Bicyclic brominated furanones: A new class of quorum sensing modulators that inhibit bacterial biofilm formation

Article abstract of DOI:10.1016/j.bmc.2014.01.004

Both natural and synthetic brominated furanones are known to inhibit biofilm formation by bacteria, but their toxicity to mammalian cells is often not reported. Here, we designed and synthesized a new class of brominated furanones (BBFs) that contained a

Full text of DOI:10.1016/j.bmc.2014.01.004

Accepted Manuscript
Bicyclic Brominated Furanones: A New Class of Quorum Sensing Modulators
that Inhibit Bacterial Biofilm Formation
Sijie Yang, Osama A. Abdel-Razek, Fei Cheng, Debjyoti Bandyopadhyay,
Gauri S. Shetye, Guirong Wang, Yan-Yeung Luk
PII:
S0968-0896(14)00022-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.01.004
BMC 11335
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 March 2013
21 December 2013
2 January 2014
Please cite this article as: Yang, S., Abdel-Razek, O.A., Cheng, F., Bandyopadhyay, D., Shetye, G.S., Wang, G.,
Luk, Y-Y., Bicyclic Brominated Furanones: A New Class of Quorum Sensing Modulators that Inhibit Bacterial
Biofilm Formation, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.01.004
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Bicyclic Brominated Furanones: A New Class of Quorum
Sensing Modulators that Inhibit Bacterial Biofilm Formation
Sijie Yang,1 Osama A. Abdel-Razek,2 Fei Cheng,1 Debjyoti Bandyopadhyay,1 Gauri S. Shetye,1
Guirong Wang,2* and Yan-Yeung Luk1,3*
1Department of Chemistry, Syracuse University, Syracuse, New York 13244; 2Department of
Surgery, Upstate Medical University, State University of New York, Syracuse, New York 13210;
3Department of Biomedical and Chemical Engineering, Syracuse University, Syracuse, New York
13244.
yluk@syr.edu
Table of Contents
A new class of synthetic bicyclic brominated furanones were synthesized and identified as quorum
sensing modulators that inhibit biofilm formation by Gram-negative bacteria.
Abstract: Both natural and synthetic brominated furanones are known to inhibit biofilm formation by
bacteria, but their toxicity to mammalian cells is often not reported. Here, we designed and
synthesized a new class of brominated furanones (BBFs) that contained a bicyclic structure having
one bromide group with well-defined regiochemistry. This class of molecules exhibited reduction in
the toxicity to mammalian cells (human neuroblastoma SK-N-SH) and did not inhibit bacteria (P.
aeruginosa and E. coli) growth, but retained the inhibitory activity towards biofilm formation of
bacteria. In addition, all the BBFs inhibited the production of virulence factor elastase B in P.
aeruginosa. To explore the effect of BBFs on quorum sensing, we used a reporter gene assay and
found that 6-BBF and 7-BBF exhibited antagonistic activities for LasR protein in the lasI quorum
sensing circuit, while 5-BBF showed agonistic activity for the rhlI quorum sensing circuit. This study
suggests that structural variation of brominated furanones can be designed for targeted functions to
control biofilm formation.
1

