Targeting Staphylococcus aureus quorum sensing with nonpeptidic small molecule inhibitors

Article abstract of DOI:10.1021/jm500215s

A series of 3-oxo-C₁₂-HSL, tetramic acid, and tetronic acid analogues were synthesized to gain insights into the structural requirements for quorum sensing inhibition in Staphylococcus aureus. Compounds active against agr were noncompetitive inhibitors of the autoinducing peptide (AIP) activated AgrC receptor, by altering the activation efficacy of the cognate AIP-1. They appeared to act as negative allosteric modulators and are exemplified by 3-tetradecanoyltetronic acid 17, which reduced nasal cell colonization and arthritis in a murine infection model.

Full text of DOI:10.1021/jm500215s

Brief Article
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Targeting Staphylococcus aureus Quorum Sensing with Nonpeptidic
Small Molecule Inhibitors
Ewan J. Murray,†,∞ Rebecca C. Crowley,†,∞,⊥ Alex Truman,† Simon R. Clarke,†,# James A. Cottam,†
Gopal P. Jadhav,‡ Victoria R. Steele,† Paul O’Shea,§ Catharina Lindholm,∥ Alan Cockayne,†
Siri Ram Chhabra,† Weng C. Chan,*,‡ and Paul Williams*,†
†School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, NG7 2RD, U.K.
‡School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, NG7 2RD, U.K.
§School of Life Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, U.K.
∥Department of Rheumatology and Inflammation Research, University of Gothenburg, Gothenburg 40530, Sweden
S
* Supporting Information
ABSTRACT: A series of 3-oxo-C12-HSL, tetramic acid, and tetronic
acid analogues were synthesized to gain insights into the structural
requirements for quorum sensing inhibition in Staphylococcus aureus.
Compounds active against agr were noncompetitive inhibitors of the
autoinducing peptide (AIP) activated AgrC receptor, by altering the
activation efficacy of the cognate AIP-1. They appeared to act as negative
allosteric modulators and are exemplified by 3-tetradecanoyltetronic acid
17, which reduced nasal cell colonization and arthritis in a murine
infection model.
genes involved in exotoxin, exoenzyme and secondary
metabolite production, as well as biofilm development.2 3-
Oxo-C12-HSL also exerts a wide spectrum of other biological
activities, for example, acting as a modulator of immune and
inflammatory responses, impacting on the cardiovascular
system, and disrupting epithelial barriers.2 As yet, the direct
target(s) for 3-oxo-C12-HSL in eukaryotic cells has not been
identified.3,4 In lymphocytes, optimal immune suppressive
activity is dependent on a C11−C13 acyl chain containing a
3-oxo or a 3-hydroxy group. Compounds lacking the L-
configuration or with polar substituents were essentially devoid
of activity.5 Further modification of the 3-oxo-C12-HSL
structure to generate compounds retaining the C12 acyl chain
length while replacing the chiral homoserine lactone with more
stable achiral heteroaryl moieties yielded compounds that
possess similar immune suppressive activity to 3-oxo-C12-HSL
but that inhibit rather than activate LasR in P. aeruginosa, thus
indicating that at the molecular level it is possible to separate
immune suppressive activity from QS activation.6
INTRODUCTION
■
Bacterial pathogens such as Pseudomonas aeruginosa and
Staphylococcus aureus employ cell-to-cell communication or
“quorum sensing” (QS) systems for coordinating collective
activities that depend on the actions of one or more chemically
distinct signal molecules.1 Such molecules largely operate as
effectors of QS-dependent gene expression through direct
activation of membrane associated sensor kinases or via
cytoplasmically located DNA-binding proteins. They are also
emerging as multifunctional signals that can influence
interactions between different bacterial species and impact
significantly the outcome of host−pathogen interactions by also
acting directly on the host.2
In P. aeruginosa, N-(3-oxododecanoyl)-L-homoserine lactone
(3-oxo-C12-HSL, 1; Table 1) activates the transcriptional
regulator LasR to drive the expression of multiple QS target
Table 1. QS and Growth Inhibitory Activities of 3-Oxo-C12-
HSL and Its Enantiomer
3-Oxo-C12-HSL also acts on other microorganisms, inhibiting
filamentation in Candida albicans7 and the growth of Gram-
positive bacteria.8,9 At subgrowth inhibitory concentrations, 3-
oxo-C12-HSL antagonizes the production of S. aureus exotoxins
(including α-hemolysin, δ-hemolysin, and toxic shock syn-
drome toxin) while enhancing cell wall protein biosynthesis
Received: December 19, 2013
Published: March 4, 2014
