Identification of γ-AApeptides with potent and broad-spectrum antimicrobial activity

Article abstract of DOI:10.1039/c1cc14476f

We report the identification of a new class of antimicrobial peptidomimetics-γ-AApeptides with potent and broad-spectrum activity, including clinically-relevant strains that are unresponsive to most antibiotics. They are also not prone to select for drug-

Full text of DOI:10.1039/c1cc14476f

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COMMUNICATION
Identification of c-AApeptides with potent and broad-spectrum
antimicrobial activityw
a
a
Youhong Niu,z Shruti Padhee,z Haifan Wu,a Ge Bai,a Lacey Harrington,b
Whittney N. Burda,b Lindsey N. Shaw,b Chuanhai Caoc and Jianfeng Cai*a
Received 22nd July 2011, Accepted 19th September 2011
DOI: 10.1039/c1cc14476f
We report the identification of a new class of antimicrobial
peptidomimetics-c-AApeptides with potent and broad-spectrum
activity, including clinically-relevant strains that are unresponsive
to most antibiotics. They are also not prone to select for drug-
resistance.
approaches that mimic antimicrobial peptides may circumvent
these impediments by introducing amide bond isosteres, and
modifying the peptide backbone so as to improve resistance to
proteolytic hydrolysis.7 To this end, non-natural antimicrobial
oligomers, such as b-peptides, peptoids, arylamides, and oil-
gourea have been developed.8 However, their rational design
sometimes turns out not to be straightforward due to the
difficulty of introducing a variety of functional groups to fine
tune their activity and selectivity, and the inconsistency of
their structure–activity-relationship.9 Furthermore, recent
research findings from many groups suggest that helical
conformations, in which lipophilic and cationic side chains
are globally segregated, are not necessary for antimicrobial
activity.10,11 Indeed, a pre-organized secondary structure
seems not required for bacterial killing;5 instead, oligomers
with a strong propensity for helical conformation or confor-
mational rigidity may lead to high hemolytic activity.4,12
Potent antimicrobial activity may actually require the presence
of flexible, or even random coiled backbones, where side
groups are segregated into hydrophobic and cationic regions
upon interaction with bacterial membranes,5,12 even if the
amphiphilic conformation is irregular and non-helical.
Antimicrobial peptides are small cationic amphiphilic peptides
found in virtually all living organisms.1 They play an important
role in innate immune defense against various infections.2 In
the last decade, there has been significant interest in the
development of antimicrobial peptides because of the emergence
of antibiotic resistance.1,2 Compared with conventional anti-
biotics, which target specific metabolic processes in bacteria,3
antimicrobial peptides are able to form amphipathic structures,
where cationic and hydrophobic groups are segregated into
two regions, so as to facilitate interaction with the negatively
charged bacterial cytoplasmic membrane.4 Such interaction is
based on the global chemical properties of peptides, rather
than their precise sequence.5 Therefore, antimicrobial peptides
are unlikely to be hindered by the resistance mechanisms
observed for current antibiotic treatments.1 Furthermore,
unlike conventional antibiotics, antimicrobial peptides exhibit
broad-spectrum activity against both Gram-positive and
Gram-negative bacteria, and even fungi and viruses.1,2 They
appear to be ideal antibiotic agents to supplement or replace
existing treatments.4
Herein, we report a new class of antimicrobial peptide
mimetics – g-AApeptides developed by a simple design strategy.
g-AApeptides were recently developed by our group (Fig. S1w)
in order to further facilitate drug discovery. Certain g-AApeptides
were able to disrupt protein-protein interactions13 and recognize
nucleic acids with high affinity and specificity,14 and were
highly resistant to protease degradation.13 Moreover, the
synthesis and diversification of g-AApeptides is efficient and
straightforward,13 strengthening their potential to generate
focused libraries for drug-lead screening.
However, despite significant enthusiasm, there are intrinsic
drawbacks associated with the development of peptide anti-
biotics due to the peptidic nature of antimicrobial peptides.
These include potential immunoreactivity and susceptibility to
enzymatic degradation, etc.6 Non-natural peptidomimetic
To design antimicrobial peptidomimetics based on g-AA-
peptides, we first followed the rationale: Global distribution of
cationic and hydrophobic groups are most important for
potent antimicrobial activity; and that secondary structure is
irrelevant.5,10–12 We have also demonstrated this principle in
the recent report of antimicrobial a-AApeptide design,15 in
which amphiphilic a-AApeptide building blocks were linked
together to display antimicrobial activity. We anticipated
g-AApeptides would also be perfect candidates as antimicrobial
agents by joining amphiphilic g-AApeptide building blocks
together, due to their high stability, unlimited derivatizing
a Department of Chemistry, University of South Florida, 4202 E.
Fowler Ave, Tampa, FL 33620, USA. E-mail: jianfengcai@usf.edu;
Fax: +1-813-974-3203; Tel: +1-813-974-9506
b Department of Cell Biology, Microbiology and Molecular Biology,
4202 E. Fowler Ave, Tampa, FL 33620, USA
c Department of Molecular Pharmacology and Physiology,
USF-Health Byrd Alzheimer’s Institute, 4001 E. Fletcher Ave,
Tampa, FL 33613, USA
w Electronic supplementary information (ESI) available: Experimental
procedures and compound characterization data. Figures for fluores-
cence microscopy and drug-resistance study. See DOI: 10.1039/
c1cc14476f
z These authors contributed equally to this work.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 12197–12199 12197

