Non-hemolytic α-AApeptides as antimicrobial peptidomimetics

Article abstract of DOI:10.1039/c1cc13684d

We report a new class of peptide mimetics, α-AApeptides, that display broad-spectrum activity against both Gram-negative and Gram-positive bacteria and fungi. With non-hemolytic activity, resistance to protease hydrolysis, and easy sequence programmability, α-AApeptides may emerge as a novel class of antibiotics. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c1cc13684d

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Non-hemolytic a-AApeptides as antimicrobial peptidomimeticsw
Shruti Padhee,a Yaogang Hu,a Youhong Niu,a Ge Bai,a Haifan Wu,a Frankie Costanza,a
Leigh West,b Lacey Harrington,c Lindsey N. Shaw,c Chuanhai Caod and Jianfeng Cai*a
Received 20th June 2011, Accepted 6th July 2011
DOI: 10.1039/c1cc13684d
We report a new class of peptide mimetics, a-AApeptides, that
display broad-spectrum activity against both Gram-negative and
Gram-positive bacteria and fungi. With non-hemolytic activity,
resistance to protease hydrolysis, and easy sequence programm-
ability, a-AApeptides may emerge as a novel class of antibiotics.
which sacrifices their selectivity.11,12,18 Achieving potent,
broad-spectrum antimicrobial activity, while retaining low
hemolytic properties, is the immediate goal of antimicrobial
peptidomimetic development.
We have recently proposed a new class of oligomeric
peptidomimetics, termed ‘‘a-AApeptides’’, based on the a-chiral
PNA backbone (Fig. 1).22,23 a-AApeptides have many advantages
over a-peptides, such as limitless diversification and resistance
to protease degradation.23 To continue to explore the potential
application of a-AApeptides, herein we report for the first time
the design, synthesis and evaluation of a-AApeptides as potential
antimicrobial agents. We show that some a-AApeptides display
potent, broad-spectrum activity against both Gram-positive and
Gram-negative bacteria as well as fungus, and are highly selective
(non-hemolytic). Coupled with straightforward solid phase
synthesis, virtually limitless structural possibilities, low cost
of production, simple tunability and programmability, and
resistance to protease hydrolysis, a-AApeptides may lead to a
new class of antimicrobial peptidomimetics.
Short cationic amphiphilic antimicrobial peptides (AMPs) are
present in almost all organisms as a component of their innate
defense against invading pathogens.1 These peptides are emerging
as a promising new generation of antibiotics because of their
potent broad-spectrum antimicrobial activity, rapid action,
ability to kill multidrug-resistant bacteria, and a low propensity
for the development of resistance.2–5 Many research findings
support that AMPs kill bacteria through bacterial membrane
disruption.1,2 This is perhaps the major reason why acquiring
resistance to AMPs is much more difficult, when compared to
that of conventional antibiotic therapies.6
Despite being one of the most promising new generations of
antimicrobial agents, AMPs face obstacles for therapeutic
development. One of the biggest impediments is their intrinsic
instability in the context of proteolytic degradation.1,2 In
contrast to conventional peptides, oligomeric peptidomimetics
are protease-resistant, which can potentially circumvent the
drawbacks of AMPs. In recent years, there has been significant
interest in the development of antimicrobial peptidomimetics such
as b-peptides,7–11 peptoids,12–15 aryl amides,16,17 and oligoureas.18
While the majority of these peptidomimetics were designed to
mimic helical amphipathic antimicrobial peptides, for example
magainin, the developments of antimicrobial peptide mimetics
which imitate unstructured peptide antibiotics such as indolicidin
are rare.17,19–21 Some peptidomimetics are active against a variety
of bacteria but are, however, hemolytic at higher concentrations,
Antimicrobial a-AApeptides were designed based on the
notion that, while globally amphipathic structures are of impor-
tance, defined secondary structures are not necessary for the
16,18,20,21
design of antimicrobial oligomers.
Gellman et al. have
demonstrated that random copolymers can display different
antibacterial activities by simply varying the ratio of hydrophobic
and cationic groups, even the polymers are not structured at all.21
Pre-organized helical oligomers can actually be more hemolytic
and less selective.12 a-AApeptides are very promising anti-
microbial agents because they are likely to adopt an extended
conformation23 due to their conformational flexibility, which
is believed to facilitate their penetration of bacterial cell
walls.16 The design of amphipathic a-AApeptide sequences is
therefore very straightforward. They can be assembled by
amphiphilic building blocks (containing both hydrophobic and
cationic groups) and they would be potential antimicrobial
agents, as the sequences can easily adjust their conformation
to become globally amphipathic due to hydrophobic and
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 Florida Center of Excellence for Drug Discovery and Innovation,
University of South Florida, 3720 Spectrum Blvd, Tampa, FL 33612,
USA
c Department of Cell Biology, Microbiology and Molecular Biology,
4202 E. Fowler Ave, Tampa, FL 33620, USA
d Dept. 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 charaterization data. Figures for struc-
tures, CD and fluorescence microscopy. See DOI: 10.1039/c1cc13684d
Fig. 1 Structures of an a-peptide and a corresponding AApeptide.