© 2014 American Chemical Society
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including the fibronectin- and immunoglobulin-binding pro-
teins.9 The mode of action of 3-oxo-C12-HSL in S. aureus
appears to involve inhibition of agr-dependent QS, which
reciprocally regulates exotoxins and cell wall colonization
factors.9,10 The agr locus consists of two divergent transcrip-
tional units, the P2 and P3 operons. The P2 operon consists of
four genes, agrBDCA, which are required for the activation of
transcription from the P2 and P3 promoters, while the
untranslated P3 transcript RNAIII is itself the effector for the
agr response. AgrA and AgrC constitute a two-component
system in which AgrC is the sensor kinase and AgrA is the
response regulator. The system is activated by the interaction of
AgrC with a 7- to 9-mer macrocyclic-containing peptide termed
the autoinducing peptide (AIP) generated from the agrD gene
product by AgrB.10 Since 3-oxo-C12-HSL binds to the S. aureus
cytoplasmic membrane in a specific saturable manner, such
membrane interactions may account for the agr inhibitory
properties of 3-oxo-C12-HSL given the membrane localization
of the AgrB and AgrC proteins.
Table 2. QS and Growth Inhibitory Activities of 3-
Acyltetramic Acids
a
Under aqueous alkaline conditions, 3-oxo-C12-HSL under-
goes lactonolysis to form the corresponding ring-opened
homoserine compound11 or an intramolecular rearrangement
reaction to afford a vinylogous acid product, 3-(1-hydrox-
ydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione [(S)-5-
hydroxyethyl-3-decanoyltetramic acid;8 5-HE-C10-TMA, 5]
(see Table 2). This compound belongs to the TMA class of
compounds, such as reutericycline which display antibacterial
properties.8,12 It is primarily active against Gram-positive
bacteria including Bacillus, Clostridium, and Staphylococcus8,13
where growth inhibition involves the dissipation of bacterial
membrane potential and pH gradient.14 The physiological
function of 5 in P. aeruginosa is not known, but it is capable of
weakly inhibiting the LasR/3-oxo-C12-HSL-dependent activa-
tion of the elastase (lasB) gene6 and reducing P. aeruginosa
viability.15 In contrast to 3-oxo-C12-HSL, 5 is also a ferric ion
chelator.8 However, while it does not function as a siderophore
for P. aeruginosa,16 iron was reported to abolish the antibacterial
activity of 5 toward P. aeruginosa15 and Clostridium difficile.13 In
common with 3-oxo-C12-HSL, 5 also exhibits immune
suppressive activity in a murine peripheral blood lymphocyte
proliferation assay and is cytotoxic toward Jurkat6 but not bone-
marrow-derived macrophage cells.14
Given the threat posed by the emergence of bacterial strains
resistant to conventional growth inhibitory antibiotics, there is
renewed interest in the discovery of agents that control
infection through the attenuation of bacterial virulence.10,17
This latter strategy has the potential advantage of expanding the
repertoire of bacterial targets and exerting reduced selective
pressure which, in turn, may retard the development of
resistance.10 In this context, the development of strategies for
inhibiting QS-dependent regulation of virulence has received
significant attention particularly for problematic multiantibiotic
resistant human pathogens such as S. aureus.10 Here major
efforts have been directed toward the inhibition of AIP/AgrC
interactions to attenuate staphylococcal virulence largely
through the synthesis and evaluation of AIP mimetics.10,18
However, such AIPs have been reported to be susceptible to
inactivation by host innate defenses.19 Given our desire to
discover new nonpeptidic agents that are capable of modulating
the AIP/AgrC interaction, we have considered simple
heterocyclic compounds. In this context, although a number
of small molecules have been reported to cause blockade of the
staphylococcal agr system, none of these compounds are
a
The asterisks indicate the following: ∗, no growth inhibition up to
100 μM; ∗∗, no inhibition of agr observed at concentrations up to 100
μM.
known to directly modulate ligand/cognate receptor inter-
actions.10 Since we have previously shown that 3-oxo-C12-HSL
can inhibit agr-dependent QS in S. aureus,9 a series of 3-oxo-
C12-HSL, TMA, and related TOA analogues were synthesized
and evaluated for their agr- and growth-inhibitory properties,
structure−activity relationships, and mechanism of action. In
addition, the efficacy of the most potent TOA analogue 17 was
investigated in a mouse staphylococcal infection model.
