Cephalosporin-3'-diazeniumdiolates: Targeted NO-donor prodrugs for dispersing bacterial biofilms

Article abstract of DOI:10.1002/anie.201202414

Just say NO to biofilms: NO-donors are used to disperse a bacterial biofilm so that co-administered antibiotics will kill the more susceptible unattached cells. The chemically stable cephalosporin-3'-diazeniumdiolate NO-donor prodrug (see scheme) is activated by bacterial β-lactamases and facilitates this two-step biofilm erradication. Copyright

Full text of DOI:10.1002/anie.201202414

Angewandte
Chemie
DOI: 10.1002/anie.201202414
Rational Drug Design
Cephalosporin-3’-diazeniumdiolates: Targeted NO-Donor Prodrugs
for Dispersing Bacterial Biofilms**
Nicolas Barraud, Bharat G. Kardak, Nageshwar R. Yepuri, Robert P. Howlin, Jeremy S. Webb,
Saul N. Faust, Staffan Kjelleberg, Scott A. Rice, and Michael J. Kelso*
Biofilms are sessile communities of microbial cells contained
within a self-produced exopolymeric matrix. Bacteria encased
in biofilms exhibit upwards of 10–1000-fold higher resistance
to biocides and traditional antibiotics than their planktonic
counterparts (i.e. floating, unattached), and they are less
susceptible to host immune defenses.[1,2] Accordingly, chronic
antimicrobial-tolerant bacterial infections are often biofilm-
based (e.g. infections on indwelling medical devices and
incurable Pseudomonas aeruginosa respiratory infections in
cystic fibrosis (CF) sufferers).[3] Currently there are few
effective therapeutics for clearing biofilm-based infections,
and a critical need exists for new agents and treatment
strategies.[2,4,5]
strategy has emerged that uses agents to first trigger biofilm
dispersion, so that the more susceptible planktonic bacteria
can be cleared by conventional antibiotics.[7] We previously
reported that picomolar and low nanomolar concentrations of
nitric oxide (NO) trigger biofilm dispersion in P. aeruginosa
and multispecies biofilms,[8,9] and that the NO-donor sodium
nitroprusside (SNP) greatly enhances the efficacy of anti-
microbial compounds (e.g. tobramycin) in removing estab-
lished P. aeruginosa biofilms.[10] While several studies have
supported our findings,[11–14] other contradictory reports
suggest that NO has no effect on biofilms or promotes
biofilm formation.[15–18] These inconsistencies may arise from
differences in experimental conditions[16] and from the fact
that only low NO concentrations induce biofilm dispersal.[17]
Nevertheless, mounting evidence supports using NO-induced
dispersion in combination with antibiotics as a new strategy
for clearing chronic biofilm infections.
Many compounds are known that spontaneously produce
NO in aqueous solution (like SNP), and many of these may be
useful as biofilm dispersants.[19] From a clinical perspective,
however, prodrugs that release NO only upon reaction with
biofilm-specific enzymes (e.g. b-lactamase) are more attrac-
tive because of their potential for targeting NO release to
biofilms, thus limiting exposure of host tissues to exogenous
NO. Herein, we report the synthesis of a cephalosporin-3’-
diazeniumdiolate NO-donor prodrug and demonstrate its
effectiveness as a b-lactamase-triggered P. aeruginosa biofilm
dispersant.
In recent years, it has emerged that many bacteria
transition between the planktonic and biofilm states, and
that given the correct environmental cues, biofilm bacteria
can be induced to undergo coordinated dispersal and
reversion to the planktonic form.[6] A putative anti-biofilm
[*] Dr. B. G. Kardak, Dr. N. R. Yepuri, Dr. M. J. Kelso
School of Chemistry, University of Wollongong, 2522 (Australia)
E-mail: mkelso@uow.edu.au
Dr. N. Barraud, Prof. S. Kjelleberg, Dr. S. A. Rice
School of Biotechnology and Biomolecular Sciences and Centre for
Marine Bio-Innovation, University of New South Wales (Australia)
Prof. S. Kjelleberg, Dr. S. A. Rice
The Singapore Centre on Environmental Life Sciences Engineering,
Nanyang Technological University (Singapore)
Cephalosporins with leaving groups at the 3’-position (e.g.
