Lipo-γ-AApeptides as a new class of potent and broad-spectrum antimicrobial agents

Article abstract of DOI:10.1021/jm300274p

There is increasing demand to develop antimicrobial peptides (AMPs) as next generation antibiotic agents, as they have the potential to circumvent emerging drug resistance against conventional antibiotic treatments. Non-natural antimicrobial peptidomimetics are an ideal example of this, as they have significant potency and in vivo stability. Here we report for the first time the design of lipidated γ-AApeptides as antimicrobial agents. These lipo-γ-AApeptides show potent broad-spectrum activities against fungi and a series of Gram-positive and Gram-negative bacteria, including clinically relevant pathogens that are resistant to most antibiotics. We have analyzed their structure-function relationship and antimicrobial mechanisms using membrane depolarization and fluorescent microscopy assays. Introduction of unsaturated lipid chain significantly decreases hemolytic activity and thereby increases the selectivity. Furthermore, a representative lipo-γ-AApeptide did not induce drug resistance in S. aureus, even after 17 rounds of passaging. These results suggest that the lipo-γ-AApeptides have bactericidal mechanisms analogous to those of AMPs and have strong potential as a new class of novel antibiotic therapeutics.

Full text of DOI:10.1021/jm300274p

Article
pubs.acs.org/jmc
Lipo-γ-AApeptides as a New Class of Potent and Broad-Spectrum
Antimicrobial Agents
Youhong Niu,† Shruti Padhee,† Haifan Wu,† Ge Bai,† Qiao Qiao,† Yaogang Hu,† Lacey Harrington,‡
Whittney N. Burda,‡ Lindsey N. Shaw,‡ Chuanhai Cao,§ and Jianfeng Cai*,†
†Department of Chemistry, CHE 205, University of South Florida, 4202 E. Fowler Avenue, Tampa, Florida 33620, United States
‡Department of Cell Biology, Microbiology and Molecular Biology, University South Florida, 4202 E. Fowler Avenue, Tampa, Florida
33620, United States
§USF College of Pharmacy, 4001 E. Fletcher Avenue, Tampa, Florida 33613, United States
S
* Supporting Information
ABSTRACT: There is increasing demand to develop antimicrobial peptides (AMPs) as next generation antibiotic agents, as
they have the potential to circumvent emerging drug resistance against conventional antibiotic treatments. Non-natural
antimicrobial peptidomimetics are an ideal example of this, as they have significant potency and in vivo stability. Here we report
for the first time the design of lipidated γ-AApeptides as antimicrobial agents. These lipo-γ-AApeptides show potent broad-
spectrum activities against fungi and a series of Gram-positive and Gram-negative bacteria, including clinically relevant pathogens
that are resistant to most antibiotics. We have analyzed their structure−function relationship and antimicrobial mechanisms using
membrane depolarization and fluorescent microscopy assays. Introduction of unsaturated lipid chain significantly decreases
hemolytic activity and thereby increases the selectivity. Furthermore, a representative lipo-γ-AApeptide did not induce drug
resistance in S. aureus, even after 17 rounds of passaging. These results suggest that the lipo-γ-AApeptides have bactericidal
mechanisms analogous to those of AMPs and have strong potential as a new class of novel antibiotic therapeutics.
prokaryotes.5 Because of the emergence of bacterial resistance
INTRODUCTION
■
to conventional antibiotics, there has been significant demand
for the development of antimicrobial peptides as therapeutic
treatments.1,2 Since the initial step of killing by AMPs is
bacterial membrane binding and disruption, followed by
aggregation, poration, and carpet-like formation,6 it is extremely
difficult for bacteria to develop resistance against antimicrobial
peptides.1 Another advantage of AMPs is that they normally
exhibit broad-spectrum activity against both Gram-negative and
Gram-positive bacteria, as well as toward fungi and viruses.1,2
As such, there is strong motivation for the development of
antimicrobial peptides into antibiotic therapeutics to supple-
ment, or even replace, existing conventional antibiotics
treatments.4
Antimicrobial peptides (AMPs) are naturally occurring, short
cationic amphiphilic peptides that are virtually ubiquitous and
form a vitally important defense against infection as part of the
innate immune system.1,2 It is thought that antimicrobial
peptides are unlikely to elicit extensive resistance observed for
conventional antibiotic treatments, as they have bactericidal
mechanism of membrane disruption, and that is probably why
they have been deployed against invading pathogens by a
variety of organisms for millions of years. Conventional
antibiotics target specific metabolic processes in bacteria,3
while antimicrobial peptides are believed to adopt amphipathic
conformations on negatively charged bacterial membranes,
leading to killing through a variety of mechanisms.4 It would be
difficult for microbes to counter AMPs multiple mechanisms of
cell killing all at once. The negatively charged membranes of
bacteria cells are distinct from zwitterionic mammalian cell
membranes, facilitating the high selectivity of AMPs toward
Received: February 27, 2012
Published: April 4, 2012
© 2012 American Chemical Society
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Figure 1. γ-AApeptides used in antimicrobial assays. The first structure shows the general structure of γ-AApeptide building blocks.
