Small molecule inhibitors of bacterial quorum sensing and biofilm formation

Article abstract of DOI:10.1021/ja0530321

Bacteria monitor their local population densities using small molecules (or autoinducers) in a process known as quorum sensing. Here, we report a new and efficient synthetic route to naturally occurring bacterial autoinducers [N-acyl L-homoserine lactones (AHLs)] that is readily amenable to the synthesis of analogues. This route has been applied in the first synthesis of a library of non-native AHLs. Evaluation of these compounds in bacterial reporter gene and biofilm assays has revealed a potent set of quorum sensing antagonists. These ligands will serve as valuable new tools to explore the role of quorum sensing in bacterial pathogenesis. Copyright

Full text of DOI:10.1021/ja0530321

Published on Web 08/26/2005
Small Molecule Inhibitors of Bacterial Quorum Sensing and Biofilm Formation
Grant D. Geske, Rachel J. Wezeman, Adam P. Siegel, and Helen E. Blackwell*
Department of Chemistry, UniVersity of WisconsinsMadison, 1101 UniVersity AVenue, Madison, Wisconsin 53706
Received May 10, 2005; E-mail: blackwell@chem.wisc.edu
Scheme 1. Microwave-Assisted Solid-Phase Synthetic Route to
Bacteria use autoinducer ligands to monitor their population
densities in a phenomenon called quorum sensing.1 At high cell
densities, bacteria use this chemical signaling process to switch
from a nomadic existence to that of multicellular community. This
lifestyle switch is significant; quorum sensing can be used to turn
on virulence pathways2 and induce the formation of drug-impervious
communities called biofilms that are the basis of myriad chronic
infections.3 Over 80% of bacterial infections in humans involve
the formation of biofilms. For example, lung infection by Pseudomo-
nas aeruginosa in the biofilm state is the primary cause of morbidity
in cystic fibrosis patients.2 As such, the development of small
molecules that attenuate bacterial quorum sensing has become an
area of intense research.4 Here, we report a highly efficient synthetic
route to new ligands that modulate quorum sensing. Application
of this route in focused library synthesis has delivered a set of potent
quorum sensing antagonists, two of which strongly inhibit biofilm
formation in P. aeruginosa.
We have focused our initial research on quorum sensing
pathways in Gram-negative bacteria, as these signaling networks
are used by numerous human pathogens, including P. aeruginosa.
Gram-negative bacteria use N-acyl L-homoserine lactone (AHL)
signal molecules to activate quorum sensing.5 At a threshold AHL
concentration (and related cell density), the AHL ligand will bind
its cognate receptor, a LuxR-type protein, and activate the transcrip-
tion of target genes involved in group behavior. An attractive
strategy for quorum sensing control is to use non-native AHL
derivatives that block natural AHL signals. The pace of AHL
analogue discovery has been slow, however. Most synthetic routes
to AHLs afford the products in low yields and purities, and the
biological activity of these compounds has been evaluated primarily
on an ad hoc basis.6 New synthetic approaches are required for the
generation of AHL analogues and the systematic evaluation of the
effects of AHL ligand structure on quorum sensing.
To meet these challenges, we developed a solid-phase synthetic
route to both natural and non-natural AHLs (Scheme 1). We chose
solid-phase methods because they routinely give improved product
purity relative to solution-phase methods and enable combinatorial
library construction.7 To date, the use of combinatorial methods to
systemically evaluate AHL analogues remains essentially un-
explored.4c To further expedite both solid-phase and library
synthesis, we incorporated microwave (MW)-assisted reactions
throughout the route.8 Our four-step synthetic approach entails first
loading amino polystyrene resin (1) with N-Fmoc-L-methionine (2)
using a MW-assisted carbodiimide coupling (DIC).9 Next, thermal
Fmoc group removal followed by a second MW-assisted DIC
coupling with various carboxylic acids (4) or protected â-keto acids
(5)10 generated acylated resin 6. Finally, we utilized the classical
reaction of cyanogen bromide (CNBr) with L-methionine in a MW-
assisted, tandem cyclization cleavage step to release AHLs 7 and
8 from the solid support.11,12
Natural and Unnatural AHLsa
a Conditions: a ) DIC, HOBT, CHCl3/DMF, MW 50 °C (2 × 10 min);
b ) DMF, MW 150 °C, 7 min; c ) CNBr, TFA, CHCl3/H2O, MW 60 °C,
30 min.
Table 1. Naturally Occurring AHLs Synthesized via Scheme 1
compound
R1 or R2
organism
purity [%]a,b
yield [%]c
7a
7b
7c
7d
7e
7f
8a
C3H7
P. aeruginosa
R. leguminosarum
Y. pseudotuberculosis
B. pseudomallei
S. meliloti
R. capsulatus
V. fischeri
A. tumefaciens
V. anguillarum
P. aeruginosa
S. meliloti
98
98
98
97
98
65
64
76
80
70
64
63
65
76
62
62
C5H11
C7H15
C9H19
C11H23
C13H27
C3H7
C5H11
C7H15
C9H19
C11H23
97
>93
>93
>93
>93
>93
8b:OOHLd
8c
8d:ODHLe
8e
a Purities of 7a-f determined by integration of GC spectra. b Purities of
8a-e determined by 1H NMR. c Isolated yields. d N-3-Oxooctanoyl L-
homoserine lactone. e N-3-Oxododecanoyl L-homoserine lactone.
to structural analogues thereof. Further, the route delivers com-
pounds in sufficient purity and quantity for biological testing. To
evaluate the scope and generality of our method, we synthesized
the majority of the natural AHLs (7a-f, 8a-e) in good yields and
excellent purities in <60 min total reaction times (Table 1). Natural
AHLs 8b (OOHL) and 8d (ODHL) from Agrobacterium tumefa-
ciens and P. aeruginosa, respectively, were used as critical control
molecules in our later quorum sensing antagonism screens (see
below). The high purities of the AHL products overall and the short
