Lipidated peptidomimetics with improved antimicrobial activity

Article abstract of DOI:10.1021/ml3001215

We report a series of lipidated α-AApeptides that mimic the structure and function of natural antimicrobial lipopeptides. Several short lipidated α-AApeptides show broad-spectrum activity against a range of clinically related Gram-positive and Gram-negative bacteria as well as fungus. Their antimicrobial activity and selectivity are comparable or even superior to the clinical candidate pexiganan as well as previously reported linear α-AApeptides. The further development of lipidated α-AApeptides will lead to a new class of antibiotics to combat drug resistance.

Full text of DOI:10.1021/ml3001215

Letter
pubs.acs.org/acsmedchemlett
Lipidated Peptidomimetics with Improved Antimicrobial Activity
Yaogang Hu,† Mohamad Nassir Amin,† Shruti Padhee,† Rongsheng E. Wang,† Qiao Qiao,† Ge Bai,†
Yaqong Li,† Archana Mathew,† Chuanhai Cao,‡ and Jianfeng Cai*,†
†Department of Chemistry and ‡College of Pharmacy, University of South Florida, 4202 East Fowler Avenue, Tampa, Florida 33620,
United States
S
* Supporting Information
ABSTRACT: We report a series of lipidated α-AApeptides
that mimic the structure and function of natural antimicrobial
lipopeptides. Several short lipidated α-AApeptides show broad-
spectrum activity against a range of clinically related Gram-
positive and Gram-negative bacteria as well as fungus. Their
antimicrobial activity and selectivity are comparable or even
superior to the clinical candidate pexiganan as well as
previously reported linear α-AApeptides. The further develop-
ment of lipidated α-AApeptides will lead to a new class of antibiotics to combat drug resistance.
KEYWORDS: antimicrobial peptides, peptidomimetics, drug resistance, lipidation, α-AApeptides
ntimicrobial peptides (AMPs) are found in most living
organisms as an integral component of their innate
peptidomimetics may overcome some of the drawbacks
associated with current lipopeptide antibiotics. Herein, we
report the development of lipidated α-AApeptides as potential
antimicrobial agents. We have recently developed a novel class
of peptidomimetics based on the α-chiral PNA (peptide nucleic
acid) backbone, which is termed “α-AApeptides” (Figure 1), as
A
defense against invading pathogens.1,2 Unlike conventional
antibiotics that target specific substrates involved in bacterial
metabolism, AMPs are believed to kill bacteria via a nonspecific
interaction with their membranes, which has less chance of
inducing the development of resistance by bacteria.1,2 Short
cationic amphiphilic AMPs tend to interact with the negatively
charged phospholipids on the bacterial membrane, which
accounts for their selectivity for bacteria against eukaryotic cells
whose membranes are more zwitterionic.3,4 Because of their
selectivity for bacteria, low propensity for development of drug
resistance, and broad-spectrum antimicrobial potency, AMPs
are considered an emerging class of antimicrobial agents.1−3
Nevertheless, the therapeutic application of AMPs is impeded
by their intrinsic instability in the context of proteolytic
degradation.1,2 Bactericidal peptidomimetics comprised of
unnatural amino acids were thereby developed to circumvent
the drawbacks of AMPs, which are protease-resistant and of
improved bioavailability.5 In recent years, several peptidomi-
metic analogues of AMPs, such as β-peptides,6−9 aryla-
mides,10,11 and peptoids,3,12,13 have received significant research
interest.
Figure 1. General structures of conventional α-peptides and α-
AApeptides.
they contain N-acylated-N-aminoethyl amino acid residues.
Chiral side chains of α-AApeptides are directly connected to
the α-carbon of the building blocks, whereas the other half of
the side chains are attached to backbone through acylation of
the central N of the building blocks.
