Inhibition of Pseudomonas aeruginosa biofilm formation with bromoageliferin analogues

Article abstract of DOI:10.1021/ja069017t

Two analogues of the marine natural product bromoageliferin have been synthesized and subsequently assayed for the ability to inhibit the formation of Pseudomonas aeruginosa biofilms. Both compounds inhibited the ability of Pseudomonas aeruginosa strains

Full text of DOI:10.1021/ja069017t

Published on Web 05/15/2007
Inhibition of Pseudomonas aeruginosa Biofilm Formation with
Bromoageliferin Analogues
Robert W. Huigens III,† Justin J. Richards,† Gina Parise,‡,§ T. Eric Ballard,† Wei Zeng,†
Rajendar Deora,‡,§ and Christian Melander*,†
Department of Chemistry, North Carolina State UniVersity, Raleigh, North Carolina 27695, and Program in
Molecular Genetics and Department of Microbiology and Immunology, Wake Forest UniVersity Health Sciences,
Winston-Salem, North Carolina 27157
Received December 16, 2006; E-mail: christian_melander@ncsu.edu
Biofilms play a significant role in infectious disease.1 Biofilms
are formed when planktonic bacteria adhere to a surface and initiate
the formation of a microcolony that exists as a community encased
in a protective extracellular matrix.1,2 It is estimated that biofilms
account for up to 80% of microbial infections in the body.3,4
Biofilms also underlie importunate infections of implanted medical
devices.2 Within a biofilm, bacteria display differential gene
expression4 and are upward of 1000-times more resistant to
conventional antibiotic treatment.5
Even though there is a pressing medical need for strategies to
control biofilm formation, there is a paucity of small molecules
that have been reported to inhibit the formation of terrestrial-based
bacterial biofilms.1 Current efforts toward small molecule-based
strategies to control biofilm formation have focused almost
Figure 1. Bromoageliferin and bromoageliferin analogues.
Scheme 1. Synthesis of TAGE (1) and CAGE (2)a
exclusively on inhibiting quorum sensing (QS), a signaling cascade
that is critical for bacterial communication6,7
To provide alternative molecular scaffolds that inhibit the
formation of terrestrial-based bacterial biofilms, we have turned to
anti-microfouling marine natural products as a source for structural
insight to guide molecular design. Microfouling is the first step of
biofouling that entails the formation of a marine-based bacterial
biofilm on the surface of a submerged object.8 Previous studies in
this field have focused on the use of brominated furanones, simple
marine natural products that accelerate the turnover of LuxR-type
proteins and disrupt QS.9 We posited that simple structural motifs
that are prevalent in the more complex, anti-microfouling natural
products would provide novel chemical architecture for inhibitors
of terrestrial-based bacterial biofilms.
The first structural motif we have investigated is based upon
the marine natural product bromoageliferin (Figure 1). Bromoag-
eliferin has been reported to possess anti-biofilm activity against
the marine R-proteobacteria R. salexigens10 and is a member of
the oroidin class of biologically active natural products that are
characterized by a 2-aminoimidazole (2-AI) subunit.11 The preva-
lence of the 2-AI subunit led us to hypothesize that this structural
motif, in tandem with the bicyclic core of bromoageliferin, was
the key pharmacophore that imparts biological activity. To test our
hypothesis, we synthesized and assayed two bromoageliferin
analogues, trans-bromoageliferin analogue 1 (TAGE) and the cis-
bromoageliferin analogue 2 (CAGE) (Figure 1) for anti-biofilm
activity. We also synthesized 4-(3-aminopropyl)-2-aminoimidazole
as a control.
a Reaction conditions (TAGE): (a) MsCl, TEA, CH2Cl2, -78 °C
-25 °C; (b) NaN3, DMF, 100 °C, 90%; (c) m-CPBA, CH2Cl2, 88%; (d)
NaN3, DMF, 120 °C, 84%; (e) H2 (1 atm), 10% Pd/C, Boc2O, DMF, 71%;
(f) PDC, DMF, 70%; (g) TFA/CH2Cl2, then HCl/MeOH; (h) NH2CN, H2O/
pH ) 4.3, 95 °C, 74%.
sodium azide to yield the diazide 4 in 90% yield. Diazide 4 was
epoxidized with m-CPBA, and the epoxide ring was subsequently
opened with sodium azide to yield the triazidoalcohol 5.
Triazidoalcohol 5 was hydrogenated in the presence of Boc2O, and
then oxidized with PDC to generate the tri-Boc protected
R-aminoketone 6. Quantitative removal of the Boc-groups were
affected with 1:4/TFA/CH2Cl2, and the resulting TFA salt was
treated with a saturated solution of HCl in methanol to give the
HCl salt of the R-aminoketone. Finally, condensation with cyana-
mide12 generated TAGE (1) in 24% overall yield in eight steps
from diol 3. Application of a similar synthetic sequence to the
known cis-diol 714 generated CAGE (2) in 18% overall yield.
With both 2-aminoimidazole derivatives in hand, we tested the
ability of these simplified bromoageliferin scaffolds to inhibit the
formation of Pseudomonas aeruginosa biofilms. P. aeruginosa is
a γ-protoeobacteria that is in the same phylum as R. salexigens
4-(3-Aminopropyl)-2-aminoimidazole was synthesized as previ-