RESULTS AND DISCUSSION
■
N-Acyl-L-homoserine lactones (AHLs) (Table 1, 1 and 2;
Supporting Information Table S1, S1−S16) were synthesized
using well-established methodologies. The synthesis of 3-oxo-
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C12-HSL 1 and its analogues has been previously reported by
Chhabra et al.5,20 and Jadhav et al.6 Similarly, the 3-acyl-5-(2-
hydroxyethyl)tetramic acids 3−13 listed in Table 2 were
prepared from their respective N-3-oxoacyl-L-homoserine
lactones by the method previously described by Kaufmann et
al.8
(S)-3-Decanoyl-5-methyltetramic acid 12 and 3-decanoyl-
tetramic acid 13 were constructed by applying the strategy
outlined in Scheme S1. Thus, the appropriate N-Boc-α-
aminoacylated Meldrum’s acid, prepared by C-acylation of
Meldrum’s acid with carbodiimide-activated N-Boc-amino acid,
was subjected to thermal cyclative elimination of Me2CO and
CO2 to yield the required 5-substituted N-Boc-tetramic acid.21
Subsequent acylation with activated decanoic acid followed by
trifluoroacetic acid mediated acidolysis delivered (S)-3-acyl-5-
methyltetramic acid 12. The 3-acyltetramic acid 13 was
similarly obtained by using N-Boc-glycine as the starting
reagent.
oring truncated 3-oxododecanamide S2 inhibited agr. More-
over, analogues in which the homoserine lactone moiety was
modified by substitution with different heteroatoms or replaced
with alternative heterocyclic ring systems failed to inhibit agr.
Removal or reduction of the 3-oxo substituent also abolished
agr inhibitory activity (Table S1). Modification of the acyl chain
by the incorporation of a double bond or partial replacement
with phenyl or cyclohexyl substituents all resulted in the loss of
agr inhibitory properties (Table S1). Apart from the two 3-oxo-
C12-HSL isomers 1 and 2, none of the other analogues
examined inhibited bacterial growth at 100 μM. Taken
together, these data show that subtle changes in 3-oxo-C12-
HSL are sufficient to abolish QS and growth inhibitory
properties.
Since 1 undergoes a base-catalyzed rearrangement to the
TMA 5, we explored the agr inhibitory activities of TMA
analogues 3−13 (Table 2) by varying the 3-acyl chain length
3−8, stereochemistry 9, and substitution at the 5-position of
the heterocyclic ring 12 and 13. Each of the TMA analogues
examined apart from 3, 10, and 11 inhibited agr (Table 2), with
The 3-acyltetronic acids 14−18 listed in Table 3 were
prepared by the general method of C-acylation of commercially
available tetronic acid.22
the most active compound being 6 (IC50 = 10
3 μM).
Switching the C5 stereochemistry from (S)-5 to (R)-9
improved agr inhibitory activity by 1.5-fold, and replacement
with Me (12) or removal (13) of the 5-(2-hydroxyethyl)
substituent in 5 also resulted in enhanced agr inhibitory activity
(Table 2), thus indicating that the 5-position can withstand
alteration.
Table 3. QS and Growth Inhibitory Activities of 3-
Acyltetronic Acids
a
Since TMAs such as 5 are known to inhibit bacterial
growth14 and since growth inhibition is an undesirable property
for agents that attenuate virulence,10 we evaluated the growth
inhibitory properties of each of the TMA compounds
synthesized. Table 2 shows that the MIC for the 3-oxo-C12-
HSL-derived TMA 5 was 100 μM.