acetate in Cefaloram 1 and Cefalotin 2, Figure 1) are known
to spontaneously release the leaving group following b-lactam
Dr. R. P. Howlin, Dr. J. S. Webb, Dr. S. N. Faust
NIHR Respiratory Biomedical Research Unit, University of South-
ampton and
University Hospital Southampton NHS Foundation Trust (UK)
Dr. R. P. Howlin, Dr. J. S. Webb
Centre for Biological Sciences, University of Southampton (UK)
Dr. S. N. Faust
NIHR Wellcome Trust Clinical Research Facility, University of
Southampton and University Hospital Southampton, NHS Foun-
dation Trust (UK)
[**] We acknowledge the Australian National Health and Medical
Research Council (NHMRC) for funding this work (Project Grant
568841). J.S.W. and S.N.F. acknowledge funding from the UK
National Institute of Health Research (NIHR) Southampton
Respiratory Biomedical Research Unit and NIHR Wellcome Trust
Clinical Research Facility.
Figure 1. Chemical formulas of cephalosporin antibiotics, Cefaloram
1 and Cefalotin 2.
ring cleavage.[20,21] It was reasoned that prodrugs could be
created which selectively release an NO-donor upon reaction
with biofilm b-lactamases (and conceivably transpeptidases,
the principal target of bactericidal cephalosporins).[22] Dia-
zeniumdiolates (also known as NONOates) seemed to be the
ideal NO-donor for inclusion in the prodrugs, because
diazeniumdiolates alkylated at their terminal oxygen are
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201202414. This information
includes experimental details for the synthesis and characterization
of compounds 3, 6, and 7, and experimental details for ampero-
metric NO-release measurements, GFP reporter assays, biofilm
dispersion studies, and minimum inhibitory concentration (MIC)
measurements.
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stable compounds that spontaneously produce NO only after
[23]
À
cleavage of the O C bond. Cephalosporin-3’-diazenium-
diolate 3, a composite of Cefaloram 1 and (Z)-1-(N,N-
diethylamino)diazen-1-ium-1,2-diolate
4 (Scheme 1), was
Scheme 1. Proposed mechanism of b-lactamase-triggered NO release
and biofilm dispersion by cephalosporin-3’-diazeniumdiolate 3/7.
the prototype prodrug targeted for study. Diazeniumdiolate
4 was chosen for its short NO-release half-life at pH 7.4 (t1/2
=
2.0 min),[24] because rapid NO release from the liberated NO-
donor is crucial for clinical applications to ensure the donor
does not diffuse away from the biofilm before releasing NO.
Cefaloram was chosen as the base cephalosporin because it is
more easily synthesized. A putative mechanism for b-
lactamase-triggered release of NO from 3 (or its K+ salt, 7)
and subsequent NO-mediated biofilm dispersion is outlined
in Scheme 1.
The synthesis of 3/7 centered around a key nucleophilic
displacement of the allylic 3’-chloride of the commercially
available p-methoxybenzyl (PMB)-protected cephalosporin
ester 5 with 4a (sodium salt of 4) to form the O-alkylated
diazeniumdiolate adduct 6 (Scheme 2). A Finkelstein con-
Figure 2. Amperometric characterization of NO release from 7. A) NO
release in the presence of penicillinase at varying pH values. Arrows
indicate addition of the following to a reaction vial at 258C containing
10 mL tris buffer at pH 9.0, 7.0, or 5.0: 1) 10 mL of 100 mm 7, 2) 5 mL
of 0.1 UmLÀ1 penicillinase, 3) 10 mL of 0.1 UmLÀ1 penicillinase, 4) 80 mL
of 10 mm PTIO. B) NO release in the presence of cell extracts of
P. aeruginosa pretreated with 50 mgmLÀ1 ampicillin or non-b-lactamase-
expressing E. coli. Arrows indicate addition of the following to a reac-
tion vial containing 10 mL tris buffer at pH 7.0: 1) 10 mL of 100 mm 7,
2) 100 mL cell extract, 3) 200 mL cell extract, 4) 80 mL of 10 mm PTIO.