1.
Introduction
Biofilms on different surfaces cause enormous detrimental effects in medical and industrial
settings1 and are the source of many diseases, including endocarditis, otitis media, chronic
prostatitis, periodontal disease, chronic urinary tract infections, and osteomyelitis,2,3 and In
particular, biofilms formed by Pseudomonas aeruginosa are often related to serious infections
in immunocompromised patients,4 particularly lung infection in cystic fibrosis patients.3,5 The
formation of biofilm is regulated by multiple genes, which results in highly complex film
structures on the surface of microbes.6 Controlling the formation of biofilm has been
challenging because inhibition of biofilm formation and dispersion of already formed biofilm
are difficult.7 Also, the bacteria reside in the biofilm often appear to be more tolerant to
antibiotic treatments than planktonic bacteria.8 One rational approach to control biofilm
formation is to interfere with the chemical communication that results in a quorum sensing
(QS) between bacteria, which is one of the key events leading to the biofilm formation.9
Several synthetic autoinducer analogs have been reported to induce or inhibit quorum sensing
and biofilm formation of P. aeruginosa.10,11 Other small molecules that are not structurally
similar to natural autoinducers have also been proven to modulate quorum sensing and inhibit
and disperse proteobacterial biofilms.12,13 The agonistic/antagonistic activities of these
molecules could be tuned by structural modifications. Chemical library screening has also been
utilized to discover biofilm formation inhibitors.14
In this work, we aim to develop new structures of inhibitors of biofilm formation that are
both nonmicrobicidal to bacteria and nontoxic to mammalian cells. Based on our previous
study on what constitute the important structural elements of brominated furanones,15 we
propose that a bicyclic version of brominated furanones, which retain the conjugated exocyclic
vinyl bromide in the furanone moiety, that could potentially reduce their toxicity while
retaining the biofilm inhibitory activities. Here, we designed a new class of bicyclic brominated
furanones (BBFs), 5-BBF, 6-BBF, and 7-BBF, with [3,3,0], [4,3,0], and [5,3,0] fused ring
structures, respectively (Figure 1). Compared to the known brominated furanones, such as
BF815 and BF4 (some literature use the name “C30”),16 the fused bicyclic systems bear only
one bromo-substitution, and introduce bulkier but semi-rigid cyclic hydrocarbon skeletons into
the molecules that can potentially increase the binding and selectivity to the receptor proteins.
Figure 1. Structures of BBFs and known brominated furanones.
In this study, the toxicity of these molecules is evaluated for bacteria P. aeruginosa and E. coli
and human neuroblastoma SK-N-SH cells. We found that BBFs exhibited reduced toxicity to
bacteria and mammalian cells compared to BF8 and BF4. It was also found that BBFs inhibited
the production of virulence factor elastase B by P. aeruginosa. To explore a mechanistic
understanding, we examined their interference (agonist or antagonist) with two quorum sensing
pathways (las and rhl) in P. aeruginosa by using reporter gene assays.
2

2.
Results and Discussion
2.1
Synthesis of BBFs
Bicyclic brominated furanones, 5-BBF, 6-BBF and 7-BBF, were synthesized via a similar
route starting with keto acids (Scheme 1). The synthesis of 7-BBF is described as an example
here. Under basic condition, coupling of cycloheptanone and dimethyl carbonate provided methyl
2-oxo-1-cycloheptanecarboxylate 2, which underwent acetoacetic ester synthesis to give 2-
oxocycloheptaneacetic acid 3. This intermediate was then subjected to bromination and
dehydration to build the fused ring framework, followed by elimination to yield the conjugated
final product 7-BBF without isolating the intermediates (Scheme 1). Known brominated
furanones BF815 and BF417 were also synthesized to compare their toxicities and biofilm
inhibition activities with BBFs.
Scheme 1. Synthesis of BBFsa
o
aReagents and conditions: (a) Br2, CH2Cl2, 0 °C to rt; (b) P2O5, DCM, 0 C to reflux; (c) Et3N,
DCM, 0 °C to reflux; (d) LiOH, THF/H2O (9:4), 23h, 1M HCl (aq.), rt; (e) NaH, benzene, rt to
85 °C; (f) ethyl bromoacetate, K2CO3, acetone, rt to reflux, 16 h; (g) 6 M HCl (aq.), AcOH, rt to
reflux, 2d.
2.2
BBFs inhibit the biofilm formation by P. aeruginosa and E. coli
We used a wild type strain PA01-GFP to study biofilm formation on steel coupons with and
without BBFs. This strain constitutively expresses green fluorescent proteins (GFP)18 and enables
easy and direct visualization of biofilm by confocal laser scanning microscopy. In the initial
screening, all three BBFs resulted in much less green fluorescence signals in the PA01 biofilms
than the BF-free control at 400 µM, with 6-BBF provided more inhibition than that by 5-BBF
and 7-BBF (Figure 2). Biofilm grown in the presence of 400 µM BF4 showed the least
fluorescence signals (see Fig S1). To characterize the biofilm inhibition quantitatively, we
accessed the biomass, mean thickness and surface area of biofilms formed with and without agents
using COMSTAT software (Figure 3).19 The results were normalized by the controls without
agents. Compound 6-BBF exhibited the strongest inhibition as it reduced the biofilm formation by
71% (P < 0.01), followed by 5-BBF with 53% inhibition (P < 0.01) and 7-BBF with 50%
inhibition (P < 0.05). The mean thickness of biofilm was reduced by 6-BBF to 60% (P < 0.01)
and by 7-BBF to 74% (P < 0.05). We note that the mean thickness of biofilm treated by 5-BBF
did not seem to reduce. One possibility is that the biofilm becomes more “fluffy” in the presence
of 400 µM 5-BBF. The surface areas of biofilm in the presence of all three BBFs were about 60%
3