Naturally occurring7−9 lipopeptides are another class of
AMPs; however, they are different from naturally occurring
host-defense peptides. They are produced as metabolites in
bacteria and fungi in the presence of various carbon sources10,11
and are cationic,9,12,13 neutral,14 or anionic.7,8 Therefore, their
mode of action is sometimes quite different from that of natural
cationic AMPs. As such, many of these natural lipoantibiotics
are not broad-spectrum in their activity. For example,
daptomycin is only active against Gram-positive bacteria,7,8
polymyxin B is active only toward Gram-negative bacteria,9,12,13
while echinocandins14 function only as antifungal drugs, as they
noncompetitively inhibit β-1,3-D-glucan synthase. Although
their antimicrobial properties are different, the lipid chain in
these natural lipopeptides has been found to be critical for
activity, as it endows lipophilicity, which facilitates bacterial
membrane interaction.10,11,15−17 Such recognition has led to
the development of cationic lipopeptides that can mimic AMP
killing of bacteria through membrane disruption.10,11,18,19 Of
note, they are generally composed of aliphatic acids attached to
the N-terminus of short cationic peptides and are broadly active
against both Gram-positive and Gram-negative bacteria, as well
as fungi. Non-natural antimicrobial peptidomimetics such as
arylamides,6,20,21 peptoids,4,22 β-peptides,23−28 and oligourea29
have been developed in recent years because they can mimic
AMP activity while proving more effective as a result of their
resistance to proteolytic hydrolysis.30 However, the develop-
ment and investigation of lipidated peptidomimetics are rare.31
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a
Table 1. Antimicrobial Activities of γ-AApeptides
MIC (μg/mL)
Gram-negative
Gram-positive
fungi
C. albicans
hemolysis
HC10/HC50
b
oligomers
E. coli
K. pneumoniae
P. aeruginosa
B. subtilis
S. epidermidis
E. faecalis
S. aureus
selectivity
1
>50
2.5
30
>50
35
>50
10
>50
1.5
1.5
1.5
2.5
2.5
>100
>100
>100
2
>50
2
>50
2.5
8
>50
2.5
5
>50
400/>500
25/40
2
2
16
3
20
20
2.5
2
2
40/300
60
4
20
>50
15
15
10
5
2
300/>500
200/>500
60/>500
250/>500
200/>500
120/>500
40/100
>100
>50
>125
5
2.5
2.5
>100
>100
>100
2.5
10
15
10
4
10
10
4
7.5
6
5
5
5
5
7
>100
>100
>100
35
>100
>100
>100
3
>100
>100
>100
2
>100
>100
>100
2.5
2.5
5
>100
>100
>100
4
>100
8
>100
9
>100
10
11
12
13
γ5
2
5
2
3
8
25
40
15
2
2
5
80/>500
250/>500
100/>500
75/300
>100
>100
>167
60
10
15
3
2.5
3
4
5
3
3
3
3
4
3
3
5
>50
2
5
5
5
a
HC10 and HC50 are the concentrations of γ-AApeptides at which 10% or 50% hemolysis was observed. The two most potent and broad-spectrum
b
lipo-γ-AApeptides, 6 and 13, are shaded in grey. Selectivity is calculated based on H50/the MIC of S. aureus.
Herein, we report lipo-γ-AApeptides as a new class of
antimicrobial peptidomimetics that mimic the bactericidal
functions of AMPs. γ-AApeptides were recently developed by
our group as a new class of peptidomimetic.32−34 The solid
phase synthesis of γ-AApeptides is straightforward, and their
potential diversification is large by the introduction of a wide
variety of functional groups.32−34 Preliminary studies have
demonstrated the ability of γ-AApeptide to modulate protein−
protein and protein−RNA interactions33,34 and their resistance
to proteolytic degradation.34 Recently, we have designed and
identified a linear γ-AApeptide, γ5,32 that has potent, broad-
spectrum activity toward a range of bacteria and fungi. In this
report, we demonstrate that lipo-γ-AApeptides, containing
hydrophobic alkyl tails and short cationic γ-AApeptide
sequences, display more potent, broad-spectrum, and highly
selective activities against fungi and a series of clinically related
Gram-positive and Gram-negative bacteria. Our mechanistic
studies indicate that the mechanism of action for lipo-γ-
AApeptides is analogous to that of AMPs, functioning via
disruption of bacterial membranes. Additionally, lipo-γ-
AApeptide does not elicit drug resistance in S. aureus, even
after 17 rounds of passaging. These results demonstrate the
strong potential of lipo-γ-AApeptide as a new class of novel
antibiotic therapeutics.
AApeptides with saturated lauric acid or palmitic acid tails.
Sequences 7 and 8 are anionic lipo-γ-AApeptides that were
prepared as negative controls. Sequence 9 contains the same γ-
AApeptide sequence as 6, yet lacks an alkyl tail. Sequences 10−
13 are cationic γ-AApeptides alkylated with unsaturated oleic
acid. These sequences were tested for their antimicrobial
activity toward a range of Gram-positive and Gram-negative
bacteria, as well as the fungus C. albicans. Their selectivity was
also evaluated via hemolytic assays. The results of these
investigations are shown in Table 1, with γ5, a linear γ-
AApeptide reported previously,32 included for comparison.