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pathogens that are unresponsive to most antibiotics (Table 1).
The results support our hypothesis very nicely, and display
clear structure-function relationships. The results support our
hypothesis very nicely, and display clear structure-function
relationships. Active antimicrobial sequences can be generated
by simply joining amphipathic building blocks together, as
seen for g3. It suggests that g-AApeptides can adopt globally
amphipathic structures upon binding onto bacterial
membranes. Interestingly, the control sequence p1, which
contains alternative Phe and Lys residues, with the functional
groups identical to g3, does not show any antimicrobial
activity at all. Such observation can be explained by peptide’s
intrinsic folding propensity.15 The results demonstrate that the
straightforward design of g-AApeptide antimicrobial agents is
not applicable to regular peptides.
Meanwhile, longer sequences are more potent and broad-
spectrum active than shorter sequences with similar structures,
as seen for the activities of g1–g3, which indicates that the
number of cationic charge is important for interaction with
bacterial membranes, consistent to what has been identified in
the development of antimicrobial a-AApeptides.15 Notably,
g3 is more potent and broad-spectrum active than a3 of same
functional groups, indicating g-AApeptides might be more
suitable than a-AApeptides for antimicrobial development.
The further development of antimicrobial g-AApeptides
based on the lead g3 yields more potent sequences. As an
initial attempt, we introduced a simple hydrophobic building
block to tune the overall hydrophobicity and hydrophilicity of
g3, in order to evaluate the feasibility to tune antimicrobial
activity of g-AApeptides in the future. Such an effort led to the
discovery of g5, in which two amphiphilic building blocks are
replaced with hydrophobic building blocks (containing two
hydrophobic side chains) compared to g3, and only one side
chain difference from g4. While g4 are not active against
Gram-negative K. pneumoniae, however, g5 potently inhibited
the growth of all tested Gram-negative and Gram-positive
bacteria, and also the fungus C. albicans. In fact, g5 is amongst
the most potent and broad-spectrum antimicrobial pepti-
domimetics reported to date. Most significantly, it arrested
the growth of the USA100 lineage MRSA strain with extreme
potency. This observation is of particular importance as this
strain displays resistance to a wealth of existing antimicrobial
Fig. 1 The structures of oligomers tested for their antimicrobial
activity. Underlined building blocks are hydrophobic building blocks
containing two hydrophobic side chains; the rest of the building blocks
in the sequences are amphiphilic with one cationic and one hydro-
phobic side chains.
potential, and capability to adopt globally amphipathic structures
by easily adjusting their conformation upon interaction with
bacterial cell membranes (Fig. S2w).
As such, we prepared some g-AApeptide sequences (Fig. 1)
and tested their antimicrobial activity against a range of
clinically-relevant Gram-negative and Gram-positive bacteria,
as well as fungus C. albicans. g1, g2, g3 are g-AApeptides of
different length that are composed of amphiphilic g-AApeptide
building blocks. g4 contains one hydrophobic building blocks
while g5 contains two hydrophobic building blocks, an
attempt to tune overall hydrophobicity of g-AApeptides. As
controls, we included AMP magainin II (m1),15 a 14-mer
conventional peptide p115 with alternative phenylalanine and
lysine residues, and the most potent antimicrobial a-AApeptide
a1 reported by us recently.15 The antimicrobial activity of
these oligomers was tested and listed in Table 1. Their
hemolytic activity was also tested to evaluate their selectivity.
As expected, a few sequences show very potent broad-
spectrum activities against fungi, and a series of clinically-
relevant Gram-positive and Gram-negative bacteria, including
Table 1 The antimicrobial and hemolytic activities of oligomers. Magainin II is a natural amphipathic antimicrobial peptide used as the
comparison here. The sequences showing broad-spectrum antimicrobial activity (g3–g5 and a1) are shaded in light grey
MIC (mg mlꢀ1
g1
)
g2
g3
g4
g5
p1
a1
m1
Organism
Gram-negative
E. coli
K. pneumoniae
Gram-positive
B. subtilis
4100
4100
25
4100
2.5–5
4100
2.5–5
4100
2.5–5
5
4100
4100
4.5
4100
40
4100
4100
5
2.5
2.5
2
4100
2
10
75
75
4100
4100
40
4100
75
4100
4100
4100
Multi-drug resistant S. epidermidis 4100
4100
4100
4100
—
6.3–12.5
12.5–25
12.5–25
—
3.1–6.3
12.5–25
3.1–6.3
—
3.1–6.3 4100
3.1–6.3 4100
5
5
5
Vancomycin-resistant E. faecalis
Methicillin-resistant S. aureus
MRSA USA100 lineage
B. anthracis
4100
4100
—
4100
—
—
—
—
25–50
25–50
Fungi
C. albicans
Hemolysis (H10/H50
4100
4100
12.5–25
12.5–25
5–10
4100
20–30
75
)
4500/4500 4500/4500 4500/4500 400/4500 75/300 300/4500 400/4500 4500/4500
c
12198 Chem. Commun., 2011, 47, 12197–12199
This journal is The Royal Society of Chemistry 2011

