Potent irreversible inhibitors of LasR quorum sensing in Pseudomonas aeruginosa

Article abstract of DOI:10.1021/ml500459f

Antagonism of quorum sensing represents a promising new antivirulence approach for the treatment of bacterial infection. The development of a novel series of non-natural irreversible antagonists of P. aeruginosa LasR is described. The lead compounds identified (25 and 28) display potent LasR antagonist activity and inhibit expression of the P. aeruginosa virulence factors pyocyanin and biofilm formation in PAO1 and PA14.

Full text of DOI:10.1021/ml500459f

Letter
pubs.acs.org/acsmedchemlett
Potent Irreversible Inhibitors of LasR Quorum Sensing in
Pseudomonas aeruginosa
Kevin T. O’Brien,† Joseph G. Noto,† Luke Nichols-O’Neill, and Lark J. Perez*
Department of Chemistry and Biochemistry, Rowan University, 201 Mullica Hill Road, Glassboro, New Jersey 08028, United States
S
* Supporting Information
ABSTRACT: Antagonism of quorum sensing represents a promising new antivirulence approach for the treatment of bacterial
infection. The development of a novel series of non-natural irreversible antagonists of P. aeruginosa LasR is described. The lead
compounds identified (25 and 28) display potent LasR antagonist activity and inhibit expression of the P. aeruginosa virulence
factors pyocyanin and biofilm formation in PAO1 and PA14.
KEYWORDS: antivirulence, Pseudomonas aeruginosa, biofilm inhibition, structure−activity relationship, quorum sensing
he common opportunistic human pathogen P. aeruginosa
Tplays a major role in infections of immunocompromised
patients and is of significant medical interest as traditional
antibiotic treatments are often ineffective.1−4 A prominent
feature in the pathogenesis and drug-resistance of this
bacterium is its ability to form a biofilm, a sessile community
of cells inhabiting an extracellular polymeric matrix.5 Inhibition
of the LasR quorum sensing circuit in P. aeruginosa has been
shown to lead to a significant decrease in overall biofilm
formation and virulence factor production.6 Further, the
importance of quorum sensing in the pathogenicity of P.
aeruginosa has been demonstrated in a number of animal
models including a Caenorhabditis elegans nematode model,7,8
Figure 1. Selected agonists and antagonists of P. aeruginosa LasR
the neonatal mouse model of pneumonia,9 and a burned mouse
quorum sensing.
model.1 In each of these cases, P. aeruginosa mutants defective
in quorum sensing were significantly less virulent than the
parent strains. Accordingly, the identification of a potent, drug-
like small molecule inhibitor of quorum sensing in P. aeruginosa
is anticipated to be of significant medical interest.10−15
homoserine lactone functionality, its agonist activity is
therapeutically undesirable. To impart the desired LasR
antagonist activity into analogues of 2, we drew inspiration
from the recent discovery of the HSL-based antagonist 3.20 In
this probe, the placement of electrophilic functionality at the
terminus of the fatty-acid tail leads to covalent modification of
LasR Cys79 and provides antagonist activity.
The P. aeruginosa LasR quorum sensing receptor is agonized
by the natural ligand N-3-oxo-dodecanoyl-L-homoserine lactone
(3-oxo-C12 HSL, 1, Figure 1).7,16 A high-throughput screen
recently identified a novel non-natural agonist 2.17 In
subsequent studies it was determined that this molecule binds
in the homoserine lactone binding pocket of the LasR receptor
and displays potent agonist activity, comparable to the natural
ligand.18,19 While this ligand lacks the metabolically labile
Received: October 10, 2014
Accepted: December 27, 2014
© XXXX American Chemical Society
A
DOI: 10.1021/ml500459f
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
In this study, we describe our discovery and optimization
efforts to identify potent drug-like inhibitors of virulence in P.
aeruginosa resulting in a series of irreversible LasR antagonists
based on the potent agonist 2.
Scheme 2. Synthesis of Isothiocyanate Analogues
Informed by the recent crystal structure of 2 complexed to
LasR,19 our analogue series focused on the incorporation of
electrophilic functionality in place of the 2-chloro benzoate
ester in 2 (see Supporting Information). The synthesis of key
intermediates 7 and 10 (Scheme 1) proceeded efficiently from
Scheme 1. Synthesis of Intermediates 7 and 10
linked analogues (Scheme 2b). Therefore, etherification of
phenol 6 followed by amine deprotection and isothiocyanate
formation yielded ether-linked isothiocyanate analogues 15 and
16.