C) NO release in the presence of supernatants from P. aeruginosa
cultures grown for 5 h in the absence of antibiotic then treated for 1 h
with imipenem (0.5 mgmLÀ1) or ampicillin (100 mgmLÀ1). Arrows
indicate addition of the following to a reaction vial containing 10 mL
tris buffer at pH 7.0: 1) 10 mL of 100 mm 7, 2) 500 mL supernatant,
3) 500 mL supernatant, 4) 80 mL of 10 mm PTIO.
pound 7 (100 mm) alone in Tris buffer (pH 5.0, 7.0, or 9.0) was
stable and did not release NO. Addition of penicillinase
(0.05 UmLÀ1) at pH 7.0 led to rapid evolution of NO from 7,
reaching a steady-state concentration of approximately 2.0 mm
NO within 15 minutes. The NO increased to approximately
4.0 mm upon addition of a further 0.1 UmLÀ1 penicillinase,
and again the steady-state concentration was reached within
15 minutes. Quenching of this response with the free-radical
scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-
oxide (PTIO) confirmed that the amperometric signal was
from NO release. A similar release profile was measured at
pH 5, while at pH 9 NO release was significantly decreased.
Release of NO from 7 was then studied in the presence of
P. aeruginosa cell lysates, grown either in the presence of sub-
inhibitory concentrations of ampicillin (which activates intra-
cellular b-lactamases localized in the periplasmic space),[25] or
non-b-lactamase-producing Escherichia coli cells. The P. aer-
Scheme 2. Synthesis of cephalosporin-3’-diazeniumdiolate free-acid 3
and K+ carboxylate salt 7.
version (NaI/acetone) of 5 to the allylic iodide, followed by
in situ addition of freshly prepared solid 4a was the optimal
procedure, providing pure 6 in 75% yield of the isolated
product. Deprotection of 6 using neat trifluoroacetic acid in
the presence of anisole for 1 hour at 08C afforded the free
acid 3 (80% yield), which was converted to the water-soluble
potassium carboxylate salt 7 (99% yield) by stirring with
1.0 equivalent of KOH in water for 20 minutes at 08C
followed by lyophilization.
Release of NO from 7 in the presence of a commercial b-
lactamase (Bacillus cereus penicillinase, Sigma) was studied
amperometrically at varying pH values (Figure 2A). Com-
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uginosa extracts were found to generate steady-state NO
concentrations of approximately 1.0 mm and 3.0 mm from 7
within 15 minutes, after successive additions of 100 mL and
200 mL cell extract, respectively (Figure 2B). A decreased, yet
measurable, release of NO was detected with E. coli extracts,
indicating that non-b-lactamase-producing bacteria can also
cleave 3, most likely by reaction with transpeptidases.
Release of NO from 7 was measured next in the presence
of supernatants from P. aeruginosa cultures which had been
pre-incubated with sub-inhibitory concentrations of either
imipenem, an inducer of extracellular b-lactamases, or
ampicillin (Figure 2C).[25] The imipenem-culture supernatants
were found to trigger robust NO generation, and the
amipicillin-culture supernatants produced no measurable
NO. These findings, together with those from Figure 2B
where NO release from 7 was demonstrated in the presence of
ampicillin-treated P. aeruginosa extracts, are consistent with
NO release being triggered by both extracellular and intra-
cellular (periplasmic) b-lactamases.
Figure 4. Dose-dependent dispersal of P. aeruginosa biofilms by 7.
A) P. aeruginosa biofilms grown in microtiter plates with shaking at
378C were pretreated with imipenem (0.5 mgmLÀ1) for 1 h and then
exposed to various concentrations of 7 (15 min) before quantifying
biofilm mass by crystal violet staining.[26] B) Stained biofilms treated
with the indicated concentrations of 7.
The ability of P. aeruginosa cells to sense and genetically
respond to NO released from 7 was tested using a strain of
P. aeruginosa that expresses green fluorescent protein (GFP)
under the control of the NO-responsive nirS promoter.[9]
Compound 7 (150 mm) was found to induce approximately
25% more GFP expression than that of an equivalent
concentration of SNP; this response was even greater in
cells grown in the presence of a sub-inhibitory concentration
of ampicillin (Figure 3). The enhanced response was equiv-
alent to the expression when 7 was co-administered with
penicillinase to cells grown in the absence of ampicillin.
Dispersal of P. aeruginosa biofilms by 7 was examined
using microtiter plate biofilm assays. Established biofilms
which had been pretreated with sub-inhibitory imipenem
(0.5 mgmLÀ1) for 1 hour showed a rapid and dose-dependent
dispersal response to 7 in the range 2–100 mm (Figure 4), with
10 mm 7 removing 70% of the biofilm mass after treatment for
15 minutes. Pretreating biofilms with imipenem before expo-
sure to 7, produced similar dispersal effects to co-administer-
ing 7 with penicillinase to cells that had not been pretreated
(Supporting Information, Figure S1).