of that formed in the absence of BFs (P < 0.05). Similar results were obtained when E.coli RP437
(pRSH103) biofilms were grown in the absence and presence of 200 µM BBFs and BF8 (see Fig
S2).
Figure 2. The effect of brominated furanones on biofilm formation by P. aeruginosa.
Representative confocal laser scan microscopy (CLSM) images of biofilm formed by PA01-GFP
(expresses green fluorescence on plasmid pSMC2) (A) in the absence of agents, and in the
presence of (B) 5-BBF, (C) 6-BBF, (D) 7-BBF. The control is supplemented with the same
amount (0.8%) of DMSO as present in the BF-treated conditions. Scale bar = 50 µm.
140
Biomass
Mean thickness
120
Surface area
100
80
*
*
*
** *
*
**
60
40
20
0
**
**
** **
1
5-BBF
2
6-BBF
3
7-BBF
4
BF4
Figure 3. Quantification of biofilm formation by PA01-GFP in the absence and presence of 400
µM brominated furanones. Biomass, mean thickness, and surface area were quantified from
fluorescence image using COMSTAT software. Z-Stack images from four different locations were
used. Values are normalized by that of the BF-free control and represent the means standard
deviation from 4 replicates. Significant differences in biofilm formation with BF-free control are
indicated by asterisks: *, P < 0.05; **, P < 0.01.
These results suggest that 6-BBF is a stronger biofilm inhibitor than 5-BBF and 7-BBF for
both P. aeruginosa and E. coli. We followed up with a dose-dependence study on inhibitory
activity of BBFs on P. aeruginosa biofilm using a colorimetric assay in 96-well plates employing a
dye molecule, crystal violet (CV) (see Fig S3).20 The half maximal inhibitory concentration (IC50)
4