While analogues with shorter 3-acyl chains (3 and 4) did not
inhibit growth at this concentration, extension of the chain by
one, two, or four carbons, 6−8, respectively, substantially
increased potency such that the C14 analogue 8 exhibited an 8-
fold lower MIC (12.5 μM). These data are broadly in
agreement with the staphylococcal MICs for 5 and 7 reported
by Lowery et al.14 Interestingly, the shorter chain compound 5-
HE-C4-TMA 3, which we found was not active against S.
aureus, has been reported to kill P. aeruginosa15 but not C.
difficile.13 TMAs with 3-acyl chains of 10−14 carbons inhibited
growth and agr with differences between the MIC and IC50
values ranging from little different (e.g., 5-HE-C14-TMA 8) to
5-fold (e.g., 5-HE-C11-TMA 6). Compound 4, 5-HE-C8-TMA,
retained reasonable agr inhibitory activity (42 13 μM) and
completely inhibited production of the agr-dependent exotoxin
α-hemolysin at 100 μM without affecting staphylococcal growth
(Figure S2). It therefore offers a platform for the further
improvement of agr antagonism independent of growth
inhibition.
a
The asterisk (∗) indicates no growth inhibition up to 100 μM. ND,
not determined.
To gain insights into the 3-oxo-C12-HSL structural require-
ments for staphylococcal agr inhibition and to discover quorum
sensing inhibitors that do not impact on staphylococcal growth,
systematic modification of 3-oxo-C12-HSL was carried out,
focusing initially on the homoserine lactone (I), 3-oxo
substituent (II), acyl side chain (III), and amide (IV) structural
units of the molecule (Figure S1). Seventeen analogues of 3-
oxo-C12-HSL were synthesized and evaluated for inhibition of
agr and bacterial growth (Table 1 and Table S1). While the L-
isomer of 3-oxo-C12-HSL 1 inhibited agr with an IC50 of 22
μM, the D-isomer 2 was approximately 2-fold less active (IC50
of 37 9 μM). However, neither the corresponding ring
opened N-(3-oxododecanoyl)-L-homoserine S1 nor the heter-
TMAs such as 5 are ferric ion chelators,8,16 and so it is
possible that the ability to inhibit staphylococcal growth is, at
least in part, a consequence of its ability to restrict the
availability of an essential bacterial nutrient.23 Since iron has
been reported to abolish the antibacterial activity of 5 toward C.
difficile and P. aeruginosa,13,15 we synthesized TOA (Table 3,
14−18; Table S2, S17−S24) variants of the TMAs in which the
ring nitrogen is replaced with oxygen. We predicted that the
tetronic acid structure would not chelate iron, as it largely exists
as a 4-enolic tautomer24 as opposed to the TMA structure
where the 3-exoenolic tautomer in (Z)-configuration predom-
6
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Journal of Medicinal Chemistry
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inates. The latter is in syn orientation with the 2-oxo group and
thus can serve as a bidentate ligand for Fe(III) binding.8 This
was confirmed experimentally, and Figure 1a shows that in
contrast to the TMA 5, the TOAs 14−16 with acyl chains
ranging from C6 to C12 do not chelate ferric iron.
and 5-HE-C14 8 TMAs, respectively, compared with 270 μM
for 3-oxo-C12-HSL 1. For the C10-, C12-, and C14-TOAs 15−
17, the dissociation constants were similar to those of the 5-
HE-TMAs, i.e., 258, 72, and 4 μM, respectively. From these
TMAs and TOAs, it is clear that the higher the membrane
affinity, the more active is the compound against agr. Hence,
these data are consistent with AgrB or AgrC as the target.
Consequently, we sought to determine whether 3-oxo-C12-
HSL 1, 5-HE-C10-TMA 5, and C12-TOA 16 are competitive
inhibitors of the interaction between the S. aureus AIP-1 and its
cognate receptor AgrC-1. Figure 2 shows that in contrast to the
Figure 1. (a) Iron-chelating properties of 5-HE-C10-TMA 5 and the
C6-, C10-, and C12-TOAs 14, 15, 16 (at 50 μM) as determined using
the CAS assay: positive control, desferrioxamine (10 μM); solvent
control, MeCN. (b) 5-HE-TMAs and TOAs disturb the membrane
dipole potential. Changes in dipole potential were determined using
di-8-ANEPPS to measure the variation in the fluorescence ratio
R(460/520) as a function of concentration. The binding profiles for 5-
■
▲
△
●
HE-C10-TMA 5 ( ), C10-TOA 15 ( ), 5-HE-C12-TMA 7 ( ),
○
C12-TOA 16 ( ), 5-HE-C14-TMA 8 (∗), and C14-TOA 17 ( )
compared with 3-oxo-C12-HSL 1 ( ) are shown.