As compared to regular α-peptides, each building block of α-
AApeptide is comparable to a dipeptide residue, and α-
AApeptides of the same chain length display an identical
number of side chains as regular peptides. As such, they can
potentially be developed to mimic the structures and functions
of peptides. There are a few advantages associated with α-
Lipidated peptides such as polymyxin B14 and daptomycin15
are lipo-antibiotics, which possess fatty acid tails that are
integral to their bactericidal activities. It has been shown that
attachment of saturated linear fatty acids to peptide termini
greatly enhanced AMPs' antimicrobial activities toward both
Gram-positive and Gram-negative strains.16−19 More recently,
short peptoid mimetics alkylated with lipids of 10 or 13 carbons
were demonstrated to bear improved selectivity, without losing
any antimicrobial activities.13 It was suspected that lipid
alkylation improves the hydrophobicity of charged peptides18
and facilitates the interaction with cytoplasmic membranes.18 It
is thereby envisioned that the development of lipidated
Received: December 19, 2012
Accepted: July 12, 2012
© XXXX American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
AApeptides. Besides their resistance to proteolysis, α-
AApeptides also feature limitless potential for diversification,
which makes it a promising agent for biological applications,
such as the inhibition of protein−protein interactions.20
Notably, a 7-mer α-AApeptide α1 was identified to kill both
Gram-negative and Gram-positive bacteria and fungi, with a
minimum hemolytic activity.21 To further explore the potential
antimicrobial application of α-AApeptide, as well as to develop
shorter peptide mimics for synthetic and pharmacological
benefits,22−26 we now report our efforts in the development of
lipidated antimicrobial α-AApeptides. Recently, we reported
lipidated antimicrobial γ-AApeptides,27 a closely related class of
peptidomimetics to α-AApeptides. Therefore, it will be very
interesting to find out the impact of lipidation of α-AApeptides
on their antimicrobial activity.
The design of α-AApeptides follows the previously
demonstrated principle21,28 that a global distribution of cationic
and hydrophobic side chains is key to antimicrobial activity and
a defined secondary structure is unnecessary, as long as side
groups can segregate into hydrophobic and cationic clusters
upon interaction with bacterial membranes. As such, α-
AApeptide building blocks were designed to be either
amphiphilic or cationic, which were assembled and alkylated
with C-16 hydrophobic lipid tail at the N terminus of the
oligomers (Figure 2). The synthesis of lipidated α-AApeptides
was carried out following a previous reported protocol,29 and
the N terminus of the last building block was capped with
palmitic acid to achieve a final lipidation.
These prepared lipidated α-AApeptides were tested for their
antimicrobial activities against a range of bacterial strains
including Gram-positive Bacillus subtilis, Staphylococcus epi-
dermidis, Enterococcus faecalis, and Staphylococcus aureus, Gram-
negative Escherichia coli, Klebsiella pneumoniae, and Pseudomo-
nas aeruginosa, and fungus Candida albicans, many of which are
multidrug-resistant strains. For the purpose of comparison, the
previously reported most active linear α-AApeptide α121 as well
as pexiganan, an AMP drug candidate,3,13,30,31 were employed
as positive controls.
The results have demonstrated the potential of lipidated α-
AApeptides as a new generation of antibiotics with broad-
spectrum activity. Being very short lipidated α-AApeptides, NA-
75 and NA-77 (Figure 2) appeared to be ineffective against
Gram-negative strains; however, they are very active against
Gram-positive strains, including multidrug-resistant strains
VREF (vancomycin-resistant E. faecalis) and MRSA (methi-
cillin-resistant S. aureus), and fungus C. albicans (Table 1).
Meanwhile, they almost had no hemolytic activity (Table 1),
which augments their potential development as antifungal
agents and antibacterial agents to treat Gram-positive bacterial
infections. The activity toward Gram-negative bacterial strains
was achieved by introducing additional α-AApeptide building
blocks. For instance, NB-119-1 and NB-127 are 3-mer
oligomers. Although NB-127 was still not active toward
Gram-negative bacteria, NB-119-1 displayed an adequate
activity against both Gram-positive and Gram-negative strains.
In addition, NB-119-1 possessed a much lower toxicity (weaker
hemolytic activity) than NB-127. Furthermore, the lipidated 3-
mer NB-119-1 already displayed an improved activity than the
linear 7-mer α121 made from the same building block,
indicating that lipidation significantly enhanced the antimicro-
bial activity of α-AApeptides. Broad-spectrum and potent
antimicrobial activity was further improved by the addition of
more α-AApeptide building blocks. NB-119-2, NB-119-3, and
NB-123 are all active toward all tested strains and are more
potent than both pexiganan and linear α1. The results are
consistent with general research findings that a certain degree
of hydrophobicity has to be reached for an antimicrobial agent
to be active. Both lipidation and addition of more α-AApeptide
building blocks increase the hydrophobicity of sequences and
thereby enhance the broad-spectrum activity against Gram-
negative stains. It is noticeable that hemolytic activity is not
always related to the overall hydrophobicity. For instance, NB-
119-3 contains more hydrophobic building blocks than NB-
127; however, it is much more active toward Gram-negative
bacteria and less hemolytic. Generally, a longer sequence of
cationic charges and hydrophobic groups can lead to improved
overall activity, which is similar to previous observations.21
They are also selective because none of them has toxicity
toward red blood cells (Table 1).