values obtained from the dose-response curves were more than 400 µM for 5-BBF, and 145.8
µM and 139.7 µM for 6-BBF and 7-BBF, respectively. These results suggest that 6-BBF and 7-
BBF are stronger biofilm inhibitors than 5-BBF for P. aeruginosa. We note that the percentage
of relative biofilm formation in the presence of 400 µM BBFs obtained from confocal laser scan
microscopy did not match exactly that obtained from the CV-dye staining assay. However, the
general trend that 6-BBF and 7-BBF is more active than 5-BBF against Pseudomonas biofilm
formation is consistent for both assays.
2.3
BBFs do not influence the growth of P. aeruginosa or E. coli
The toxicity of brominated furanones to the growth of bacteria is unpredictable based on
structures. For example, BF8 does not inhibit the growth of E. coli, whereas similar structures
do.15 Here, we compared the toxicity of BBFs and BF4 to the growth of P. aeruginosa and E.
coli to study if the biofilm inhibition was due to bactericidal effect. At 400 µM, none of the three
BBFs inhibited the growth of P. aeruginosa PA01 (Figure 4), and bacteria grown in the presence
of BBFs reached the same optical density (OD600) as those in the absence of BBFs after 24 h.
Under the same conditions, BF4 completely inhibited bacterial growth (P < 0.01), which
suggested that the significant biofilm inhibition observed for BF4 at this concentration was due to
bactericidal effect. The effect of the BBFs on the growth of E. coli strain RP437 was also
studied, with BF8 (a known biofilm inhibitor to this strain at 200 µM) as a positive control. At
200 µM, none of the BBFs exhibited obvious impact on the growth of E. coli RP437 (see Fig S4).
The growth curve of bacteria in the presence of BF8 deviated from that of the control for up to 8
h and then the OD600 values were essentially the same to the control after then.
1.4
1.2
1
control
0.8
5-BBF
0.6
6-BBF
7-BBF
0.4
BF4
0.2
**
0
0
2
4
6
8
10 12 14 16 18 20 22 24
Time/h
Figure 4. Growth curves of P. aeruginosa PA01 in the absence (control) and presence of 400 µM
brominated furanones. Values represent the means standard deviation from six replicates.
Significant differences in the optical density with BF-free control are indicated by asterisks: **, P
< 0.01.
2.4
BBFs interfere with the quorum sensing in P. aeruginosa.
To investigate whether BBFs inhibit biofilm formation via interference with QS, we studied
the effects of BBFs on the QS systems in P. aeruginosa. There are two identified N-acyl
homoserine lactone (AHL)-mediated quorum sensing circuits in P. aeruginosa. One is the las
circuit that includes an AHL synthase gene lasI responsible for the synthesis of N-(3-oxo-
dodecanoyl)-L-homoserine lactone (3-oxo-C12-HSL or PAI1);21 the PAI1 binds to LasR to
activate a range of quorum sensing genes. The other is the rhl circuit that includes rhlI gene
responsible for synthesis of N-butanoyl-L-homoserine lactone (C4-HSL, or PAI2);22 the PAI2
binds to RhlR to further activate other genes.
Reporter strain PA01 (plasI-LVAgfp)12 was used to evaluate the las quorum sensing system.
The GFP production is quantified by correcting the measured fluorescence signal for cell density.
5