Chem. Commun., 2011, 47, 9729–9731 9729
c
This journal is The Royal Society of Chemistry 2011

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also investigated to measure their selectivity (Table 1). As
expected, the numbers of hydrophobic and cationic groups are
important for antimicrobial activity, since longer a-AApeptides
are found to be more potent (Fig. S2, ESIw). a-AApeptide 6
shows the best activity toward both Gram-positive and
Gram-negative bacterial strains, and is amongst the most potent
antimicrobial oligomeric peptidomimetics reported.10,12 It also
shows strong activity against the multi-drug resistant
S. epidermidis strain RP62A, which is insensitive to a variety of
conventional antibiotics. Two natural antimicrobial peptides,
magainin 7 (helical)24 and indolicidin (unstructured),19,25–27 are
both less active against all of the strains tested. Interestingly, the
control peptide 8, bearing exactly the same functional groups
and backbone length as a-AApeptide 6, did not show any
activity at the concentrations tested. We hypothesize that such
a distinct difference must result from the different backbones,
and global amphipathic structures of the two peptides.28
Surprisingly, control a-AApeptide 9 only shows very weak
antibacterial activity, probably due to the existence of bulky
chiral aromatic groups, which prevent the formation of an
extended structure upon binding to bacterial cell membrane.
To test the selectivity of the oligomers, 1–9 were incubated
with human red blood cells. None of them showed detectable
hemolysis up to a concentration of 100 mg mlÀ1. Then, the
most active a-AApeptides 4, 5 and 6, along with control
peptides magainin 7, 8, and 9, were also tested at higher
concentrations. Again, each of them effectively displayed
Fig. 2 a-AApeptide building blocks (m1 and m2) used for the
preparation of antimicrobial a-AApeptides.
electrostatic interactions when binding onto bacterial membranes,
owing to their flexible backbone. We also predicted longer
sequences would be more potent antimicrobial agents because
more positive charges and hydrophobic groups can be involved
in the interaction with bacterial membranes. To test our
hypothesis, two amphiphilic a-AApeptide building blocks
(Fig. 2) were synthesized, and a variety of a-AApeptide
sequences with different lengths (1–6) were prepared (Fig. 3
and Fig. S1, ESIw) by assembling amphipathic building blocks
on the solid phase via the reported protocol.23 As controls,
we prepared magainin II (a natural antimicrobial peptide) 7, a
14-mer conventional peptide 8 with alternative phenylalanine
and lysine residues and peptidomimetic 9, the opposite
a-AApeptide where the amino acid is Phe and the lysine like
side chain comes off the amine (Fig. 3 and Fig. S1, ESIw).
The antimicrobial activities of 1–9 were tested against
the Gram negative E. coli and Gram positive B. subtilis,
S. epidermidis and fungus C. albicans (Table 1). Hemolytic
activities of the oligomers toward human red blood cells were
almost 0% hemolysis at concentrations of up to 250 mg mlÀ1
,
with the exception of peptide 8, which showed 5% hemolysis
at this concentration (Fig. S3, ESIw). It seems that the a-
AApeptide backbone is slightly less hemolytic than that of a
conventional peptide, as seen by comparing a-AApeptide 6 to
peptide 8, in which the same functional groups are present.
Meanwhile, the evidence suggests that hemolytic activity is
more likely related to functional groups present on the oligo-
mer backbone than to the nature of the backbone, since
indolicidin, containing multiple hydrophobic tryptophan
residues, causes 10% hemolysis at 30 mg mlÀ1 and 100%
Fig. 3 Oligomers for the antimicrobial assay. 1–6 are a-AApeptides; 7 is
magainin II; 8 is the 14-mer regular peptide with alternative phenylalanine
and lysine residues. 9 is the opposite a-AApeptide where the amino acid is
Phe and the lysine-like side chain comes off the amine.
hemolysis at approximately 200 mg mlÀ1 25
.