Our results demonstrate that lipo-γ-AApeptides appear to be
an effective and novel class of antimicrobial agents. As shown in
Table 1, almost all the cationic lipo-γ-AApeptides display
broad-spectrum antimicrobial activity against an array of Gram-
negative and Gram-positive bacteria, as well as toward the
fungus C. albicans. Sequences 7, 8, and 9, however, are not
active at all under these experimental conditions. This is likely
because, with a positively charged surface post-self-assembly,
cationic lipo-γ-AApeptides selectively target bacteria that have
negatively charged membranes. Sequences 7 and 8, which are
negatively charged lipo-γ-AApeptides, should not, and do not,
present any activity because of electrostatic repulsion with
bacterial membranes. Linear sequence 9, although positively
charged, cannot strongly interact with bacterial membranes
because of lack of lipid tail. In order to shed light on the further
development of lipo-γ-AApeptide based antimicrobial agents,
the structure−function relationship of these compounds was
investigated. Sequence 1, bearing one cationic γ-AApeptide
building block and a lauric acid alkyl tail, is not active toward
any microorganism. However, simply changing this tail to a
palmitic alkyl chain renders 2 a potent antimicrobial agents
toward most bacteria, as well as the fungus C. albicans. This
indicates that lipophilicity of the alkyl tail is highly important
for interaction with cell membranes and that increased cationic
charge does not necessarily lead to more potent antimicrobial
agents. Sequences 3 and 4, which have many more cationic
charges than 2, are seemingly less active toward bacteria and
fungus. As such, the balance of hydrophobicity and cationic
charge appears to be critical for antimicrobial activity. This led
RESULTS AND DISCUSSION
■
The lipo-γ-AApeptides were designed based on the widely
accepted understanding that globally amphipathic structures are
important for bacterial membrane disruption, which can be
easily achieved by attaching hydrophobic alkyl tails to cationic
γ-AApeptides. The synthesis of lipo-γ-AApeptides was carried
out on solid phase (Figure S1) and purified by HPLC adapted
from previously reported protocols (see Supporting Informa-
tion for details).32−34 Briefly, the necessary γ-AApeptide
building blocks were synthesized and assembled on the solid
support. Next, the fatty acids, in our experiments lauric acid,
palmitic acid, or oleic acid, were coupled to the N-terminus of
γ-AApeptide sequences. Lastly, sequences were cleaved from
the resin and purified by HPLC to give the desired lipidated γ-
AApeptides (Figure 1). Sequences 1−6 are cationic lipo-γ-
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to the discovery of 5 and 6, with two more hydrophobic γ-
AApeptide building blocks, which are more broadly active.
Sequence 6, which is a lipidated version of 9, effectively arrests
the growth of all tested bacteria and fungi. However, as
lipophilicity increases, the hemolytic activity also increases,
compromising selectivity. To enhance selectivity, sequences
10−13, which contain similar or identical sequences to 2−6 but
with unsaturated oleic tails, were prepared. We hypothesized
that unsaturated tail would have less propensity for aggregation
but still possess the same hydrophobicity compared to their
saturated counterparts. Surprisingly, a number of even more
potent and broad-spectrum-active lipo-γ-AApeptides were
obtained, some of which are much less hemolytic, as seen
with 10, 12, and 13. For example, 3 and 12 only differ in their
alkyl tails, yet 3 is somewhat hemolytic, while 12 is virtually
nonhemolytic, and is generally more active toward bacteria.
Sequence 13, being the most potent and broad-spectrum
sequence, has similar and better antimicrobial activity than 6
toward all microorganisms, while it is again much less
hemolytic. Such increased potency and enhanced selectivity
may indicate that bacterial membranes are more sensitive to
unsaturated alkyl tails than mammalian cells. This is an
important finding and would be of considerable significance for
the development of lipoantibiotics in the future. It is noticeable
that both 13 and 6 are more potent and broad-spectrum than
the previously reported linear sequence γ5,32 particularly
toward the Gram-negative bacterium P. aeruginosa and fungus
C. albicans, although they contain shorter lengths of γ-
AApeptide fragments. Furthermore, the liposequence 13 is
less hemolytic than γ5. These results may indicate that bacteria
and fungi are more sensitive and susceptible to hydrophobic
lipid chains, which augments the potential of lipo-γ-AApeptides
as novel antibiotic agents.
Figure 2. Fluorescence micrographs of E. coli and B. subtilis treated
with 10 μg/mL 13 for 2 h: (a1−a4) E. coli; (a1) control, no treatment,
DAPI stained; (a2) control, no treatment, PI stained; (a3) 13
treatment, DAPI stained; (a4) 13 treatment, PI stained; (b1−b4) B.
subtilis; (b1) control, no treatment, DAPI stained; (b2) control, no
treatment, PI stained; (b3) 13 treatment, DAPI stained; (b4) 13
treatment, PI stained.