Both 6-BBF and 7-BBF decreased fluorescent signals as the concentration was increased from 50
to 150 to 300 µM (Figure 5), indicating an inhibition of GFP production. At 300 µM , the
presence of 6-BBF or 7-BBF caused a reduction in GFP production to 78 1.1% (P < 0.05) and
69 4.0% (P < 0.001), respectively, as compared to that of the BF-free controls. These results
suggest that 6-BBF and 7-BBF are weak antagonists of LasR protein. The known biofilm
inhibitor BF815 resulted in less GFP production, 46 4.8%, compared to that of the BF-free
control (P < 0.001). Compound 5-BBF inhibited the GFP production slightly at 50 and 150 µM,
and reduced the GFP expression by approximately half at 300 µM. These results suggest that the
BBFs are weak to moderate antagonists of the las QS circuit, possibly by binding to LasR
receptor protein. The BBFs were also examined for antagonism of QS using a double-knockout
reporter strain P. aeruginosa PAO-JP223 (∆lasI∆rhlI) harboring the plasmid plasI-LVAgfp.24
When bacteria are growing in the presence of exogenously introduced autoinducer 3-oxo-C12-
HSL or analogues, LasR receptor binds to the AI and activates transcription of the lasI promoter
which controls green fluorescence protein (GFP) expression. Similar inhibition of GFP production
was obtained when PAO-JP2 (plasI-LVAgfp) was used (see Fig S5). We note that the inhibition
of GFP production within the concentration range tested is not due to bactericidal effect.
50 µM
120
100
80
60
40
20
0
150 µM
300 µM
*
*
*
***
*
***
***
5-BBF
6-BBF
7-BBF
BF8
Figure 5. GFP expression by PA01 (plasI-LVAgfp) in the absence or presence of 5-BBF, 6-BBF,
7-BBF, or BF8 at 50, 150, and 300 µM. Fluorescence signals were corrected for cell density by
dividing by OD600 of cell culture and the results were normalized to that of the BF-free control.
Values represent the means standard deviation of the mean. Data shown is a representative of at
least three separate experiments. Significant differences in the GFP expression with the control are
indicated by asterisks: *, P < 0.05; ***, P < 0.001.
We also tested the effects of BBFs on the rhl quorum sensing system using PA01 (prhlI-
LVAgfp). Interestingly, 5-BBF promoted, instead of suppressed, the GFP expression significantly
as its concentration was increased (See Fig S5). At 150 µM and 300 µM, 5-BBF increased the
GFP expression by 82.6 4.4% and 165 17.6%, respectively (P < 0.01). On the contrary, 6-
BBF, 7-BBF and BF8 had no effect on the GFP expression at all concentrations tested. These
results suggest that 5-BBF is a strong agonist of RhlR, while 6-BBF and 7-BBF are not. Similar
results were also obtained when reporter strain PAO-JP2 (prhlI-LVAgfp) was used (see Fig S6).
The expression of the virulence factor metalloprotease elastase B (LasB) in P. aeruginosa is
also controlled by the QS.25 This highly toxic virulence factor facilitates the invasion and
destruction of host tissues,26 induces inflammatory responses from the host27 and also signals the
biofilm formation.28 Thus, molecules that inhibit virulence factor production without bactericidal
effect are of potential use without invoking the bacterial resistance. Because BBFs appeared to be
potent antagonists of LasR, we also tested their ability to inhibit elastase B production. At 300
µM, all BBFs significantly reduced the production of elastase B by ~80% (see Fig S7). In a dose-
dependent study, the expression of elastase B decreased as the concentration of 6-BBF increased.
These results are consistent with BBFs being antagonists of LasR in P. aeruginosa.
These results suggest that the ring size of BBFs have an impact on their ability to modulate
QS in P. aeruginosa. For the las system, BBFs with larger rings (6-BBF and 7-BBF) seemed to
6