▽
When assayed for staphylococcal agr and growth inhibitory
properties (Table 3 and Table S2), each of the TOAs apart
from the C6-TOA 14 exhibited similar activities to the TMAs
with potency increasing as a function of acyl chain length. For
agr inhibition, C14-TOA 17 was the most potent compound
with an IC50 of 3 1 μM, approximately 8 times lower than the
MIC (25 μM). In fact, when S. aureus USA300 was exposed to
10 μM C14-TOA 17, production of the agr-dependent exotoxin
α-hemolysin was substantially reduced but growth was
unaffected (Figure S3). This TOA was also the most active
agr inhibitor among the 3-oxo-C12-HSL, TMA, and TOA
compounds evaluated in the present investigation. Two
additional TOA analogues S18 and S19 incorporating an
unsaturated acyl chain were also synthesized. While the
presence of a trans double bond in S18 had little effect on
activity (cf., 15), it was almost abolished in the cis analogue S19
(cf., 16). Further modifications of the 3-acyl chain by
introducing aryl substituents or 3-oxo substituent did not
yield active analogues (Table S2). Taken together, these data
demonstrate that TOAs can also be effective agr inhibitors and
that iron chelation is not required for growth or agr inhibition.
Since two of the major proteins involved in agr-dependent
QS (AgrC and AgrB) are located in the cytoplasmic membrane,
we sought to determine whether the TMAs and TOAs bind
directly to staphylococcal membranes. This was achieved using
the fluorescent probe di-8-ANEPPS in a dual-wavelength
ratiometric method that detects changes in membrane dipole
potential and yields binding information, such as affinity and
overall binding capacity.25 Figure 1b shows the binding profiles
for the 5-HE-C10-, 5-HE-C12-, and 5-HE-C14-TMA and the
C10-, C12, and C14-TOA compared with 3-oxo-C12-HSL 1.
For each of the 5-HE-TMAs 5, 7, 8 and TOAs 15−17, the data
fit a hyperbolic function that is consistent with a non-
cooperative, single-site binding model (Figure 1b). The
TMAs and TOAs bind to the staphylococcal membrane with
higher affinity than 3-oxo-C12-HSL with affinity increasing as
chain length increases. In these experiments, Kd values of 222,
23, and 2 μM were estimated for the 5-HE-C10 5, 5-HE-C12 7,
Figure 2. 3-Oxo-C12-HSL 1, 5-HE-C10-TMA 5, and C12-TOA 16 do
not compete with AIP-1 for cognate AgrC. Dose−response curves
showing the inhibition of a blaZ-based agrP3 reporter by (a) 3-oxo-
C12-HSL 1, (b) 5-HE-C10-TMA 5, (c) C12-TOA 16, and (d) the
competitive antagonist (Ala5)AIP-1.
rightward shift in the concentration−response curve for AIP-1
(i.e., the EC50 of AIP-1 increased by about 200-fold, from 5 nM
to 1.10 μM) in the presence of increasing concentrations of the
competitive agr inhibitor, (Ala5)AIP-1,26 neither 3-oxo-C12-
HSL 1, 5-HE-C10-TMA 5, nor C12-TOA 16 competitively
inhibits the activation of AgrC-1 by AIP-1. Consequently, agr
inhibition by these compounds is likely to involve an allosteric
interaction with AgrC, preventing efficient activation by the
AIP signal molecule. It is worth noting that C12-TOA 16 is at
least 5-fold more potent than 3-oxo-C12-HSL 1 and 5-HE-C10-
TMA 5 in preventing the AIP-mediated activation of AgrC
(Figure 2). Significantly, the three classes of negative allosteric
modulators appeared to act by altering the efficacy of the
natural ligand AIP-1 with no noticeable effect on the affinity of
AIP-1 to cognate AgrC receptor. For example, in the presence
of increasing concentrations of 5-HE-C10-TMA 5 (from 0 to
100 μM), the maximum level of activation was decreased by
over 10-fold while the EC50 of AIP-1 remained broadly in the
2−4 nM range (Figure 2b). The consequence(s) of the
allosteric interaction mediated by these agr inhibitors could be
prevention of AgrC receptor dimerization or interference with
the interactions between the AgrC cytoplasmic domain and the
response regulator protein AgrA which activates the agr P2 and
P3 promoters.27 To date, we have not been able to express and
purify functional recombinant AgrC protein, and therefore,
further elucidation of this inhibitory mechanism and/or binding
mode awaits resolution of this technical hurdle.