To probe the antimicrobial mechanism of lipo-γ-AApeptides
activity, we first performed fluorescence microscopy to assess
the ability of our two most potent sequences 13 (Figure 2) and
6 (Figure S3) to cause membrane leakage, since membrane
disruption is a general function of AMPs. A double staining
method with DAPI and PI was used, where DAPI stains all
bacterial cells irrespective of their viability and PI only stains
injured or dead cells with compromised membranes.19,35 Parts
a1 and b1 of Figure 2 show that, without treatment by 13, both
E. coli and B. subtilis stain with DAPI but not PI, indicating that
their membranes are intact. When cells were treated with 13 for
2 h, however, both E. coli and B. subtilis strongly stain with both
DAPI (parts a3 and b3 of Figure 2) and PI (parts a4 and b4 of
Figure 2), demonstrating membrane disruption. The aggrega-
tion of dead cells is also observed because of the loss of
membrane ζ potential, which is consistent with the findings of
previous studies on antimicrobial peptide amphiphiles.19
Similar membrane disruption was also seen with the treatment
of 6 (Figure S3).
there is no perfect relationship between the MIC and the
capability for depolarization (Figure 3). However, the treat-
ment of MRSA with lipo-γ-AApeptides led to dramatically
increased fluorescence, which was maximal after 10 min (Figure
3). Meanwhile, there is a general trend that lipo-γ-AApeptides
with higher MICs require higher concentrations to cause the
The antimicrobial mechanism of membrane disruption for
lipo-γ-AApeptides was further investigated using a membrane
depolarization assay. MRSA, a clinically relevant and widely
drug resistant bacterial pathogen, was selected for this study
using the membrane potential-sensitive dye DiSC3 to test
membrane integrity.21 In this assay, fluorescence intensity
increases dramatically if there is significant membrane
disruption or permeation, resulting from a loss in membrane
potential.21 Consistent with previous reports,21 our analysis
shows that the concentrations of lipo-γ-AApeptides required for
complete depolarization are much higher than their MICs, and
Figure 3. Depolarization of the membrane of S. aureus. The
fluorescence intensity of membrane potential-sensitive dye DiSC3
was used as the positive control.
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same degree of depolarization than those with lower MICs.
Taken together, these data further suggest that lipo-γ-
AApeptides kill bacteria via membrane disruption.
demonstrating the feasibility of lipo-γ-AApeptides for use as
antimicrobial therapeutics.
The emergence of drug resistance in bacteria for conven-
tional antibiotic treatments is a major public-health concern. To
investigate the potential of lipo-γ-AApeptides to select for drug
resistant isolates, meticillin-resistant S. aureus was serially
passaged on half-MIC concentrations of 6, with new MIC
values determined every 24 h. Sequence 6 was chosen as a
representative sequence as a result of its broad spectrum of
activity against test microorganisms. As a positive control,
parallel cultures were exposed to 2-fold dilutions of the
antibiotic norfloxacin (Figure 4).21 We determined that, while
CONCLUSION
■
In summary, we have successfully designed and synthesized
lipo-γ-AApeptides, revealing them to be a new and important
class of antimicrobial agents. These lipo-γ-AApeptides show
very potent, broad-spectrum activity against fungi and a series
of Gram-positive and Gram-negative bacteria, including
clinically relevant pathogens that are resistant to most
antibiotics. More importantly, analogue 6, a representative
potent example, does not induce drug resistance, possibly
because of its membrane disruption properties. Our results also
show that unsaturated lipid chain can decrease the hemolytic
activity, thereby enhancing the selectivity of lipo-γ-AApeptides.
With the additional advantage of their resistance to proteolytic
degradation and limitless potential for derivatization, lipo-γ-
AApeptides may lead to a new generation of antibiotic agents to
circumvent emerging drug resistance. Further optimization of
lipo-γ-AApeptides and in vivo studies with lead compounds 6
and 13 in mouse models are currently under investigation.
EXPERIMENTAL SECTION
■
Solid Phase Synthesis, Purification, and Characterization of
Lipidated γ-AApeptides.32−34 Lipo-γ-AApeptides were prepared on
the Rink amide resin in peptide synthesis vessels on a Burrell wrist-
action shaker following the standard Fmoc chemistry of solid phase
peptide synthesis protocol. Each coupling cycle included an Fmoc
deprotection using 20% piperidine in DMF and 4 h coupling of 1.5
equiv of γ-AApeptide building blocks32−34 onto resin in the presence 2
equiv of DIC (diisopropylcarbodiimide)/DhBtOH (3-hydroxy-1,2,3-
benzotriazin-4(3H)-one) in DMF. The lipidation was accomplished by
reacting lauric acid, palmitic acid, or oleic acid with the N-terminus of
γ-AApeptides using DIC/DhBtOH as activation agents on the solid
phase. After the desired sequences were assembled, the resin was
transferred into a 4 mL vial and the sequences were cleaved from solid
support in 50:45:5 TFA/CH2Cl2/triisopropylsilane overnight. The
solvent was evaporated and the residues were analyzed and purified on
a Waters HPLC instrument installed with both analytic module (1
mL/min) and preparative module (20 mL/min). Both modules had
the same methods, which were using 5−100% linear gradient of
solvent B (0.1% TFA in acetonitrile) in solvent A (0.1% TFA in water)
over 40 min, followed by 100% solvent B over 10 min. The desired
fractions were generally over 70% in crude (determined by HPLC)
and eluted as single peaks at >95% purity. They were collected and
lyophilized. The molecular weights of lipo-γ-AApeptides were obtained
on a Bruker AutoFlex MALDI-TOF mass spectrometer using α-cyano-
4-hydroxycinnamic acid as the matrix.