Table 1 The antimicrobial and hemolytic activities of oligomers 1–8. The microbial strains used were E. coli (JM109), B. subtilis (BR151),
S. epidermidis (RP62A), and C. albicans. S. epidermidis is a multi-drug resistant isolate which is insensitive to b-lactam antibiotics, and a number of
other antimicrobial agents. The minimum inhibitory concentration (MIC) is defined as the lowest inhibitor concentration that completely inhibits
the growth of microbes during a 24 h incubation period at 37 1C. MHC (the minimum hemolytic concentration) is defined as the lowest
concentration at which hemolysis is detected. H10 is defined as the concentration that causes 10% hemolysis of human red blood cells. Selectivity is
defined as H10/MIC towards B. subtilis. Activities of the extended antimicrobial peptide indolicidin (ILPWKWPWWPWRR) were obtained
previously by other research groups19,25–27
MICa mM (mg mlÀ1
)
MHCa/mg mlÀ1
Selectivity (H10/B. subtilis MIC)
E. coli
B. subtilis
S. epidermidis
C. albicans
Oligomers
1
2
3
4
5
6
7
8
293(90)
64(60)
16(20)
243(75)
43(40)
15(18)
8(12.5)
6.8(12.5)
0.9(2)
16(40)
451(100)
38(75)
5(10)
4162(50)
453(50)
440(50)
20(30)
427(50)
4.6(10)
420(50)
451(100)
4100
—
—
—
—
4100
4100
4100
4250
4250
4250
4250
B250
4250
B30
41.3
42.5
45.6
420
420
4125
46.3
o2.5
43.3
B3
13(20)
9.8(18)
2.1(4.5)
16(40)
451(100)
38(75)
13(25)
—
9.3(20)–14(30)
420(50)
451(100)
451(100)
433(64)
9
Indolicidin
10(20)
a
For concentrations shown as ‘‘4x’’, x = 50, 100 or 250 mg mlÀ1, which are the highest concentrations tested for that sequence.
c
9730 Chem. Commun., 2011, 47, 9729–9731
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Circular dichroism (CD) spectroscopy was employed to
study the conformational structure of 1–8 in both aqueous
buffer and in lipid vesicles that mimic bacterial membranes
(Fig. S4, ESIw).13 a-AApeptides have a different backbone
compared to peptides; therefore CD cannot specifically elucidate
a specific secondary structure. However, results suggest that
a-AApeptides are probably unstructured in buffer solution,
since their CD spectra are quite similar to monomer 1, which is
unlikely to have any secondary structure at all (Fig. S4a, ESIw).
Magainin 7 is also randomly coiled in buffer, while peptide 8
displays a very weak minimum at 222 nm (Fig. S4a, ESIw),
indicating the presence of some a-helical character. Upon
binding to lipid vesicles, the CD of a-AApeptide 6 shows a
minimum at 207 nm (Fig. S4b, ESIw), which is not observed in
PBS. The reason is unclear, possibly because the amphipathic
structure of a-AApeptide 6 is more defined on the lipid
vesicles. Magainin 7 becomes highly helical (Fig. S4b, ESIw)
under similar conditions, which is consistent with previous
reports.24 Interestingly, 8 completely lost its helical conformation
when exposed to lipid vesicles, and turned into a random coil.
We believe a-AApeptides are not structured in aqueous
solution due to their flexible backbone. However, upon binding
to negatively charged bacterial membranes, they can easily adopt
globally amphipathic structures to facilitate membrane disruption
(Fig. S5, ESIw). Indeed, the flexibility of the AApeptide backbone
leads to a stronger bacterial membrane-disruptive capability
than conventional peptides,16 which accounts for the potent
antimicrobial activity of a-AApeptides. Control peptide 8,
containing alternative cationic and hydrophobic residues,
displays positive charges all over the helical backbone in water
solution. Forming an amphipathic structure for bacterial cell
wall penetration is not favorable because regular peptide
backbones have limited conformational freedoms, which lead
to very weak antimicrobial activity. The same assumption can
be used to explain the antibacterial activity of a-AApeptide 9.
The antimicrobial mechanism of a-AApeptides in disrupting
bacterial membranes was further assessed by fluorescence
microscopy (Fig. S6, ESIw) 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.29,30 After incubation with a-AApeptide
6 for 2 h, strongly PI-stained red fluorescent E. coli and B.
subtilis were observed, demonstrating that the membranes of
those bacteria had been disrupted.
than to pre-defined secondary structures. The number (not just
the ratio) and nature of cationic and hydrophobic groups also
have profound impact on activity. The simple design and easy
modular programmability should facilitate quick identification
of more potent and selective antimicrobial a-AApeptides in
the near future.
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In conclusion, this is the first report of water-soluble a-
AApeptides as antimicrobial peptidomimetics. Three a-AA-
peptides (4, 5 and 6) exhibit high selectivity, broad-spectrum,
and potent antibacterial activity against fungus, Gram-
positive and Gram-negative bacteria, including clinically-relevant
multi-drug resistant strains. a-AApeptide 6 was of significant
interest as it displayed antimicrobial activities, activity that was
equal to or better than the most antimicrobial oligomeric
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all, under tested experimental conditions. Such high selectivity is
attributed to the intrinsic properties of the flexible a-AApeptide
backbone in adopting amphipathic structures. Our results
support recent findings that antimicrobial activity of peptides,
or peptide mimics, is related to the presence of global amphi-
pathicity upon interaction with bacterial membranes rather
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Chem. Commun., 2011, 47, 9729–9731 9731