ously described.12 The synthesis of TAGE (1) is outlined in Scheme
1. The known diol 313 was bismesylated and then reacted with
† North Carolina State University.
‡ Program in Molecular Genetics, Wake Forest University Health Sciences.
§ Department of Microbiology and Immunology, Wake Forest University Health
Sciences.
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J. AM. CHEM. SOC. 2007, 129, 6966-6967
10.1021/ja069017t CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Chart 1. Relative Biofilm Formation of PAO1 and PA14 in the
Presence of TAGE (1) or CAGE (2) after 24 h.
the formation of P. aeruginosa biofilms. Although both compounds
inhibit the formation of P. aeruginosa biofilms, we note differential
toxicity between TAGE (1) and CAGE (2) toward planktonic P.
aeruginosa bacteria. This activity is driven by the inversion of one
stereocenter. At the higher concentrations of CAGE (2) (400 and
500 µM) where we observe significant antimicrobial activity, the
observed reduction of biofilm mass may be due to a combination
of bactericidal and biofilm inhibition activity. Further experiments
are necessary to decouple these two effects. Both compounds have
no structural homology to any of the known inhibitors of P.
aeruginosa biofilm formation1 and may be operating by a unique
mechanism that can be exploited for future drug development
efforts. We are currently establishing the mechanism of action for
both these compounds and will report our findings in due course.
Acknowledgment. Financial support is gratefully acknowledged
from NCSU, Burroughs-Wellcome (predoctoral fellowship to
J.J.R.), and GSK (predoctoral fellowship to T.E.B.). Research in
the laboratory of R.D. is supported by funds from WFU Health
Sciences and by grants from the USDA (CSREES-USDA Grant
No. 2006-35604-16874) and the NIH (Grant R21 AI071054). Mass
spectra were obtained at the Mass Spectrometry Laboratory for
Biotechnology at NCSU. We thank Professor Daniel Wozniak
(WFU) for providing PAO1 and PA14 and for helpful discussions.
We also thank Professor Reza Ghiladi (NCSU) for spectroscopic
assistance.
and is a serious threat to cystic fibrosis (CF) patients.15,16 As CF
patients age, P. aeruginosa becomes the predominant pulmonary
pathogen and is present in ca. 85% of cultures isolated from patients
with advanced disease.15,16 Despite significant progress in the
management of CF symptoms, virtually all CF patients succumb
to chronic pulmonary infections.
The P. aeruginosa bacterial strains PAO1 or PA14 were allowed
to form biofilms in a 96-well plate in the absence or presence of
TAGE (1) or CAGE (2). After 24 h,17 the media and planktonic
bacteria were removed, the wells were washed vigorously, and
crystal violet was added. Crystal violet stains the bacterial biofilm
that forms on the inside wall of the well, which, following ethanol
solubilization, can be quantitated by spectrophotometry (A540).21
Chart 1 summarizes the results of this assay. We determined IC50
values of biofilm inhibition for TAGE (1) against PAO1 (100 µM)
and PA14 (190 µM) and IC50 values for CAGE (2) against PAO1
(100 µM) and PA14 (180 µM). 4-(3-Aminopropyl)-2-aminoimid-
zole displayed only marginal activity at 500 µM (20% against PAO1
and 15% against PA14) and did not inhibit biofilm development at
100, 200, 300, or 400 µM (data not shown).
Supporting Information Available: Experimental procedures and
characterization data for all new compounds; planktonic growth curves
and colony counts for PAO1 and PA14 in the presence and absence of
TAGE and CAGE. This material is available free of charge via the
Internet at http://pubs.acs.org.
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(17) We determined that both PAO1 and PA14 reached maximum cellular
density (in the absence of TAGE or CAGE) after 24 hours of growth
(see Supporting Information).
A fundamental question underlying the inhibition assays was the
effect each compound had on planktonic growth. Growth curves
were determined for both PAO1 and PA14 in the absence and
presence of TAGE (1) or CAGE (2) (Supporting Information).
TAGE (1) showed no effect on the growth of either PAO1 or PA14
over 24 h at 100, 200, or 300 µM. At 400 µM TAGE (1), we
observed a modest reduction in bacterial growth (ca. 25%), for both
PAO1 and PA14, while we observed significant reduction in
planktonic growth (>50%) at 500 µM TAGE (1). For CAGE (2),
we observed no effect on bacterial growth (for either PAO1 or
PA14) at 100 or 200 µM, modest reduction at 300 µM (e25%),
and significant reduction (>50%) at both 400 and 500 µM. Colony
counts were also performed for both PAO1 and PA14 grown in
the absence or presence of TAGE (1) and CAGE (2) that verified
that no reduction in the growth curve correlated with no reduction
in viable colonies (Supporting Information).
In conclusion, we have identified two analogues of the marine
natural product bromoageliferin that have biological activity against
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