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agents, and is broadly multi-drug resistant. As such, this may
satisfy urgent needs in hospitals for new therapeutics, since
this strain has been identified as the most prevalent cause of
hospital-associated infections in the United States, and almost
no current antibiotics can effectively target it. g5 also potently
inhibits B. anthracis, which is known to cause the highly lethal
condition, anthrax. The findings may suggest that g5 has the
potential for use in bio-defence in the future. In the mean time,
all the g-AApeptides have excellent selectivity for bacteria
than human blood cells, since the majority of them has H50
of more than 500 mg mlꢀ1; even the most hemolytic sequence
g5 still have a selectivity of at least 60-fold. This is because
mammalian cell membranes are almost neutrally charged,
while bacterial membranes are negatively charged, which
favors more for electrostatic interactions with cationic
g-AApeptide sequences.
which is unique in the development of antimicrobial AApeptides.
Such approach avoids tedious and sometimes ineffective
secondary structure development in the design of other classes
of antimicrobial peptidomimetics. Coupled with virtually
limitless side chain variation, bioavailability and enhanced
stability, it is very straightforward to further tune the anti-
microbial activity and selectivity of g-AApeptides by changing
hydrophobicity and hydrophilicity of building blocks and
backbone lengths. This would be particularly effective if
resistance to these compounds were ever observed, allowing
rapid change and alteration to circumnavigate any such
occurrence. These findings will also shed further light on the
design and optimization of other non-natural antimicrobial
oligomers in the future. For example, other class of pepti-
domimetics with alternative hydrophobic and hydrophilic
residues could be potential antimicrobial agents since their
backbones are different from natural a-peptides. Comprehensive
One of the biggest challenges for conventional antibiotics is
their susceptibility to the development of resistance, which
quickly abolishes their efficacy. This situation has become
more severe in recent years, and may lead to outbreaks of
deadly infectious diseases that are untreatable. To investigate
the potential for bacteria to develop resistance against the
treatment of g-AApeptides, methicillin-resistant S. aureus
(ATCC 33592) was serially passaged in half-MIC concentrations
of g5, and new MIC values were determined every 24 h. As a
positive control, parallel cultures were exposed to serial 2-fold
dilutions of the antibiotic norfloxacin (Fig. S3w).16 After 17
days, virtually no change in the MIC occurred for g5 over the
17 passages, whereas the MIC for norfloxacin started to
increase after just three passages, and developed to profound
resistance after 17 (MIC increased 4 20-fold). These results
demonstrate that MRSA strains do not readily develop resistance
to g-AApeptide g5. Although additional studies to assess the
ability of bacteria to evolve resistance to g5 are required, we
suggest that this preliminary data indicates that g5 may
actually mimic natural antimicrobial peptides which are
notoriously difficult to develop resistance towards. This
augments the potential of g5 as a new generation of antibiotic
lead compounds.
studies of
a wide variety of g-AApeptides containing
hydrophobic and amphiphilic building blocks of different
functional groups, positions, and ratio are currently
underway. Further investigation of bactericidal mechanisms
is also ongoing.
This work is supported by USF start-up fund (JC) and NIH
1R01AI080626-01A2 (LNS).
Notes and references
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The antimicrobial mechanism of g-AApeptides in disrupting
bacterial membranes was also assessed by fluorescence micro-
scopy (Fig. S4w) using a double staining method with DAPI
and PI. DAPI stains all bacterial cells irrespective of their
viability, whereas PI only stains injured or dead cells with
damaged membranes.17,18 After incubation with g-AApeptide
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these bacteria had been disrupted. The aggregation of dead
cells is consistent to previously reported observation.18
In summary, we reported the identification of a new class of
antimicrobial peptidomimetics-g-AApeptides, with potency
and broad-spectrum activity far superior to previous reported
a-peptides.15 These g-AApeptides likely inhibit bacterial
growth by mimicking conventional antimicrobial peptides
through membrane disruption. This is an important observation
as it suggests the potential for virtually no development of
resistance towards these agents. Additionally, although more
extensive studies are needed, our initial attempt has demonstrated
the ease and effectiveness of building block-joining strategy,
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c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 12197–12199 12199