Evaluation of the LasR antagonist assay of these analogues
revealed that incorporation of a pendant isothiocyanate
provided a series of LasR antagonists with IC50 values greater
than 145 μM (Table 1). Full inhibition of LasR with all of the
Table 1. Biological Activity of Isothiocyanate Analogues
a
b
compd
IC50 (μM)
% inhibition at 100 μM
commercially available material in three and five steps,
respectively. Briefly, 2-methoxyphenylacetic acid (5) was
reacted with 2-nitroaniline in the presence of SOCl2 to provide
6, which was subsequently deprotected and brominated to give
key intermediate phenol 7. Intermediate 10 was prepared from
bromination and benzyl protection of salicylamide (8). The
resulting amide 9 was reduced to the amine, coupled with 2-
nitrobenzoic acid and deprotected to provide intermediate
phenol 10. With these two core scaffolds in hand, we began to
investigate the incorporation of pendant electrophilic function-
ality through reaction of the phenol.
11
12
15
16
148 66.6
>200
69
26
55
61
>200
145 105
a
Values determined employing the GFP-based LasR antagonist
bioassay (see Supporting Information). All IC50 values are reported
as the mean of triplicate analysis with the range defining the 95%
confidence interval. Br-HSL (4) was used as a control for antagonism
with an IC50 = 16.4 7.4 μM. Percent inhibition of GFP at 100 μM
with respect to Br-HSL (4) as a positive control for LasR antagonism,
b
set at 100% inhibition.
Building on precedent for the antagonist activity of
isothiocyanate containing ligands (e.g., 3), we evaluated a
series of ligands bearing this electrophilic functionality tethered
to the free phenol present in 7 and 10 (Scheme 2). EDC
coupling of an isothiocyanate-containing carboxylic acid 14
with 7 or 10 provided analogues 11 and 12 containing a three-
carbon linker. The synthesis of analogues with shorter linkers
was not successful using this route. Isothiocyanate preparation
(e.g., 13 to 14) with shorter carbon linkers led a complex
mixture of unidentified reaction products. Recognizing the
potential for reaction of the carbonyl group with the proximal
isothiocyanate in these analogues bearing a shorter linker, we
turned our attention to the preparation of a series of ether-
ligands in this series was observed with no effect on bacterial
growth up to 200 μM, as monitored by evaluating OD600
.
Further, replacement of the ester linkage in analogue 11 with
an ether linker (16) was well tolerated with the two analogues
showing comparable IC50 values and % inhibition. To evaluate
if these analogues were binding in the HSL ligand-binding
pocket of LasR and to examine the mechanism of inhibition, we
performed a competition-binding assay. As previously shown
for HSL analogue 3,20 isothiocyanate-containing analogue 16 is
a noncompetitive antagonist of LasR (see Supporting
Information).
B
DOI: 10.1021/ml500459f
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
In an effort to enhance the potency (IC50) of our antagonists
we evaluated an alternate series of electrophilic analogues with
the potential to react with cysteine.21 Preparation of
chloroacetamide-containing analogues 17 and 18 proceeded
in three steps from intermediate 7 (Scheme 3a). Biological
Evaluation of the biological activity of the maleimide series of
analogues revealed several potent LasR antagonists (Table 2).
Within this series strict dependence on the length of the linker
was observed. In the analogues derived from intermediate 7, the
optimal linker length was two-carbon atoms (i.e., 25), with an
IC50 value of 3.6 μM. This analogue represents one of the most
potent antagonists of LasR yet discovered.20,22−28 Ligands with
either a one-carbon linker or a three-carbon linker demon-
strated diminished potency, with more than a 22-fold increase
in IC50 values. A similar dependence on linker length is found
with analogues derived from intermediate 10. In this series,
however, the optimal linker length is a three-carbon linker (i.e.,
28) with best-in-class antagonist activity of 1.5 μM. The
analogue containing a two-carbon linker (27) is 34-fold less
potent.