Figure 4. Development of resistance by S. aureus ATCC 33592 toward
6 and norfloxacin.
there are almost no changes in the MIC for 6 after 17 days with
17 passages, an increase in MIC for norfloxacin was found after
just three passages, with a more than 20-fold increase in MIC
observed after 17 days. This is yet further support that lipo-γ-
AApeptides do not readily permit the development of drug
resistance.
In order to further assess the potential of γ-AApeptide
amphiphiles as novel antibiotics, we also evaluated the toxicity
of 6 toward mammalian cells using a MTT assay (Figure 5). At
concentrations up to 50 μg/mL, almost no toxicity was
observed, while at 100 μg/mL, only around 50% of N2a/APP
cell viability was compromised. These results show selectivity
that is 10- to 50-fold lower than for bacteria, further
Antimicrobial Assays.32 The bacterial strains used in the
experiment were E. coli (JM109), B. subtilis (BR151), multidrug
resistant S. epidermidis (RP62A), vancomycin-resistant E. faecalis
(ATCC 700802), methicillin-resistant S. aureus (ATCC 33592), K.
pneumoniae (ATCC 13383), and multidrug resistant P. aeruginosa
ATCC 27853. The fungal strain used was C. albicans (ATCC 10231).
The antimicrobial activities of the lipo- γ-AApeptides were determined
in sterile 96-well plates by broth microdilution method. Bacterial
cells36 and fungi37 were grown overnight at 37 °C in 5 mL of medium,
after which a bacterial suspension (approximately 106 CFU/ml) or
fungal suspension Candida albicans (ATCC 10231) (approximately
103 CFU/ml) in Luria broth or trypticase soy was prepared. Aliquots
of 50 μL of bacterial or fungal suspension were added to 50 μL of
medium containing the γ-AApeptides for a total volume of 100 μL in
each well. The γ-AApeptides were prepared in PBS buffer in 2-fold
serial dilutions, with a final concentration range of 0.5−100 μg/mL.
Plates were then incubated at 37 °C for 24 h (for bacteria) or 48 h (for
Candida albicans (ATCC 10231). The lowest concentration at which
the complete inhibition of bacterial growth (determined by a lack of
Figure 5. MTT assay of N2a/APP cells treated with different
concentrations of 6.
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turbidity) is observed throughout the incubation time is defined as the
minimum inhibitory concentration (MIC). The experiments were
carried out independently three times in duplicate.
(approximately 106 CFU/mL) for the next experiment. These bacterial
suspensions were then incubated with 6 and norfloxacin, respectively.
After incubation at 37 °C for 24 h, the new MIC was determined. The
experiment was repeated each day for 17 passages.
Hemolytic Assay.32 Freshly drawn human red blood cells
(hRBCs) with additive K2 EDTA (spray-dried) were washed with
PBS buffer several times and centrifuged at 1000g for 10 min until a
clear supernatant was observed. The hRBCs were resuspended in 1×
PBS to make a 5% v/v suspension. Two-fold serial dilutions of lipo-γ-
AApeptides dissolved in 1× PBS from 1 mg/mL through 6.3 μg/mL
were added to a sterile 96-well plate to make up a total volume of 50
μL in each well. Then 50 μL of 5% v/v hRBC solution was added to
make up a total volume of 100 μL in each well. The 0% hemolysis
point and 100% hemolysis point were determined in 1× PBS and 0.2%
Triton-X-100, respectively.36 The plate was then incubated at 37 °C
for 1 h and centrifuged at 3500 rpm for 10 min. The supernatant (30
μL) was diluted with 100 μL of 1× PBS, and absorption was detected
by measuring the optical density at 360 nm by Biotek Synergy HT
microtiter plate reader. The percent hemolysis was determined by the
following equation:
MTT Assay. N2a APP cells were used to access the cell viability
after treatment of 6, daptomycin, and polymyxin B (see Supporting
Information). Typically, stock concentration of lipo-γ-AApeptide 6 (1
mg/mL) was diluted in medium in a 96-well plate to make different
concentrations and then incubated at 37 °C. In another 96-well plate,
N2a APP cells were seeded to 1 × 104 cells/well, each of which
contained 100 μL of medium. After incubation for 12 h, an amount of
100 μL of different concentrations of lipo-γ-AApeptide 6 was added
and the plate was incubated for another 36 h. At 1 h before time is due,
MTT reagent (Roche) was incubated at 37 °C degree water bath. The
medium in the 96-well plate was removed and washed with fresh
medium once, followed by adding 110 μL of MTT reagent, and then
incubated for another 4 h, after which 100 μL of prewarmed
solubilization solution was added. The plate was then incubated at 37
