ꢀ
S.R. Chhabra, M. Camara, P. Williams, Functional genetic analysis reveals a 2-
4.2.4.3. Binding studies. The ACoA preincubation studies were
performed as previously described using a constant flow rate of
alkyl-4-quinolone signaling system in the human pathogen Burkholderia
pseudomallei and related bacteria, Chem. Biol. 13 (2006) 701e710.
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required for extracellular quinolone signaling by Pseudomonas aeruginosa,
J. Bacteriol. 184 (2002) 6472e6480.
25
HEPES, pH 7.4, 150 mM NaCl, 5% DMSO (v/v), 0.05% Polysorbat 20
(v/v)) [20]. ACoA (100 M) was injected for approximately 40 min
with a constant flow of 5 L/min to reach saturation of the ACoA
binding site. Afterwards, the flow rate was increased to 50 L/min
mL/min and HEPES buffer as instrument running buffer (10 mM
m
m
m
for 30 min in order to flush all CoA away. Once the baseline is stable
again the compounds were consecutively injected and the re-
sponses at equilibrium were compared to those obtained with the
untreated surface. Experiments were performed in duplicate. For B
[13] D.S. Wade, M. Worth Calfee, E.R. Rocha, E.A. Ling, E. Engstrom, J.P. Coleman,
E.C. Pesci, Regulation of Pseudomonas quinolone signal synthesis in Pseudo-
monas aeruginosa, J. Bacteriol. 187 (2005) 4372e4380.
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A.P. Tampakaki, S.E. Stachel, L.G. Rahme, MvfR, a key Pseudomonas aeruginosa
pathogenicity LTTR-class regulatory protein, has dual ligands, Mol. Microbiol.
62 (2006) 1689e1699.
a concentration series of 250, 125 and 62.5
stronger binding signal was observed for 21, the concentration
series was decreased to 100, 50 and 25 M, whereas a series of 25,
12.5, and 6.25 M was measured for 22. The compounds were
mM were used. Since
m
[15] A. Bandyopadhaya, M. Kesarwani, Y.-A. Que, J. He, K. Padfield, R. Tompkins,
L.G. Rahme, The quorum sensing volatile molecule 2-amino acetophenon
modulates host immune response in a manner that promotes life with un-
wanted guests, PLoS Pathog. 8 (2012) e1003024.
m
injected for 120 s association times and 300 s dissociation times.
ꢀ
[16] K. Kim, Y.U. Kim, B.H. Koh, S.S. Hwang, S.-H. Kim, F. Lepine, Y.-H. Cho, G.R. Lee,
4.3. Computational chemistry
HHQ and PQS, two Pseudomonas aeruginosa quorum-sensing molecules,
down regulate the innate immune responses through the nuclear factor-kB
pathway, Immunology 129 (2010) 578e588.
4.3.1. Docking
[17] C.E. Dulcey, V. Dekimpe, D.-A. Fauvelle, S. Milot, M.-C. Groleau, N. Doucet,
Inhibitors were built in MOE. The receptor was derived from
crystal structure of PqsD in complex with ACoA (PDB Code: 3H77)
[10] The residuals of CoA, the covalently bound anthranilate and
H2O were removed and Cys112 was restored considering its
conformation in 3H76 [10]. AutoDockTools V.1.5.6 was used to add
polar hydrogens and to save the protein in the appropriate file
formate for docking with Vina. AutoDockVina was used for docking
calculations [38]. The docking parameters were kept at their default
values. The docking grid was sized 18 Å ꢁ 24 Å ꢁ 24 Å, covering the
entire ACoA channel.
ꢀ
ꢀ
L.G. Rahme, F. Lepine, E. Deziel, The end of an old hypothesis: the Pseudo-
monas signaling molecules 4-hydroxy-2-alkylquinolines derive from fatty
acids, not 3-ketofatty acids, Chem. Biol. 20 (2013) 1481e1491.
[18] C. Lu, C.K. Maurer, B. Kirsch, A. Steinbach, R.W. Hartmann, Overcoming un-
expected functional inversion of a PqsR antagonist in Pseudomonas aerugi-
nosa: an in vivo potent antivirulence agent targeting pqs quorum sensing,
Angew. Chem. Int. Ed. 53 (2014) 1109e1112.
[19] M.P. Storz, C.K. Maurer, C. Zimmer, N. Wagner, C. Brengel, J.C. de Jong, S. Lucas,
€
M. Müsken, S. Haussler, A. Steinbach, R.W. Hartmann, Validation of PqsD as an
anti-biofilm target in Pseudomonas aeruginosa by development of small-
molecule inhibitors, J. Am. Chem. Soc. 134 (2012) 16143e16146.
[20] D. Pistorius, A. Ullrich, S. Lucas, R.W. Hartmann, U. Kazmaier, R. Müller,
Biosynthesis of 2-alkyl-4(1H)-quinolones in Pseudomonas aeruginosa: po-
tential for therapeutic interference with pathogenicity, ChemBioChem 12
(2011) 850e853.
Acknowledgments
[21] J.H. Sahner, C. Brengel, M.P. Storz, M. Groh, A. Plaza, R. Müller, R.W. Hartmann,
Combining in silico and biophysical methods for the development of Pseu-
domonas aeruginosa quorum sensing inhibitors: an alternative approach for
structure-based drug design, J. Med. Chem. 56 (2013) 8656e8664.
[22] E. Weidel, J.C. de Jong, C. Brengel, M.P. Storz, A. Braunshausen, M. Negri,
A. Plaza, A. Steinbach, R. Müller, R.W. Hartmann, Structure optimization of 2-
Benzamidobenzoic acids as PqsD inhibitors for Pseudomonas aeruginosa in-
fections and elucidation of binding mode by SPR, STD NMR, and molecular
docking, J. Med. Chem. 56 (2013) 6146e6155.
[23] M. Storz, G. Allegretta, B. Kirsch, M. Empting, R.W. Hartmann, From in vitro to
in cellulo: structure-activity relationship of (2-nitrophenyl)methanol de-
rivatives as inhibitors of PqsD in Pseudomonas aeruginosa, Org. Biomol. Chem.
12 (2014) 6094e6104.
The authors thank Carina Scheidt and Simone Amman for per-
forming the in vitro tests. We also appreciate Patrick Fischer's help
in performing the synthesis. Furthermore we thank Dr. Werner
Tegge (Helmholtz Centre for Infection Research (HZI), Braunsch-
weig, Germany) for kindly providing the cell penetrating peptide. J.
Henning Sahner gratefully acknowledges a scholarship from the
“Stiftung der Deutschen Wirtschaft” (SDW). This project was fun-
ded by BMBF through grant 1616038B.
[24] M. Storz, C. Brengel, E. Weidel, M. Hoffmann, K. Hollemeyer, A. Steinbach,
R. Müller, M. Empting, R.W. Hartmann, Biochemical and biophysical analysis
of chiral PqsD inhibitor revealing tight-binding behavior and enantiomers
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.04.007.
with contrary thermodynamic signatures, ACS Chem. Biol.
8 (2013)
2794e2801.
[25] S. Hinsberger, Johannes C. de Jong, M. Groh, J. Haupenthal, R.W. Hartmann,
Benzamidobenzoic acids as potent PqsD inhibitors for the treatment of
Pseudomonas aeruginosa infections, Eur. J. Med. Chem. 76 (2014) 343e351.
[26] F.A. Cotton, V.W. Day, E.E. Hazen Jr., S. Larsen, S.T.K. Wong, Structure of
bis(methylguanidinium) monohydrogen orthophosphate. A model for the
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