Scheme 3. Synthesis of Chloroacetamide and Maleimide
Analogues
Having identified potent antagonists of LasR (25 and 28), we
examined their mechanism of inhibition. We first evaluated if
these analogues bind in the HSL binding pocket of LasR and
act as noncompetitive antagonists as previously observed with
16. In this competition-binding assay, the concentration of the
antagonist is held constant while evaluating the agonist dose−
response curve of the native agonist, 3-oxo-C12 HSL (Figure 2a
assay of these analogues showed improvement in potency with
IC50 values between 74 and 100 μM (Table 2). Additionally, a
series of maleimide-containg analogues were prepared (24−28)
employing a BOP coupling reaction between intermediate
phenol 7 or 10 and the appropriately functionalized carboxylic
acid 21−23 (Scheme 3b).
Figure 2. (a) Competition assay characteristic of irreversible LasR
inhibition by 28. (b) Antagonist bioassay for 28 and succinimide 29
with (S)-2-(4-bromophenyl)-N-(2-oxotetrahydrofuran-3-yl) acetamide
(Br-HSL) as a positive control for antagonism. (c) The structures and
biological activity of succinimide containing ligands 29 and 30.
Table 2. Biological Activity of Chloroacetamide and
Maleimide Analogues
and Supporting Information). We observe consistent EC50
values for the native agonist; however, higher concentrations
of 25 or 28 lead to diminished maximal activation of LasR. This
feature is consistent with ligands 25 and 28 acting as
irreversible antagonists of LasR.
We then looked to differentiate between two potential
mechanisms of irreversible inhibition. In the first, irreversible
modification of LasR by an inhibitor renders the antagonist
activity of the ligand noncompetitive. In contrast, formation of
a covalent bond between ligand and receptor may represent a
key feature in both the ligand binding kinetics and the
antagonist activity of the molecule. If the first mechanism is
operative, covalent attachment to LasR simply serves to render
an antagonist ligand irreversible. In contrast, in the second
mechanism, the bond formation process is responsible for
antagonism.
a
b
compd
IC50 (μM)
% inhibition at 100 μM
17
18
24
25
26
27
28
73.8 19.4
99.5 21.0
86.8 57.4
3.6 1.9
91
79
56
98
30
32
101
78.1 69.3
51.9 35.1
1.5 0.5
a
Values determined employing the GFP-based LasR antagonist
bioassay (see Supporting Information). All IC50 values are reported
as the mean of triplicate analysis with the range defining the 95%
confidence interval. Br-HSL (4) was used as a control for antagonism
b
with an IC50 = 16.4 7.4 μM. Percent inhibition of GFP at 100 μM
with respect to Br-HSL (4) as a positive control for LasR antagonism,
set at 100% inhibition.
C
DOI: 10.1021/ml500459f
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
To differentiate between the two possible mechanisms of
inhibition we prepared succinimide-containing ligands 29 and
30 (Figure 2c). These ligands are nearly identical to lead
antagonists 25 and 28 in terms of their steric and electronic
properties; however, because of the absence of the maleimide,
these ligands would be unable to covalently modify LasR.
Evaluation of the biological activity of 29 and 30 reveals that
these ligands do not display LasR antagonist activity and
instead are LasR agonists (Figure 2b,c). The agonist activity
observed in the absence of a cysteine-reactive maleimide
supports a mechanism in which covalent modification of LasR
is critical for the antagonist activity of this class of molecules.
Having established that the potent LasR antagonists 25 and
28 are noncompetitive and that the presence of an electrophilic
maleimide is critical for the inhibitory activity of these
molecules, we examined computationally the binding of these
ligands with LasR.29 Docking of these ligands with LasR reveals
that the maleimide is in close proximity to Cys79 within the
HSL ligand-binding pocket (Figure 3a). The reactivity of a free
28). This observation suggests that linker length to the
electrophile is not an independent activity determinant but
rather linker length is coupled to an additional critical binding
determinant(s).
Analysis of the ligands with potent activity provides
guidelines for the development of a pharmacophore model to
describe the observed activity (Figure 3b,c). The pharmaco-
phore model was built by identifying the key binding
determinants in the most potent analogue (28) in comparison
to the remaining analogues in this series (24−27).30 Using a
pharmacophore model derived from the low energy docking
pose for 28 (Figure3a and see Supporting Information) we
evaluated a conformer library of ligands 24−27 to determine
the fit of these ligands to our model. As anticipated by the
antagonist activity of all ligands in this series, structural
deviation from the pharmacophore model defined by 28 is
subtle. The analogues displaying the lowest potency (24 and
26) maintain structural overlap for much of the model;
however, they deviate in their positioning of the critical
maleimide functionality (Figure 3b). In contrast, the potent
antagonist 25 (gray, Figure 3c) maintains complete structural
overlap with the pharmacophore model generated from 28,
supporting the accuracy of this model. We note, however, that
ligand 27 (pink, Figure 3c), which has median potency within
this series also appears to fully satisfy our pharmacophore
model.