As AMPs usually employ a membrane disruption mechanism
to circumvent drug resistance occurring in the use of
conventional antibiotics, it is essential to evaluate whether
lipidated α-AApeptides can also adopt a similar bactericidal
mechanism. Lipopeptide antibiotics polymyxin B and dapto-
mycin do not adopt such mechanisms because they are only
active toward either Gram-negative or Gram-positive bacterial
strains, respectively. As such, one of the most potent lipidated
α-AApeptide, NB-119-2, was assessed by fluorescence micros-
copy. Both Gram-negative E. coli and Gram-positive B. subtilis
Figure 2. Structures of synthesized lipidated α-AApeptides.
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ACS Medicinal Chemistry Letters
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a
Table 1. Antimicrobial and Hemolytic Activities of Oligomers
MIC (μg/mL)
organism
B. subtilis
NA-75
NA-77
NB-119-1
NB-127
NB-119-2 NB-119-3
NB-123
pexiganan3,13,30,31
linear α121
Gram-positive
2
4
2
8
4
4
2
4
2
4
4
4
4
4
2
S. epidermidis (MRSE)
E. faecalis (VREF)
S. aureus (MRSA)
10
1
15
3
20
10
4
10
20
8
8
10
10
32
16
>50
>50
5
8
8
8
Gram-negative
>50
E. coli
>50
>50
>50
>50
>50
>50
8
4
30
8
4
8
4.5
K. pneumoniae
P. aeruginosa
>50
20
>50
8
20
10
8
>50
>50
>50
12
8
8−16
fungus
>50
C. albicans
2
2
3
4
20
10
124
20−30
hemolysis (H10/H50)
>500/>500
>500/>500
50/300
15/150
20/250
40/400
100/ >500
181/495
400/>500
a
The minimum inhibitory concentration (MIC) for bacteria is the lowest concentration that completely inhibits growth after 24 h, and the MIC for
fungus C. albicans is the lowest concentration that completely inhibits growth after 48 h. Pexiganan and Linear α1 were used as controls. H10 is the
concentration of α-AApeptides at which 10% hemolysis was observed, and H50 is the concentration of α-AApeptides at which 50% hemolysis was
observed.
were treated with NB-119-2 and then stained by 4′,6-
diamidino-2-phenylindole (DAPI), which stains all bacterial
cells, and propidium iodide (PI) dye, which selectively
penetrates dead or injured cells with damaged membranes32,33
(Figure 3). Without incubation with NB-119-2, both E. coli and
B. subtilis were stained in blue by DAPI, while little of them had
PI staining (red fluorescence). However, both bacteria showed
a strong red fluorescence as a result of PI staining after
incubation with NB-119-2 for 2 h, suggesting significant
membrane damage. The aggregation of injured or dead cells
in both oligomer-treated E. coli and B. subtilis is consistent with
previous reports,32 which indicates a loss of membrane
potential. Collectively, the lipidated α-AApeptide effectively
inhibits bacteria, via a membrane-lysis mechanism similar to
natural AMPs but different from lipo-antibiotics such as
polymyxin B34 and daptomycin.35 This result further augments
the potential of lipo-α-AApeptide as a new class of
peptidomimetics to combat drug resistance occurring in
bacteria during treatment of conventional antibiotics.
Taken together, we have reported a series of lipidated α-
AApeptides that feature significant antimicrobial activities.
Through preliminary structure and activity studies, several
short lipidated α-AApeptides, such as NB-123, NB-119-2, and
NB-119-3, have been identified to have comparable or even
superior activity and selectivity to the clinical candidate
pexiganan, as well as the previously reported linear α-
AApeptide α1. Because of the activity enhancement by
lipidation, these oligomers have a relatively shorter backbone
than the traditional linear α1. Mechanistic study shows that
these lipidated α-AApeptides can mimic AMPs by disrupting
bacterial membranes, which potentially circumvents drug
resistance. Among all of the tested lipidated and regular α-
AApeptides, NB-123, NB-119-2, and NB-119-3 bear the most
potent and broad-spectrum antimicrobial activity and the
highest selectivity. They are better than pexiganan in terms of
the overall performance and thus could be promising novel
antibiotic candidates in the future.
ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed experimental procedure and characterizations. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: 813-974-9506. Fax: 813-974-3203. E-mail: jianfengcai@
usf.edu.
■
Figure 3. Fluorescence micrographs of E. coli and B. subtilis treated
with 10 μg/mL of NB-119-2 for 2 h. a1−a4, E. coli; b1−b4, B. subtilis.
A1 and b1, control, no treatment, DAPI stained; a2 and b2, control, no
treatment, PI stained; a3 and b3, NB-119-2 treatment, DAPI stained;
and a4 and b4, NB-119-2 treatment, PI stained.
Funding
This work was supported by USF Start-Up fund.
C
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Notes
The authors declare no competing financial interest.
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