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To determine whether the TMAs and TOAs have potential
as antistaphylococcal agents, we first examined the ability of the
naturally produced P. aeruginosa TMA, 5-HE-C10-TMA 5, and
the most potent TOA C14-TOA 17 to reduce the adherence of
S. aureus to human nasal squamous cells, since a key risk factor
for staphylococcal disease is nasal carriage.28 Figure 3a shows
that C14-TOA 17 and 5-HE-C10-TMA 5 at 1 and 10 μM,
respectively, substantially reduced attachment to human
squamous cells.
rather than growth. In S. aureus, cyclic peptide mimetics of
the native QS signal molecules have shown promise as agr-
inhibitory virulence attenuators. These findings stimulated our
search for simplified chemical structures capable of blocking
agr-dependent QS. Previously, we discovered that the P.
aeruginosa signal molecule 3-oxo-C12-HSL 1 inhibited agr, and
so we utilized this compound as a starting point to investigate
the key structural features involved, mechanism of action, and
in vivo efficacy.
Intriguingly, changes in 1 resulted mostly in the complete
loss of activity. These subtle structural changes in 1 that
resulted in the loss of QS inhibitory properties require further
investigation and may involve differential access via the cell wall
to the cytoplasmic membrane. Fatty acids such as oleate
(nontoxic) and palmitoleate (growth inhibitor), for example,
show structure-specific antistaphylococcal activity that depends
on the cell wall teichoic acids.30
Furthermore, we have identified two new related nonpeptidic
classes of compounds, the TMAs and TOAs, as noncompetitive
AgrC inhibitors that are more potent than 3-oxo-C12-HSL 1.
The TMAs are rearrangement products of their corresponding
3-oxoacylhomoserine lactones. These compounds act as
negative allosteric modulators by affecting cognate ligand
efficacy. The most potent compound identified, C14-TOA 17,
reduced colonization of human nasal epithelial cells and
showed efficacy in a staphylococcal experimental mouse
infection model without obvious toxicity. Consequently, our
findings offer additional opportunities to exploit this chemical
architecture for the discovery of more potent analogues for
probing agr-dependent QS and for evaluation as antivirulent
agents.
Figure 3. Impact of C14-TOA 17 on adherence of S. aureus to
desquamated human nasal epithelial cells and experimental infection.
(a) Binding of S. aureus to squamous epithelial cells before or after
treatment with 5-HE-C10-TMA 5 (A, 10 μM) or C14-TOA 17 (B, 1
μM). Counts represent the number of bacterial cells adhered to 100
nasal cells. Results are expressed as a mean of three experiments
performed in duplicate. (b) Frequency of arthritis and (c) arthritic
index of mice treated with 17 and challenged with S. aureus. White bars
represent data from the control animals (PBS treated) and gray bars
animals treated with 17 (10 mg/kg body weight). Data are presented
as median (horizontal lines), interquartile ranges (bars), and ranges
(error bars). An arthritic index was calculated by scoring all four limbs
of each animal. Comparisons of groups for weight change and arthritis
score were done by Mann−Whitney U test (∗, p < 0.05). Fischer exact
probability test was used to calculate statistical differences in the
frequency of arthritis.
EXPERIMENTAL SECTION
■
Compounds and Chemistry. Synthesis procedures are in
Supporting Information. The chemical structures of the presented
1
compounds were verified by H and 13C NMR and high-resolution
mass spectrometry. The purity (>95%) was established by RP-HPLC
and 1H NMR spectrometry. 1H NMR spectra were recorded in CDCl3
or DMSO-d6 on a Bruker Avance-400 operating at 400 MHz. 13C
NMR spectra were recorded on a Bruker Avance-400 or Bruker
Avance(III)-500 operating at 100 or 125 MHz, respectively. High-
resolution electrospray (ES) mass spectra were recorded using Waters
Micromass LCT spectrometer. Analytical RP-HPLC was used to
establish purity and was performed using a Waters setup comprising
two 510 pumps, a 2487 dual λ absorbance detector, and Millennium
software. The separation was performed using a Phenomenex Onyx
monolithic C18 column (4.6 mm × 100 mm) and a linear gradient of
30−70% solvent B in 16.0 min, then 70−100% B in 1.0 min at a flow
rate of 3.0 mL/min. Solvent A was 0.06% TFA in H2O, and solvent B
was 0.06% TFA in MeCN/H2O (90:10). UV detection was at 214 and
254 nm.