Having identified two potent antagonists of LasR quorum
sensing, we evaluated the ability of these analogues to inhibit
virulence factor expression in wild-type clinical isolates of P.
aeruginosa. We focused on quantifying the inhibition of the
virulence factor pyocyanin and the inhibition of biofilm
formation using P. aeruginosa strains PAO1 (ATCC 10145)
and PA14.31
The inhibition of pyocyanin in these strains was first
evaluated. Pyocyanin is a blue-green redox-active pigment and
a prominent virulence factor in PAO1 and PA14. Lead quorum
sensing inhibitor, 28, provided potent dose-dependent
inhibition of this virulence factor (Figure 4). We observed a
Figure 3. (a) Lead inhibitor 28 docked in the ligand binding pocket of
LasR highlighting amino acid residues Y56, W60, D73, and C79. (b)
Pharmacophore model showing the low energy docking pose of 28
(black) compared to lower activity ligands 24 (gray) and 26 (yellow).
(c) Pharmacophore model showing the low energy docking pose of 28
(black) compared to high activity ligand 25 (gray) and median activity
ligand 27 (pink).
cysteine with an electrophilic maleimide is well-known,21 and
the reactivity of LasR Cys79 with an HSL-based ligand bearing
a pendant isothiocyanate (i.e., 3) has been established.20
Together, our results are consistent with the mechanism of
inhibition for 25 and 28 involving covalent modification of
Cys79 in the LasR HSL-binding pocket.
Figure 4. Inhibition of pyocyanin expression in P. aeruginosa PAO1
and PA14 by 28. Pyocyanin production was determined in triplicate by
direct detection of pyocyanin absorbance (A695). No inhibition of
growth was observed.
Given the requirement of covalent bond formation with
Cys79 for antagonism, the observation of a strict dependence of
IC50 on linker length is not unexpected. Variation of linker
length would be expected to most directly influence placement
of the electrophilic maleimide in an orientation suitable for
reaction with Cys79; however, the effect of linker length is not
independent of the structure of the analogue core. The optimal
linker length for a maleimide ligand derived from phenol 6 is
two carbon atoms (i.e., 25) whereas the optimal linker length
for the maleimide derived from 9 is three carbon atoms (i.e.,
decrease in absorbance (A695) in cell-free supernatants from P.
aeruginosa cultures that is characteristic of decreasing levels of
pyocyanin. To further support that this change in absorbance is
pyocyanin dependent, we performed an extraction and
acidification of the cell-free supernatants selective for pyocyanin
before measuring absorbance (A520, see Supporting Informa-
tion). Using both methods, we similarly observe a decrease in
absorbance, which is consistent with a decrease in pyocyanin
D
DOI: 10.1021/ml500459f
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
expression in the presence of 28. Importantly, in this assay, no
effect on growth is observed in either strain as assessed by
measurement of cell density (A600). Higher levels of pyocyanin
expression in untreated samples of PA14 are noted when
compared to PAO1 and the inhibition of pyocyanin expression
in PAO1 is achieved at somewhat lower antagonist
concentrations, consistent with the enhanced virulence of the
PA14 strain.8,31,32
The ability of 28 to inhibit biofilm formation in static
cultures of P. aeruginosa PA14 was evaluated using a crystal
violet staining protocol with 48 h biofilms. Similar to the
inhibition of the virulence factor pyocyanin, potent, dose-
dependent inhibition of biofilm formation is observed in the
presence of lead antagonist 28 (Figure 5a).
analogues 25 and 28, together with enhanced metabolic
stability in comparison to other reported LasR antagonists
(e.g., 3) provides for the expectation that this series will serve as
new leads in the development of an antivirulence therapy for
the prominent Gram-negative bacterial pathogen P. aeruginosa.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis, characterization (1H NMR, 13C NMR, and LC−MS
spectra and chromatograms along with full tabulated data),
bioassay results with dose−response curves for all new
compounds, competition assay for 16 and 25, pyocyanin
analysis, additional confocal fluorescence microscopy data,
additional docking poses, and supplemental discussion. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 856-256-4502. E-mail: perezla@rowan.edu.