°C for 12 h before absorbance at 550 nm was read. Percentage of cell
viability was calculated based on the following equation:
% hemolysis = (Abssample − AbsPBS)/(AbsTriton − AbsPBS) × 100
% cell viability = (A/Acontrol) × 100
Fluorescence Microscopy.32 A double staining method with
DAPI (4′,6-diamidino-2-phenylindole dihydrochloride, Sigma, >98%)
and PI (propidium iodide, Sigma) as fluorophores was used to
visualize and differentiate the viable from the dead E. coli or B. subtilis
cells. DAPI as a double stranded DNA binding dye stains all bacterial
cells irrespective of their viability. Propidium iodide (PI) is capable of
passing through only damaged cell membranes and intercalates with
the nucleic acids of injured and dead cells to form a bright red
fluorescent complex.35 The cells were first stained with PI and then
with DAPI. Bacterial cells were grown until they reached mid-
logarithmic phase, and then they (∼2 × 106 cells) were incubated with
the lipo-γ-AApeptides at a concentration of 2 MIC (10 μg/mL) for 2
h. Then the cells were pelleted by centrifugation at 3000g for 15 min in
an Eppendorf microcentrifuge. The supernatant was decanted, and the
cells were washed with 1× PBS several times and then incubated with
PI (5 μg/mL) in the dark for 15 min at 0 °C. The excess PI was
removed by washing the cells with 1× PBS several times. Then the
cells were incubated with DAPI (10 μg/mL in water) for 15 min in the
dark at 0 °C. The DAPI solution was removed, and cells were washed
with 1× PBS several times. Controls were performed following the
exact same procedure for bacteria without the addition of γ-
AApeptides. The bacterial cells were then examined by using the
Zeiss Axio imager Z1 optical microscope with an oil-immersion
objective (100×).38
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures, representative HPLC traces, NMR
spectra, and MALDI-TOF MS results. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 813-974-9506. Fax: 813-974-3203. E-mail:
jianfengcai@usf.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work is supported by startup funds from University of
South Florida.
ABBREVIATIONS USED
■
Lipid Depolarization.21,39,40 The lipid depolarization of the
bacterial cell membrane was conducted using the membrane potential
sensitive dye 3,5′-dipropylthiacarbocyanine iodide (DiSC3-5) that
distributes between the cells and the medium depending on the
membrane potential gradient. S. aureus (ATCC 33592) cells were
grown in Trypticase soy broth medium to reach a mid-logarithmic
phase (OD600 = 0.5−0.6). The bacterial cells were then collected by
centrifugation at 3000 rpm for 10 min and then washed once with
buffer (5 mM HEPES and 5 mM glucose, pH 7.2). The cells were
resuspended to OD600 = 0.05 with 100 mM KCl, 2 μM DiSC3-5, 5
mM HEPES, and 5 mM glucose and were incubated for 30 min at 37
°C for maximal dye uptake and fluorescence self-quenching. This
bacterial suspension (50 μL) and 50 μL of lipo-γ-AApeptide stock
solution or control drug solution were added to white flat bottomed
polypropylene 96-well plate (Costar). The fluorescence reading was
recorded every 2 min for 30 min using the microplate reader (Biotek)
at an excitation wavelength of 622 nm and an emission wavelength of
670 nm. Valinomycin (final concentration 250 μg/mL) was used as a
positive control, and the blank with only cells and dye was used as the
background.
AMP, antimicrobial peptide; MIC, minimum inhibitory
concentration; APP, amyloid precursor protein
REFERENCES
■
(1) Marr, A. K.; Gooderham, W. J.; Hancock, R. E. Antibacterial
peptides for therapeutic use: obstacles and realistic outlook. Curr.
Opin. Pharmacol. 2006, 6, 468 −472.
(2) Hancock, R. E.; Sahl, H. G. Antimicrobial and host-defense
peptides as new anti-infective therapeutic strategies. Nat. Biotechnol.
2006, 24, 1551−1557.
(3) Alekshun, M. N.; Levy, S. B. Molecular mechanisms of
antibacterial multidrug resistance. Cell 2007, 128, 1037−1050.
(4) Chongsiriwatana, N. P.; Patch, J. A.; Czyzewski, A. M.; Dohm, M.
T.; Ivankin, A.; Gidalevitz, D.; Zuckermann, R. N.; Barron, A. E.
Peptoids that mimic the structure, function, and mechanism of helical
antimicrobial peptides. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2794−
2799.
(5) Takahashi, D.; Shukla, S. K.; Prakash, O.; Zhang, G. Structural
determinants of host defense peptides for antimicrobial activity and
target cell selectivity. Biochimie 2010, 92, 1236−1241.
(6) Scott, R. W.; DeGrado, W. F.; Tew, G. N. De novo designed
synthetic mimics of antimicrobial peptides. Curr. Opin. Biotechnol.