General Procedure for the Synthesis of (S)-3-Acyl-5-(2-
hydroxyethyl)tetramic Acids. A solution of sodium methoxide in
MeOH (0.5 M, 2.0 mL, 1.0 mmol) was added to a stirred solution of
N-(3-oxoacyl)-L-homoserine lactone (1.0 mmol) in MeOH (3 mL)
under nitrogen.8 The mixture was stirred for 3 h at 55 °C and then
overnight at 50 °C. The mixture was cooled to rt and then passed
through an acidic ion-exchange resin (Dowex 50 WX2-200). The resin
was eluted with MeOH (30 mL). The eluents were combined and
concentrated in vacuo to afford the tetramic acids as solids.
To investigate the in vivo activity of C14-TOA 17 and 5-HE-
C10-TMA 5, we used the established murine arthritis infection
model.29 In these experiments, mice treated with C14-TOA 17
were challenged with S. aureus. The frequency and severity of
induced arthritis were assessed over a 10 day period. The
compound C14-TOA 17 significantly reduced the frequency of
arthritis and the arthritic index compared with the control
group over the first 3−7 days (Figure 3b,c). While a reduction
in synovitis and joint destruction was noted (Figure S4),
treatment with C14-TOA 17 did not reduce weight loss or the
numbers of viable staphylococci in the kidneys (Figure S4).
These results suggest that although 17 did not reduce bacterial
growth in vivo at the dose tested, it exhibits antivirulence and/
or anti-inflammatory activities in vivo which impact the course
of staphylococcal disease. In contrast, 5-HE-C10-TMA 5 was
inactive in this murine infection model (data not shown).
(S)-3-Decanoyl-5-(2-hydroxyethyl)tetramic Acid (5-HE-C10-
TMA, 5). Using the above general procedure and N-(3-oxododeca-
noyl)-L-homoserine lactone gave the desired compound as a cream
CONCLUSIONS
■
1
Given the global health threat posed by multiantibiotic resistant
bacteria, there is considerable interest in the discovery of
antibacterial compounds that attenuate bacterial virulence
solid (68%). H NMR (CDCl3) δ 0.90 (3H, t, Me), 1.28−1.41 (12H,
m, (CH2)6Me), 1.68 (2H, m, CH2(CH2)6Me), 1.81 and 2.14 (2H, 2m,
CH2CH2OH), 2.87 (2H, m, CH2(CH2)7Me), 3.84−4.01 (3H, m, ring
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CH and CH2CH2OH), 6.33 (1H, b, NH). 13C NMR (CDCl3) δ 14.11,
22.66, 25.91, 29.25, 29.27, 29.33, 29.40, 31.85, 33.00, 34.11, 60.91,
61.81, 100.53, 175.13, 189.98, 195.51. ES-MS m/z 298.2002 [M + H],
ASSOCIATED CONTENT
* Supporting Information
■
S
Scheme S1, Figures S1−S4, Tables S1 and S2, and experimental
procedures for the chemical synthesis and characterization of
N-acyl-L-homoserine lactone, TMA, and TOA analogues and
the biochemical, biological, and in vivo infection model tests.
This material is available free of charge via the Internet at
http://pubs.acs.org.
+
C16H28NO4 requires 298.2018. RP-HPLC tR = 9.13 min.
(S)-3-Dodecanoyl-5-(2-hydroxyethyl)tetramic Acid (5-HE-
C12-TMA, 7). Using the above procedure and N-(3-oxotetradeca-
noyl)-L-homoserine lactone gave 7 as an off-white solid (75%). [α]D
1
−7.1 (c 1.42, CHCl3). H NMR (CDCl3) δ 0.90 (3H, t, Me), 1.28−
1.40 (16H, m, (CH2)8Me), 1.68 (2H, m, CH2(CH2)8Me), 1.83 and
2.15 (2H, 2m, CH2CH2OH), 2.86 (2H, m, CH2(CH2)9Me), 3.84−
4.01 (3H, m, ring CH and CH2CH2OH), 6.33 (1H, b, NH). 13C NMR
(CDCl3) δ 14.12, 22.69, 23.36, 25.91, 28.99, 29.28, 29.33, 29.44, 29.53,
29.60, 29.80, 31.91, 32.96, 34.09, 60.96, 61.85, 65.95, 100.57, 175.10,
189.94, 195.47. ES-MS m/z 326.2345 [M + H], C18H32NO4+ requires
326.2331. RP-HPLC tR = 13.17 min.
AUTHOR INFORMATION
Corresponding Authors
*W.C.C.: phone, +44-115-9515080; e-mail, weng.chan@
nottingham.ac.uk.