■
Author Contributions
†These authors contributed equally to this work.
Funding
This work was financially supported by Rowan University CSM
and SEED grants and by a Rowan University New Faculty
Research Startup Grant.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge Dr. Bonnie L. Bassler (Princeton
University) for providing bacterial strains used in this study, Dr.
Venkat Venkataraman and Ms. Hao Wu (Rowan University
School of Osteopathic Medicine) for assistance with the
confocal fluorescence microscopy, Dr. Martin F. Semmelhack
and Dr. Christian Ventocilla (Princeton University) for
collection of HRMS data, Mr. Joseph N. Capilato and Ms.
Amy L. Warren (Rowan University) for assistance with
compound synthesis, and Ms. Lydia M. Hanna (Rowan
University) for assistance with the virulence factor assays.
Figure 5. Inhibition of biofilm formation in P. aeruginosa PA14 in the
presence of 28. (a) PA14 biofilm formation evaluated by crystal violet
staining (A550). (b) Biofilm biomass and thickness determined from
confocal fluorescence microscopy images using the program
Comstat2.34,35 (c) Confocal fluorescence microscopy images of
PA14 biofilms. Confocal images were collected in duplicate and
imaging was repeated three times, representative images are shown.
Scale bar = 25 μm.
To further evaluate the potent inhibitory effect on biofilm
formation observed in the presence of 28, we imaged a series of
36 h P. aeruginosa PA14 biofilms grown in rich media (Luria
broth) using confocal fluorescence microscopy after staining
with SYTO-9 nucleic acid stain.33 The images collected display
a dose-dependent reduction in biofilm thickness and overall
biomass (Figure 5b,c). Repeating this assay using defined media
similarly shows a striking decrease in biofilm biomass, thickness,
and overall morphology, confirming the virulence factor
inhibitory activity of 28 (see Supporting Information).
We have identified a series of potent irreversible inhibitors of
P. aeruginosa LasR, which display antivirulence activity against
PAO1 and the hyper-virulent strain PA14. The mechanism of
inhibition for our lead analogues in this series has been
characterized enabling the development of a pharmacophore
model to describe the potent irreversible antagonist activity
observed. Within this series, we find that antagonist activity is
dependent on the presence of a pendant electrophile (e.g.,
maleimide). Our lead inhibitor shows potent, dose-dependent
inhibition of the P. aeruginosa virulence factor pyocyanin and
biofilm formation while possessing no bactericidal or
bacteriostatic activity. The potent biological activity of
REFERENCES
■
(1) Rumbaugh, K. P.; Griswold, J. A.; Iglewski, B. H.; Hamood, A. N.
Contribution of Quorum Sensing to the Virulence of Pseudomonas
aeruginosa in Burn Wound Infections. Infect. Immun. 1999, 67, 5854−
5862.
(2) Lambert, P. A. Mechanisms of Antibiotic Resistance in
Pseudomonas Aeruginosa. J. R. Soc. Med. 2002, 95, 22.
(3) Driscoll, J.; Brody, S.; Kollef, M. The Epidemiology, Pathogenesis
and Treatment of Pseudomonas Aeruginosa Infections. Drugs 2007,
67, 351−368−368.
(4) Hirsch, E. B.; Tam, V. H. Impact of Multidrug-Resistant
Pseudomonas Aeruginosainfection on Patient Outcomes. Expert Rev.
Pharmacoeconomics Outcomes Res. 2010, 10, 441−451.
(5) Musk, J.; Dinty, J.; Hergenrother, P. J. Chemical Counter-
measures for the Control of Bacterial Biofilms: Effective Compounds
and Promising Targets. Curr. Med. Chem. 2006, 13, 2163−2177.
(6) Smith, R. S.; Harris, S. G.; Phipps, R.; Iglewski, B. The
Pseudomonas aeruginosa Quorum-Sensing Molecule N-(3-
Oxododecanoyl)homoserine Lactone Contributes to Virulence and
Induces Inflammation in Vivo. J. Bacteriol. 2002, 184, 1132−1139.