The biofilm dispersing properties of 7 were confirmed
using a continuous-flow biofilm culture assay. P. aeruginosa
PAO1 biofilms were established in glass microfermenters,
receiving a continuous flow of fresh M9 minimal medium.
The inlet was switched to vessels containing fresh medium,
with or without 7 (100 mm), and OD600 measurements of the
effluent were taken. A rapid and significant increase in
released cells was observed after addition of 7, while the
amount of cells released from untreated biofilms remained
unchanged (Supporting Information, Figure S2).
Our final experiments used microtiter-plate assays to
examine the effects of 7 on P. aeruginosa biofilms when used
in combination with two front-line antibiotics. We found that
co-administration of 7 (10 mm) with tobramycin or ciproflox-
acin resulted in further decrease of log 1.8 and log 1.5,
respectively, in the number of colony-forming units (cfu)
remaining in biofilms when compared to the equivalent
antibiotic treatments in the absence of 7 (Table 1).
Table 1: Compound 7 potentiates the anti-biofilm efficacy of tobramycin
and ciprofloxacin.[a]
Antibiotic
Compound 7 [mm]
log decrease [cfu]
Tobramycin
0
10
100
2.15Æ0.16
3.92Æ0.06
3.58Æ0.15
80 mgmLÀ1
Ciprofloxacin
0
10
100
3.54Æ0.09
5.06Æ0.02
4.90Æ0.13
5 mgmLÀ1
[a] Established P. aeruginosa biofilms grown in microtiter plates with
shaking at 378C were pretreated with sub-inhibitory imipenem
(10 mgmLÀ1) for 1 h. Tobramycin or ciprofloxacin were then added either
with or without compound 7, or biofilms were left untreated (control).
Biofilm bacteria were further incubated for 1 h, followed by resuspension
and quantitation of cfu. Values represent the decrease in cfu on a log
scale, compared to control biofilms and are the mean of two
independent experiments (ÆSEM). Bacteria in the supernatants were
fully eradicated (below the detection limit; 10 cfumLÀ1) after treatment
with either antibiotic alone or antibiotic in combination with 7.
Figure 3. Compound 7 induces an NO-dependent genetic response
(nirS) in a P. aeruginosa NSGFP reporter strain. NSGFP cells grown in
the presence or absence of ampicillin (50 mgmLÀ1) were exposed to
SNP (150 mm), compound 7 (150 mm), compound 7 (150 mm) plus
penicillinase (0.2 UmLÀ1), cefalotin 2 (150 mm) or penicillinase
(0.2 UmLÀ1), or left untreated.
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In summary, we have synthesized a novel cephalosporin-
based NO-donor prodrug and demonstrated its potential for
use in dispersing P. aeruginosa biofilms. Selective release of
NO from the prodrug through contact with biofilm b-
lactamases is an attractive feature, which should allow
targeted enhancement of bacterial killing by conventional
antimicrobials at sites of biofilm infections, while also
minimizing NO-mediated toxicity. Given that cephalosporins
have a long history of safe and effective use in humans, we
anticipate that cephalosporin-3’-diazeniumdiolates will have
acceptable drug-like properties, facilitating their develop-
ment into clinically useful anti-biofilm therapeutics. Accord-
ingly, compound 7 (and related analogues) are currently being
tested as new treatments for chronic biofilm-mediated
infections.
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[17] M. L. Falsetta, A. G. McEwan, M. P. Jennings, M. A. Apicella,
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Received: March 28, 2012
Revised: June 18, 2012
Published online: && &&, &&&&
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Keywords: antibiotics · biofilms · cephalosporins · nitric oxide ·
prodrugs
.
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4
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Communications
Rational Drug Design
N. Barraud, B. G. Kardak, N. R. Yepuri,
R. P. Howlin, J. S. Webb, S. N. Faust,
S. Kjelleberg, S. A. Rice,
M. J. Kelso*
&&&& — &&&&
Cephalosporin-3’-diazeniumdiolates:
Targeted NO-Donor Prodrugs for
Dispersing Bacterial Biofilms
Just say NO to biofilms: NO-donors are
used to disperse a bacterial biofilm so
that co-administered antibiotics will kill
the more susceptible unattached cells.
The chemically stable cephalosporin-3’-
diazeniumdiolate NO-donor prodrug
(see scheme) is activated by bacterial b-
lactamases and facilitates this two-step
biofilm erradication.
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!