2008, 19, 620−627.
Drug Resistance Study.21,32 The initial MIC of lipo-γ-AApeptide
6 and control antibiotics norfloxacin against S. aureus was obtained as
described above. Bacteria from duplicate wells at a concentration of
one-half MIC were then used to prepare the bacterial dilution
4008
dx.doi.org/10.1021/jm300274p | J. Med. Chem. 2012, 55, 4003−4009

Journal of Medicinal Chemistry
Article
(7) Wu, G.; Abraham, T.; Rapp, J.; Vastey, F.; Saad, N.; Balmir, E.
Daptomycin: evaluation of a high-dose treatment strategy. Int. J.
Antimicrob. Agents 2011, 38, 192−196.
antimicrobial foldamers with heterogeneous backbones. J. Am. Chem.
Soc. 2004, 126, 6848−6849.
(28) Raguse, T. L.; Porter, E. A.; Weisblum, B.; Gellman, S. H.
Structure−activity studies of 14-helical antimicrobial beta-peptides:
probing the relationship between conformational stability and
antimicrobial potency. J. Am. Chem. Soc. 2002, 124, 12774−12785.
(29) Claudon, P.; Violette, A.; Lamour, K.; Decossas, M.; Fournel, S.;
Heurtault, B.; Godet, J.; Mely, Y.; Jamart-Gregoire, B.; Averlant-Petit,
M. C.; Briand, J. P.; Duportail, G.; Monteil, H.; Guichard, G.
Consequences of isostructural main-chain modifications for the design
of antimicrobial foldamers: helical mimics of host-defense peptides
based on a heterogeneous amide/urea backbone. Angew. Chem., Int.
Ed. 2010, 49, 333−336.
(8) Beiras-Fernandez, A.; Vogt, F.; Sodian, R.; Weis, F. Daptomycin:
a novel lipopeptide antibiotic against Gram-positive pathogens. Infect.
Drug Resist. 2010, 3, 95−101.
(9) Kvitko, C. H.; Rigatto, M. H.; Moro, A. L.; Zavascki, A. P.
Polymyxin B versus other antimicrobials for the treatment of
pseudomonas aeruginosa bacteraemia. J. Antimicrob. Chemother.
2011, 66, 175−179.
(10) Makovitzki, A.; Baram, J.; Shai, Y. Antimicrobial lipopolypep-
tides composed of palmitoyl di- and tricationic peptides: in vitro and in
vivo activities, self-assembly to nanostructures, and a plausible mode of
action. Biochemistry 2008, 47, 10630−10636.
(11) Makovitzki, A.; Avrahami, D.; Shai, Y. Ultrashort antibacterial
and antifungal lipopeptides. Proc. Natl. Acad. Sci. U.S.A. 2006, 103,
15997−6002.
(12) Urakawa, H.; Yamada, K.; Komagoe, K.; Ando, S.; Oku, H.;
Katsu, T.; Matsuo, I. Structure−activity relationships of bacterial outer-
membrane permeabilizers based on polymyxin B heptapeptides. Bioorg.
Med. Chem. Lett. 2010, 20, 1771−1775.
(13) Tsubery, H.; Ofek, I.; Cohen, S.; Fridkin, M. N-Terminal
modifications of polymyxin B nonapeptide and their effect on
antibacterial activity. Peptides 2001, 22, 1675−1681.
(14) Morris, M. I.; Villmann, M. Echinocandins in the management
of invasive fungal infections, part 1. Am. J. Health-Syst. Pharm. 2006,
63, 1693−1703.
(15) Avrahami, D.; Shai, Y. Bestowing antifungal and antibacterial
activities by lipophilic acid conjugation to D,L-amino acid-containing
antimicrobial peptides: a plausible mode of action. Biochemistry 2003,
42, 14946−14956.
(16) Malina, A.; Shai, Y. Conjugation of fatty acids with different
lengths modulates the antibacterial and antifungal activity of a cationic
biologically inactive peptide. Biochem. J. 2005, 390, 695−702.
(17) Makovitzki, A.; Shai, Y. pH-dependent antifungal lipopeptides
and their plausible mode of action. Biochemistry 2005, 44, 9775−9884.
(18) Zhao, X. B.; Pan, F.; Xu, H.; Yaseen, M.; Shan, H. H.; Hauser, C.
A. E.; Zhang, S. G.; Lu, J. R. Molecular self-assembly and applications
of designer peptide amphiphiles. Chem. Soc. Rev. 2010, 39, 3480−3498.
(19) Chen, C.; Pan, F.; Zhang, S.; Hu, J.; Cao, M.; Wang, J.; Xu, H.;
Zhao, X.; Lu, J. R. Antibacterial activities of short designer peptides: a
link between propensity for nanostructuring and capacity for
membrane destabilization. Biomacromolecules 2010, 11, 402−411.
(20) Tew, G. N.; Scott, R. W.; Klein, M. L.; Degrado, W. F. De novo
design of antimicrobial polymers, foldamers, and small molecules:
from discovery to practical applications. Acc. Chem. Res. 2009, 43, 30−
39.