(7) Pearson, J. P.; Gray, K. M.; Passador, L.; Tucker, K. D.; Eberhard,
A.; Iglewski, B. H.; Greenberg, E. P. Structure of the Autoinducer
E
DOI: 10.1021/ml500459f
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Required for Expression of Pseudomonas aeruginosa Virulence Genes.
Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 197−201.
(8) Tan, M. W.; Rahme, L. G.; Sternberg, J. A.; Tompkins, R. G.;
Ausubel, F. M. Pseudomonas aeruginosa Killing of Caenorhabditis elegans
Used to Identify P. aeruginosa Virulence Factors. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 2408−2413.
(9) Tang, H. B.; DiMango, E.; Bryan, R.; Gambello, M.; Iglewski, B.
H.; Goldberg, J. B.; Prince, A. Contribution of Specific Pseudomonas
aeruginosa Virulence Factors to Pathogenesis of Pneumonia in a
Neonatal Mouse Model of Infection. Infect. Immun. 1996, 64, 37−43.
(10) Rasmussen, T. B.; Givskov, M. Quorum-Sensing Inhibitors as
Anti-Pathogenic Drugs. Int. J. Med. Microbiol. 2006, 296, 149−161.
(11) Rasko, D. A.; Sperandio, V. Anti-Virulence Strategies to Combat
Bacteria-Mediated Disease. Nat. Rev. Drug Discovery 2010, 9, 117−128.
(12) Clatworthy, A. E.; Pierson, E.; Hung, D. T. Targeting Virulence:
a New Paradigm for Antimicrobial Therapy. Nat. Chem. Biol. 2007, 3,
541−548.
(13) Khmel, I. A.; Metlitskaya, A. Z. Quorum Sensing Regulation of
Gene Expression: a Promising Target for Drugs Against Bacterial
Pathogenicity. Mol. Biol. 2006, 40, 169−182.
(14) Rutherford, S. T.; Bassler, B. L. Bacterial Quorum Sensing: Its
Role in Virulence and Possibilities for Its Control. Cold Spring Harbor
Perspect. Med. 2012, 2.
(15) LaSarre, B.; Federle, M. J. Exploiting Quorum Sensing to
Confuse Bacterial Pathogens. Microbiol. Mol. Biol. Rev. 2013, 77, 73−
111.
of Potent Quorum Sensing Modulators. Bioorg. Med. Chem. Lett. 2008,
18, 5978−5981.
(27) Ni, N.; Li, M.; Wang, J.; Wang, B. Inhibitors and Antagonists of
Bacterial Quorum Sensing. Med. Res. Rev. 2009, 29, 65−124.
(28) O’Loughlin, C. T.; Miller, L. C.; Siryaporn, A.; Drescher, K.;
Semmelhack, M. F.; Bassler, B. L. A Quorum-Sensing Inhibitor Blocks
Pseudomonas aeruginosa Virulence and Biofilm Formation. Proc. Natl.
Acad. Sci. U.S.A. 2013, 110, 17981−17986.
(29) Grosdidier, A.; Zoete, V.; Michielin, O. SwissDock, a Protein-
Small Molecule Docking Web Service Based on EADock DSS. Nucleic
Acids Res. 2011, 39, W270−W277.
(30) Wolber, G.; Langer, T. LigandScout: 3-D Pharmacophores
Derived From Protein-Bound Ligands and Their Use as Virtual
Screening Filters. J. Chem. Inf. Model. 2005, 45, 160−169.
(31) Rahme, L. G.; Stevens, E. J.; Wolfort, S. F.; Shao, J.; Tompkins,
R. G.; Ausubel, F. M. Common Virulence Factors for Bacterial
Pathogenicity in Plants and Animals. Science 1995, 268, 1899−1902.
(32) Mahajan-Miklos, S.; Tan, M. W.; Rahme, L. G.; Ausubel, F. M.
Molecular Mechanisms of Bacterial Virulence Elucidated Using a
Pseudomonas aeruginosa-Caenorhabditis elegans Pathogenesis Model.
Cell 1999, 96, 47−56.
(33) Musken, M.; Di Fiore, S.; Romling, U.; Haussler, S. A 96-Well-
̈
̈
̈
Plate−Based Optical Method for the Quantitative and Qualitative
Evaluation of Pseudomonas aeruginosa Biofilm Formation and Its
Application to Susceptibility Testing. Nat. Protoc. 2010, 5, 1460−1469.