have slightly stronger antagonistic activity than that with smaller rings (5-BBF). For the rhl
system, the agonistic activity seemed to be more sensitive to the ring size, as 5-BBF was able to
enhance the activity of RhlR protein while 6-BBF or 7-BBF had no effect. We believe that the
interference of only one quorum sensing signaling pathway will likely elicit an effect on biofilm
formation, and response in an in vivo environment, but does not ensure a therapeutic
development.29
2.5
BBFs are less cytotoxic than other BFs to human cells
To avoid invoking bacterial resistance,30-33 nonmicrobicidal agents that do not kill bacteria or
influence bacterial growth are being sought over. However, for any potential use of the agents, the
toxicity of the molecules towards mammalian or human cells should be minimized. Although many
natural and non-natural brominated furanones inhibit biofilm formation by a wide range of
bacteria, few studies have addressed their toxicity to mammalian or human cells.11,34 Here, we
compare the toxicity of BBFs to a nonmicrobicidal brominated furanone, BF8, against human
neuroblastoma SK-N-SH. This human cell line were allowed to grow in 96-well tissue culture-
treated microtiter plates for 24 h, after which they were treated with at 400 µM (the highest
concentration tested for biofilm inhibition) of each agent (5-BBF, 6-BBF, 7-BBF and BF8) for 1
h. The agent-containing medium was replaced with fresh medium and the cells were then allowed
to recover for 0, 24, and 48 h (recovery time), at which time the number of live cells was
determined by the CCK 8 assay.35 Survival (%) was calculated based on BF-free control.
After 1 h treatment of agents and 0 h recovery time, BF8 exhibited the strongest cytotoxic
effect toward human neuroblastoma SK-N-SH cells; 29.0 14.1 % of cells were alive compare to
the BF-free control (P < 0.001). Bicyclic brominated furanones 5-BBF and 6-BBF showed mild
toxicity (~ 76% cell survival for both), and 7-BBF resulted in ~ 44% of cell survival (Figure 6).
After 24 h and 48 h of recovery time, survival (%) for cells treated with 5-BBF dropped to ~ 21%
and ~ 6%, respectively. For cells treated with BF8, almost no live cell remained (< 2%) after 24
and 48 h. In the presence of 7-BBF, cell survival dropped to ~ 7 % after 24 h of recovery time,
but appeared to increase to 13 % after 48 h. In contrast, 6-BBF exhibited the least cytotoxic
effect to this human cell line. After 24 h and 48 h of recovery time, about 50 % survival was
observed for cells treated with 6-BBF. We note this cytotoxicity assay was also performed at
lower concentration (100 µM). At this lower concentration, all three BBFs showed little to no
toxicity, with 6-BBF being the least toxic one. Brominated furanone, BF8, was still toxic at this
low concentration (see Fig S8). The cytotoxicity exhibited by the BBFs to the human
neuroblastoma limited their use in further internal clinical study.
100
0 h
90
24 h
80
48 h
70
60
******
50
**
40
**
30
***
20
10
0
***
***
**
** ***
5-BBF
6-BBF
7-BBF
BF8
Figure 6. Survival (%) of human neuroblastoma SK-N-SH cells at 0, 24, and 48 h after 1 h
treatment of 400 µM 5-BBF, 6-BBF, 7-BBF and BF8, respectively. Values represent the means
standard deviation from six replicates. Significant differences between BF-treated conditions in the
survival (%) and BF-free control at each time point are indicated by asterisks: *, P < 0.05; **, P <
0.01; ***, P < 0.001.
7

3.
Conclusion
The bicyclic brominated furanones are a new series of synthetic brominated furanones that
interfere with quorum sensing and QS-controlled activities in Gram-negative bacteria. They inhibit
biofilm formation by E. coli and P. aeruginosa and inhibit elastase B production by P. aeruginosa
at nonmicrobicidal concentrations. The BBFs are also less cytotoxic to human neuroblastoma cells
compared to other known brominated furanones. This work indicates some structure-bioactivity
relationship for brominated furanones. The relative high IC50 of biofilm inhibition and moderate
cytotoxicity to human cells of these BBFs need further improvement for pharmaceutical area.
However, they may be useful in industrial and other ex vivo settings. The exploration of structures
that give high potency for biofilm inhibition while maintaining a low toxicity is an ongoing subject
of our research.
Acknowledgment
This work was partially supported by NSF-CAREER (#0845686, PI Y. Y. L), NSF-EFRI
(#1137186, coPI, Y. Y. L), and NIH HL096007 (G. W.). We thank Bonnie B. Toms (Upstate
Medical University, SUNY) for the generous donation of human neuroblastoma cell line SK-N-
SH. We thank Dr. Helen E. Blackwell (Univ. of Wisconsin-Madison) for the generous donation of
strains PAO-JP2 (plasI-LVAgfp) and PAO-JP2 (prhlI-LVAgfp), Dr. Dacheng Ren (SU) for the
generous donation of strains E. coli RP437, E. coli RP437(pRSH103), and Dr. Hiroaki Suga (The
University of Tokyo) for the generous donation of plasmids plasI-LVAgfp and prhlI-LVAgfp. We
also thank Dr. James L. Hougland (SU) for the use of fluorescence plate reader and the review of
the manuscript.
Supplementary Information Available
Supplementary information including the synthesis procedures, NMR characterization,
protocols for biological assays, and supplemental graph of data for biological activities are
included with this article online.
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Graphical Abstract:
10