(30) Violette, A.; Fournel, S.; Lamour, K.; Chaloin, O.; Frisch, B.;
Briand, J. P.; Monteil, H.; Guichard, G. Mimicking helical antibacterial
peptides with nonpeptidic folding oligomers. Chem. Biol. 2006, 13,
531−538.
(31) Chongsiriwatana, N. P.; Miller, T. M.; Wetzler, M.; Vakulenko,
S.; Karlsson, A. J.; Palecek, S. P.; Mobashery, S.; Barron, A. E. Short
alkylated peptoid mimics of antimicrobial lipopeptides. Antimicrob.
Agents Chemother. 2011, 55, 417−420.
(32) Niu, Y.; Padhee, S.; Wu, H.; Bai, G.; Harrington, L.; Burda, W.
N.; Shaw, L. N.; Cao, C.; Cai, J. Identification of gamma-AApeptides
with potent and broad-spectrum antimicrobial activity. Chem.
Commun. (Cambridge, U. K.) 2011, 47, 12197−12199.
(33) Niu, Y.; Jones, A. J.; Wu, H.; Varani, G.; Cai, J. gamma-
AApeptides bind to RNA by mimicking RNA-binding proteins. Org.
Biomol. Chem. 2011, 9, 6604−6609.
(34) Niu, Y.; Hu, Y.; Li, X.; Chen, J.; Cai, J. [gamma]-AApeptides:
design, synthesis and evaluation. New J. Chem. 2011, 35, 542−545.
(35) Matsunaga, T.; Okochi, M.; Nakasono, S. Direct count of
bacteria using fluorescent dyes: application to assessment of
electrochemical disinfection. Anal. Chem. 1995, 67, 4487−4490.
(36) Patch, J. A.; Barron, A. E. Helical peptoid mimics of magainin-2
amide. J. Am. Chem. Soc. 2003, 125, 12092−12093.
(37) Karlsson, A. J.; Pomerantz, W. C.; Weisblum, B.; Gellman, S. H.;
Palecek, S. P. Antifungal activity from 14-helical β-peptides. J. Am.
Chem. Soc. 2006, 128, 12630−12631.
(38) Williams, S. C.; Hong, Y.; Danavall, D. C. A.; Howard-Jones, M.
H.; Gibson, D.; Frischer, M. E.; Verity, P. G. Distinguishing between
living and nonliving bacteria: evaluation of the vital stain propidium
iodide and its combined use with molecular probes in aquatic samples.
J. Microbiol. Methods 1998, 32, 225−236.
(39) Wu, M.; Maier, E.; Benz, R.; Hancock, R. E. W. Mechanism of
interaction of different classes of cationic antimicrobial peptides with
planar bilayers and with the cytoplasmic membrane of Escherichia coli.
Biochemistry 1999, 38, 7235−7242.
(40) Friedrich, C. L.; Moyles, D.; Beveridge, T. J.; Hancock, R. E.
Antibacterial action of structurally diverse cationic peptides on Gram-
positive bacteria. Antimicrob. Agents Chemother. 2000, 44, 2086−2092.
(21) Choi, S.; Isaacs, A.; Clements, D.; Liu, D.; Kim, H.; Scott, R. W.;
Winkler, J. D.; DeGrado, W. F. De novo design and in vivo activity of
conformationally restrained antimicrobial arylamide foldamers. Proc.
Natl. Acad. Sci. U.S.A. 2009, 106, 6968−6973.
(22) Chongsiriwatana, N. P.; Miller, T. M.; Wetzler, M.; Vakulenko,
S.; Karlsson, A. J.; Palacek, S. P.; Mobashery, S.; Barron, A. E. Short
alkylated peptoid mimics of antimicrobial lipopeptides. Antimicrob.
Agents Chemother. 2010, 55, 417−420.
(23) Liu, D.; DeGrado, W. F. De novo design, synthesis, and
characterization of antimicrobial beta-peptides. J. Am. Chem. Soc. 2001,
123, 7553−7559.
(24) Porter, E. A.; Wang, X.; Lee, H. S.; Weisblum, B.; Gellman, S. H.
Non-haemolytic beta-amino-acid oligomers. Nature 2000, 404, 565.
(25) Porter, E. A.; Weisblum, B.; Gellman, S. H. Mimicry of host-
defense peptides by unnatural oligomers: antimicrobial beta-peptides.
J. Am. Chem. Soc. 2002, 124, 7324−7330.
(26) Porter, E. A.; Weisblum, B.; Gellman, S. H. Use of parallel
synthesis to probe structure−activity relationships among 12-helical
beta-peptides: evidence of a limit on antimicrobial activity. J. Am.
Chem. Soc. 2005, 127, 11516−11529.
(27) Schmitt, M. A.; Weisblum, B.; Gellman, S. H. Unexpected
relationships between structure and function in alpha,beta-peptides:
4009
dx.doi.org/10.1021/jm300274p | J. Med. Chem. 2012, 55, 4003−4009