reaction times used in our approach underscore the value of MW-
assisted solid-phase chemistry for AHL synthesis.8a
We next applied our solid-phase route to the parallel synthesis
of a small test library of non-natural AHLs (7g-q, 8f-h). The
acyl substituents and stereochemistry of the AHL products were
chosen to probe broadly the sterics and functionality present in the
AHL binding site of LuxR-type proteins, as revealed in a recent
X-ray structure of TraR from A. tumefaciens.13 Our synthetic route
again proved robust and delivered the non-native AHLs in good
yields (ca. 70%) and high purities (>93%) (Table S-1, Supporting
Information).
The AHL library was screened in two well-characterized bacterial
reporter strains for antagonism of quorum sensing: P. aeruginosa
PAO-JP2(plasI-LVAgfp)14 and A. tumefaciens WCF47(pCF372).6c
Examination of these strains was valuable for two reasons: (1) the
direct clinical relevance of P. aeruginosa, and (2) the extensive
body of biochemical and structural data for TraR in A. tumefa-
This solid-phase route to AHLs is the first to provide access to
the ca. 15 known natural AHLs from Gram-negative bacteria and
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J. AM. CHEM. SOC. 2005, 127, 12762-12763
10.1021/ja0530321 CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. Composite 3-D micrographs of P. aeruginosa biofilms grown
on glass slides after 48 h in the presence of synthetic ligands (at 50 µM).
Scale bar ) 50 µm. (a) Untreated. (b) Compound 7h. (c) Compound 7o.
have direct clinical impact.2,3 The discovery of potent inhibitors
from a small library highlights the potential utility of focused
combinatorial methods for the discovery of additional small
molecule modulators of quorum sensing.
In summary, we have developed a robust synthetic route to AHL
autoinducers that provides access to both natural and unnatural
AHLs in high purity. We have implemented this route to identify
a set of non-native AHLs that are among the most potent inhibitors
of bacterial quorum sensing reported to date. These molecules
represent powerful new tools to elucidate the role of bacterial
communication in pathogenesis.
Figure 1. (a) Quorum sensing antagonists identified in this study (7h, 7k,
and 7o). (b) Known quorum sensing antagonists (7g, 8f, and 9).4c,6b,c (c) A.
tumefaciens reporter strain agonism (gray bar: 100 nM 8b) and antagonism
data (red and black bars: against 100 nM 8b). Miller units report relative
â-galactosidase activity. (d) P. aeruginosa reporter strain agonism (gray
bar: 1 µM 8d) and antagonism data (red and black bars: against 1 µM
8d). Error bars reflect at least three experiments.
ciens.13 Both of these reporter strains lack their native AHL
synthases, yet retain active LuxR-type receptors (LasR and TraR
proteins, respectively); exogenous ligand is required for receptor
activation, which can be measured by fluorescence (green fluores-
cent protein (GFP) for LasR) or absorbance (via â-galactosidase
activity for TraR) measurements.15
Acknowledgment. This work was supported by the NSF (CHE-
0449959), Greater Milwaukee Foundation Shaw Scientist Program,
and UWsMadison. We thank Profs. Laura Kiessling, Samuel
Gellman, and Jo Handelsman for helpful discussions, and Profs.
Barbara Iglewski and Stephan Winans for generous donations of
bacterial reporter strains.
Supporting Information Available: Full details of solid-phase
synthesis, compound characterization, and assay protocols. This material
is available free of charge via the Internet at http://pubs.acs.org.
The antagonism screens revealed a suite of new quorum sensing
inhibitors. In these experiments, the strains were treated with non-
native AHL in the presence of native AHL ligand (8b or 8d), and
a reduction in absorbance or fluorescence signal indicated that the
non-native AHL was able to antagonize LuxR-type protein activity.
Three compounds (7h, 7k, and 7o) showed significant activity
against TraR in A. tumefaciens and were 1-2 orders of magnitude
more active than the previously reported LuxR-type protein
antagonists examined as controls (7g,6c 8f,6b and 94c at 10 µM;
Figure 1a-c). Impressively, bromophenyl AHL 7o displayed 50%
inhibition at an equimolar concentration of 8b (100 nM). Interest-
ingly, the same three ligands were also identified as potent
antagonists against LasR in P. aeruginosa (Figure 1d). Here, indol
AHL 7h and bromophenyl AHL 7o were 2-fold as active as the
three controls (at 400 µM), with indol AHL 7h displaying 50%
inhibition at a 12.5:1 ratio with native ligand 8d.16 Notably, all
three ligands contain bulky, hydrophobic acyl groups. This structural
similarity, coupled with their cross activity, suggests that the ligands
could bind the TraR and LasR receptors in analogous manners;
efforts to characterize these interactions are currently underway in
our laboratory.
As biofilm formation is largely under the control of LasR in P.
aeruginosa,2,3,14 we hypothesized that antagonists 7h and 7o could
disrupt P. aeruginosa biofilm formation. We performed standard
static biofilm assays using a P. aeruginosa (PAO1(pLVAgfp)) strain
that constitutively produces GFP to facilitate visualization.14
Biofilms were grown in the presence of ligand (50 µM) for 48 h
and visualized using scanning laser confocal microscopy (Figure
2). The treated biofilms were significantly less fluorescent relative
to the untreated control, which indicates that the treated biofilms
have reduced cell densities and are weakly organized.2,14 These data
show that compounds 7h and 7o strongly inhibit P. aeruginosa
biofilm formation. This finding is important; few inhibitors of
bacterial biofilm formation are known, yet such compounds should
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