*P.W.: phone, +44-115-9515047; e-mail, paul.williams@
nottingham.ac.uk.
■
General Procedure for Synthesis of 3-Acyltetronic Acids.
N,N′-Dicyclohexylcarbodiimide (2.3 mmol) was added to a stirred
solution of an alkanoic acid (2.0 mmol) and 4-(dimethylamino)-
pyridine (3.0 mmol) in dry CH2Cl2 (20 mL) at rt under nitrogen
atmosphere. Tetronic acid (2.1 mmol) was then added, and the
mixture was stirred overnight at rt. The mixture was filtered, and the
filtrate was extracted with 1 M aq HCl (2 × 10 mL). The organic
extract was dried over MgSO4 and concentrated in vacuo to dryness to
give, following crystallization, the desired 3-acyltetronic acids.
3-Dodecanoyltetronic Acid (C12-TOA, 16). The use of
dodecanoic acid in the above procedure gave 16 as a cream solid in
64% yield. 1H NMR (CDCl3) δ 0.90 (3H, t, Me), 1.28−1.41 (16H, m,
(CH2)8Me), 1.72 (2H, m, CH2(CH2)8Me), 2.95 (2H, m,
CH2(CH2)9Me), 4.58 and 4.70 (2H, 2s, ring CH2). 13C NMR
(CDCl3) δ 14.11, 22.65, 25.22, 29.19, 29.32, 29.38, 29.49, 29.56, 29.58,
Present Addresses
⊥R.C.C.: Department of Microbiology, Cornell University,
Wing Hall, Ithaca, NY, U.S.
#S.R.C.: School of Biological Sciences, University of Reading,
Whiteknights, Reading, RG6 6AJ, U.K.
Author Contributions
∞E.J.M. and R.C.C. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was funded by the Medical Research Council UK
(Grant G9219778), which is gratefully acknowledged. We are
grateful to Dr. Stephan Heeb for his assistance with figures
preparation.
31.90, 49.25, 68.80, 99.96, 168.34, 192.67, 197.99. ES-MS m/z
−
281.1743 [M − H], C16H25O4 requires 281.1753. RP-HPLC tR
=
11.71 min.
3-Tetradecanoyltetronic Acid (C14-TOA, 17). The use of
tetradecanoic acid in the above procedure gave 17 as a pale gray solid
in 73% yield. 1H NMR (CDCl3) δ 0.90 (3H, t, Me), 1.28−1.41 (20H,
m, (CH2)10Me), 1.72 (2H, m, CH2(CH2)10Me), 2.95 (2H, m,
CH2(CH2)11Me), 4.58 and 4.70 (2H, 2s, ring CH2). 13C NMR
(CDCl3) δ 14.12, 22.69, 24.92, 25.59, 29.19, 29.22, 29.35, 29.39, 29.57,
29.64, 29.66, 31.92, 38.73, 68.16, 128.81, 177.17, 192.31, 197.88. ES-
MS m/z 309.2061 [M − H], C18H29O4− requires 309.2066. RP-HPLC
tR = 16.78 min.
Bacterial Strains and Growth. Staphylococci were cultured in LB
or Mueller−Hinton (MH) or CYGP broth26 and incubated with
shaking at 37 °C. For agr inhibition assays, the agrP3::blaZ reporter
strain S. aureus 6390B (pRN6683) was employed. Growth curves in
the presence or absence of test compounds were derived by
determining optical density at 600 nm (OD600) over 18 h.
ABBREVIATIONS USED
■
agr, accessory gene regulator; AIP, autoinducing peptide; CAS,
chrome azurol S; MIC, minimum inhibitory concentration; 3-
oxo-C12-HSL, N-(3-oxododecanoyl)-L-homoserine lactone; QS,
quorum sensing; SAR, structure−activity relationship; di-8-
ANEPPS, 1-(3-sulfonatopropyl)-4-[β-2-(di-n-octylamino)-6-
naphthylvinyl]pyridinium betaine; TMA, tetramic acid; TOA,
tetronic acid
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