(34) Heydorn, A.; Nielsen, A. T.; Hentzer, M.; Sternberg, C.;
Givskov, M.; Ersbøll, B. K.; Molin, S. Quantification of Biofilm
Structures by the Novel Computer Program COMSTAT. Microbiology
2000, 146 (Pt 10), 2395−2407.
(16) Jimenez, P. N.; Koch, G.; Thompson, J. A.; Xavier, K. B.; Cool,
R. H.; Quax, W. J. The Multiple Signaling Systems Regulating
Virulence in Pseudomonas aeruginosa. Microbiol. Mol. Biol. Rev. 2012,
76, 46−65.
(35) Vorregaard, M.; Ersbøll, B. K.; Yang, L.; Haagensen, J. A. J.;
Heydorn, A.; Molin, S.; Sternberg C. Personal communication; http://
www.comstat.dk.
(17) Muh, U.; Hare, B. J.; Duerkop, B. A.; Schuster, M.; Hanzelka, B.
̈
L.; Heim, R.; Olson, E. R.; Greenberg, E. P. A Structurally Unrelated
Mimic of a Pseudomonas aeruginosa Acyl-Homoserine Lactone
Quorum-Sensing Signal. Proc. Natl. Acad. Sci. U.S.A. 2006, 103,
16948−16952.
(18) Zakhari, J. S.; Kinoyama, I.; Struss, A. K.; Pullanikat, P.; Lowery,
C. A.; Lardy, M.; Janda, K. D. Synthesis and Molecular Modeling
Provide Insight Into a Pseudomonas aeruginosa Quorum Sensing
Conundrum. J. Am. Chem. Soc. 2011, 133, 3840−3842.
(19) Zou, Y.; Nair, S. K. Molecular Basis for the Recognition of
Structurally Distinct Autoinducer Mimics by the Pseudomonas
aeruginosa LasR Quorum-Sensing Signaling Receptor. Chem. Biol.
2009, 16, 961−970.
(20) Amara, N.; Mashiach, R.; Amar, D.; Krief, P.; Spieser, S. A. H.;
Bottomley, M. J.; Aharoni, A.; Meijler, M. M. Covalent Inhibition of
Bacterial Quorum Sensing. J. Am. Chem. Soc. 2009, 131, 10610−
10619.
(21) Sletten, E. M.; Bertozzi, C. R. Bioorthogonal Chemistry: Fishing
for Selectivity in a Sea of Functionality. Angew. Chem., Int. Ed. 2009,
48, 6974−6998.
(22) Muh, U.; Schuster, M.; Heim, R.; Singh, A.; Olson, E. R.;
̈
Greenberg, E. P. Novel Pseudomonas aeruginosa Quorum-Sensing
Inhibitors Identified in an Ultra-High-Throughput Screen. Antimicrob.
Agents Chemother. 2006, 50, 3674−3679.
(23) Geske, G. D.; O’Neill, J. C.; Miller, D. M.; Wezeman, R. J.;
Mattmann, M. E.; Lin, Q.; Blackwell, H. E. Comparative Analyses of
N-Acylated Homoserine Lactones Reveal Unique Structural Features
That Dictate Their Ability to Activate or Inhibit Quorum Sensing.
ChemBioChem 2008, 9, 389−400.
(24) Geske, G. D.; O’Neill, J. C.; Miller, D. M.; Mattmann, M. E.;
Blackwell, H. E. Modulation of Bacterial Quorum Sensing with
Synthetic Ligands: Systematic Evaluation of N-Acylated Homoserine
Lactones in Multiple Species and New Insights Into Their
Mechanisms of Action. J. Am. Chem. Soc. 2007, 129, 13613−13625.
(25) Mattmann, M. E.; Blackwell, H. E. Small Molecules That
Modulate Quorum Sensing and Control Virulence in Pseudomonas
aeruginosa. J. Org. Chem. 2010, 75, 6737−6746.
(26) Geske, G. D.; Mattmann, M. E.; Blackwell, H. E. Evaluation of a
Focused Library of N-Aryl L-Homoserine Lactones Reveals a New Set
F
DOI: 10.1021/ml500459f
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX