Regulatory Peptides 129 (2005) 85 – 91

www.elsevier.com/locate/regpep

Design of potent, non-toxic antimicrobial agents based upon the structure

of the frog skin peptide, pseudin-2
Tibor Pála, Ágnes Sonnevenda, Sehamuddin Galadarib, J. Michael Conlonb,T
a
Department of Medical Microbiology, Faculty of Medicine and Health Sciences, United Arab Emirates University,
PO Box 17666 Al-Ain, United Arab Emirates
b
Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, PO Box 17666 Al-Ain, United Arab Emirates

Received 3 November 2004; received in revised form 1 January 2005; accepted 27 January 2005
Available online 26 February 2005

Abstract

Pseudin-2, a naturally occurring 24 amino-acid-residue antimicrobial peptide first isolated from the skin of the South American
paradoxical frog Pseudis paradoxa, has weak hemolytic and cytolytic activity but also relatively low potency against microorganisms. In a
membrane-mimetic environment, the peptide exists in an amphipathic a-helical conformation. Analogs of the peptide with increased
cationicity and a-helicity were chemically synthesized by progressively substituting neutral and acidic amino acid residues on the hydrophilic
face of the a-helix by lysine. Analogs with up to three L-lysine substitutions showed increased potency against a range of gram-negative and
gram-positive bacteria (up to 16-fold) whilst retaining low hemolytic activity. The analog [d-Lys3, d-Lys10, d-Lys14]pseudin-2 showed
potent activity against gram-negative bacteria (minimum inhibitory concentration, MIC=5 AM against several antibiotic-resistant strains of
Escherichia coli) but very low hemolytic activity (HC50N500 AM) and cytolytic activity against L929 fibroblasts (LC50=215 AM). Increasing
the number of l-lysines to four and five did not enhance antimicrobial potency further but increased hemolytic activity towards human
erythrocytes. Time-kill studies demonstrated that the analog [Lys3, Lys10, Lys14, Lys21]pseudin-2 at a concentration of 1MIC was
bacteriocidal against E. coli (99.9% cell death after 96 min) but was bacteriostatic against S. aureus. Increasing the hydrophobicity of
pseudin-2, while maintaining the amphipathic character of the molecule, by substitution of neutral amino acids on the hydrophobic face of the
a-helix by l-phenylalanine, had only minor effects on antimicrobial and hemolytic activities.
D 2005 Elsevier B.V. All rights reserved.

Keywords: Pseudin-2; Antimicrobial; Hemolytic; Cytolytic; Amphibian

1. Introduction
The emergence in all regions of the world of strains of
pathogenic bacteria and fungi with resistance to commonly
used antibiotics has necessitated a search for novel types of
antimicrobial agent to which the microorganisms have not
been exposed. Peptide-based anti-infectives are being
increasingly considered as potential therapeutic agents [1–
4]. On the positive side, because of their relatively non-
specific mechanism of action (either a detergent-like
disruption of the bacterial cell membrane into peptide-
coated vesicles or formation of transient transmembrane
T Corresponding author. Tel.: +971 3 7039 484; fax: +971 3 7672 033.
E-mail address: jmconlon@uaeu.ac.ae (J.M. Conlon).
0167-0115/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.regpep.2005.01.015
pores [5,6]), the peptides show broad spectrum activity and
development of resistance occurs at rates that are orders of
magnitude lower than those observed for conventional
antibiotics [7]. The major obstacles to their development
as useful drugs are their toxicities, particularly if they are to
be administered systemically, and their short half-lives in the
circulation. Thus, future therapeutic applications are more
likely to involve topical rather than systemic administration,
for example, in treatment of infected foot ulcers of diabetic
patients [8].
The synthesis of peptides with antimicrobial activity in
granular glands located in the skin is a feature of several
anuran (frog and toad) species, particularly those belonging
to the families Bombinatoridae, Hylidae, Hyperoliidae,
Leptodactylidae, Myobatrachidae, Pipidae, and Ranidae
86 T. Pál et al. / Regulatory Peptides 129 (2005) 85–91
(reviewed in [4,9–11]). These antimicrobial peptides com-
prise between 12 and 48 amino acid residues and are
characterized by a remarkable degree of structural diversity,
which is considered to be important in protecting the
organism against invasion by a wide range of pathogenic
microorganisms [12]. The peptides lack any consensus
amino acid sequences that are associated with biological
activity but, with few exceptions, they are cationic,
relatively hydrophobic and have the propensity to form an
amphipathic a-helix in a membrane-mimetic solvent such as
trifluoroethanol [4,13].
Pseudin-2 (GLNALKKVFQ10GIHEAIKLIN20NHVQ) is
a 24 amino-acid-residue antimicrobial peptide first isolated
from an extract of the skin of the paradoxical frog, Pseudis
paradoxa (Hylidae) [14]. The peptide shows moderately
high potency against the gram-negative bacterium Escher-
ichia coli (Minimum Inhibitory Concentration, MIC=20
AM) but is only very weakly active (MICN100 AM) against
the gram-positive bacterium Staphylococcus aureus How-
ever, in comparison with most other antimicrobial peptides
from frog skin [4,9], pseudin-2 shows very low hemolytic
activity against human erythrocytes (concentration produc-
ing 50% hemolysis, HC50=360 AM).
A previous study using circular dichroism spectroscopy
[14] has shown that, in aqueous solution, pseudin-2 exists
predominantly as a random coil but in 50 % trifluoroetha-
nol/water, a solvent that mimics the hydrophobic environ-
ment of the cell membrane, the peptide adopts an a-helical
conformation. A Schiffer–Edmundson helical wheel projec-
tion [15] of the pseudin-2 structure indicates that this a-
I19
G1 I12 V23
V8
A15 L5
H22
A4
I16
G11
F9
L18
L2
N20
K7
H13
E14 K6
K17
N3 Q10
Q24
N21
Fig. 1. A Schiffer–Edmundson helical wheel projection [15] of the pseudin-
2 structure. The polar residues are shown in bold type. Amino acid residues
at positions 3, 10, 14, 21 and 24 were progressively substituted by lysine.
helix has considerable amphipathic character with the
hydrophilic residues Lys6, Lys7, Glu14, and Lys17 segregat-
ing on one face and the hydrophobic residues Leu2, Leu5,
Val8, Phe9, Ile12, Ile16, and Val23 segregating on the opposite
face (Fig. 1). The aim of the present study was to design
analogues of pseudin-2 that maintain the amphipathic a-
helical character and the low hemolytic activity of the
naturally-occurring peptide but display increased potencies
towards a range of pathogenic microorganisms. The strategy
adopted was to increase cationicity by progressively
substituting neutral and acidic residues on the hydrophilic
face by either l-lysine or d-lysine.
2. Materials and methods
2.1. Peptide synthesis
All synthetic peptides were supplied in crude form either
by Sigma Genosys (USA) or by GL Biochem (Shanghai)
(China). The peptides were purified to near homogeneity by
reverse-phase HPLC on a (255-cm) Vydac 218TP1022 (C-
18) column (Separations Group) equilibrated with acetoni-
trile/water/trifluoroacetic acid (28.0/71.9/0.1) at a flow rate
of 6 ml/min. The concentration of acetonitrile was raised to
56 % (v/v) over 60 min using a linear gradient. Absorbance
was measured at 214 and 280 nm and the major peak in the
chromatogram was collected by hand. The identities of the
synthetic peptides were confirmed by MALDI mass
spectrometry. For all peptides, the average molecular masses
determined by mass spectrometry were consistent with the
masses calculated from the proposed structures. The purity
of all peptides tested was N95%.
2.2. Antimicrobial assays
Reference strains were purchased from the American
Type Culture Collection (Rockville, MD), from the National
Collection of Type Cultures (London, UK), and from the
Hungarian National Type Culture Collection (Budapest,
Hungary) and have been described previously [16]. Clinical
isolates of antibiotic-resistant strains of E. coli were
recovered from the urines of patients from Tawam Hospital
(Al-Ain, U.A.E.) with urinary tract infections.
MIC testing of the peptides was determined by a
standard microdilution method using 96-well microtiter
cell-culture plates [17]. Serial dilutions of the peptides in
Mueller-Hinton broth (50 Al) were mixed with an
inoculum (50 Al of 106 CFU/ml) from a log-phase culture.
Bacteria were incubated for 18 h at 37 0C in a humidified
atmosphere of air. Incubations with C. albicans were
carried out in RPMI 1640 medium for 48 h at 35 8C. After
incubation, the absorbance at 630 nm of each well was
determined using a microtiter plate reader. In order to
monitor the validity of the assay, incubations with
bacitracin were carried out in parallel with increasing
 T. Pál et al. / Regulatory Peptides 129 (2005) 85–91 87

concentrations of bacitracin and incubations with C. Table 1

albicans in parallel with amphotericin B. Minimum inhibitory concentrations against reference strains of Escherichia
coli and Staphylococcus aureus (AM) and hemolytic activities against
In order to determine the rate at which the analog [Lys3, human erythrocytes (AM) of lysine- and phenylalanine-substituted analogs
Lys10, Lys14, Lys21]pseudin-2 caused cell death, log-phase of pseudin-2
cultures of E. coli and S. aureus, at a concentration of Net charge E. coli S. aureus HC50
5105 CFU/ml, were incubated in Eppendorf tubes at 37 8C
Pseudin-2 +2 20 160 360
with the peptides at a concentration of 1MIC. A control Lys3 +3 10 80 340
incubation in the absence of peptide was carried out. Lys10 +3 20 80 240
Aliquots (10 Al) were removed at 0, 0.5, 1, 3, 4, 5 and 24 Lys14 +4 20 40 240
h, and the CFU of the serially diluted samples was Lys21 +3 20 80 270
Lys24 +3 20 80 380
determined on tryptic-soy agar plates. The time required
Lys3,14 +5 5 40 350
for 99.9% cell death was calculated by linear regression Lys3,10,14 +6 5 20 330
analysis of a plot of log10CFU versus time. d-[Lys3,10,14] +6 5 N160 N500
Lys3,10,14,21 +7 5 20 180
2.3. Hemolysis assay Lys3,10,14,21,24 +8 5 20 95
Phe5 +2 20 N160 440
Phe8 +2 20 N160 270
Peptides in the concentration range 1–200 AM were Phe12 +2 N80 N160 370
incubated with washed human erythrocytes (2107 cells) Phe16 +2 20 160 250
from healthy donors in Dulbecco’s phosphate-buffered Phe19 +2 10 160 430
saline, pH 7.4 (100 Al) for 1 h at 37 8C with constant
agitation. After centrifugation (12,000 g for 30 s), the
absorbance at 450 nm of the supernatant was measured. A Gln24YLys increased potency against S. aureus only. The
parallel incubation in the presence of 1% v/v Tween-20 was amino acid asparagine has the property of destabilizing an
carried out to determine the absorbance associated with a-helix [19] so that replacement of Asn3 and Asn21 by Lys
100% hemolysis. The HC50 value was taken as the mean will increase the a-helical character of pseudin-2 as well as
concentration of peptide producing 50% hemolysis for three increasing its cationicity. The effect of the single lysine
independent experiments. substitution had no effect on activity against C. albicans
and the HC50 value of all analogs was N200 AM.
2.4. Cytolysis assay
3.2. Effect of substitutions by multiple lysine residues
L929 mouse fibroblast cells (3105 cells per well) were
seeded onto 24-well culture plates and allowed to grow for Progressively increasing the cationicity of pseudin-2
48 h as previously described [18]. Peptides in the further by substituting multiple residues in the hydrophilic
concentration range 1–400 AM were incubated with the face of the peptide with l-lysine resulted in an enhance-
cells for 1 h at 37 8C in RPMI 1640 medium (200 Al) and ment of antimicrobial potency (Table 1). Introduction of
cell viability was assessed by measurement of lactate two lysine residues at positions 3 and 14 led to 4-fold
dehydrogenase release using a CytoTox 96 assay kit increases in potency against E. coli and S. aureus without
(Promega). The LC50 value was taken as the mean changing hemolytic activity (HC50=360 AM). The tri-
concentration of peptide producing 50% cell death in substituted analogue [Lys3, Lys10, Lys14]pseudin-2 showed
three independent experiments. an 8-fold increase against E. coli, an 8-fold increase
against S. aureus, and showed comparable hemolytic
activity to the native peptide (HC50=330 AM). The
3. Results corresponding trisubstituted analog containing D-lysine
retained high potency against the reference strain of E.
3.1. Effect of substitutions by single lysine residues coli (MIC=5 AM) but was inactive against S. aureus
(MICN160 AM). This analog was almost completely
In order to increase the cationicity of the peptide, while devoid of hemolytic activity (HC50H500 AM), with no
still maintaining the amphipathic nature of the a-helix, significant hemolysis below 300 AM. The LC50 value of
analogs containing substitutions of the neutral and acidic [Lys3, Lys10, Lys14]pseudin-2 against L929 fibroblasts was
residues on the hydrophilic face by l-lysine were 215F5 AM compared with an LC50 value of 75F3 AM for
synthesized. The effects of these substitutions on anti- pseudin-2 (meanFSD; n=3), with no significant cytolysis
microbial activity against reference strains of E. coli below 100 AM. Increasing cationicity even further by
(ATCC 25922) and S. aureus (NCTC 8325) are shown introduction of a fourth l-lysine residue at position 21 did
in Table 1. The substitution Asn3YLys resulted in a 2-fold not increase antimicrobial potency appreciably but led to
increase in potency against both microorganisms whereas an increase in hemolytic and cytolytic activity. Thus, the
the substitutions Gln10YLys, Glu14YLys, Asn21YLys and tetra-substituted analogue [Lys3, Lys10, Lys14, Lys21]pseu-
88 T. Pál et al. / Regulatory Peptides 129 (2005) 85–91

CFU / ml
The multi-l-lysine-substituted analogs showed relatively
109 high potency against reference strains of several pathogenic
A bacteria Enterobacter cloacae, Klebsiella pneumoniae,
108 Pseudomonas aeruginosa, Staphylococcus epidermidis
and Streptococcus Group B (Table 2). The potency of
107 [Lys3, Lys10, Lys14, Lys24]pseudin-2 against Enterobacter
cloacae was enhanced 32-fold compared with the native
peptide (MIC=2.5 AM vs 80 AM for pseudin-2). No peptide
106
tested was active against Proteus mirabilis and Enter-
ococcus faecalis at concentrations V 80 AM. The d-lysine
105 substituted analog [d-Lys3, d-Lys10, d-Lys14]pseudin-2 was
active only against gram-negative bacteria but was the only
104 analog tested that inhibited growth of the opportunistic yeast
pathogen C. albicans, albeit a relatively high concentration
1010
(MIC=80 AM). In particular, this analog was active (MIC=5
109 B AM) against five clinical isolates of E. coli with well-
documented resistance to a wide range of commonly used
108
antibiotics (Table 3).
107
3.3. Effect of substitutions by single phenylalanine residues
106

105 The effect on antimicrobial and hemolytic activities of

104
increasing the hydrophobicity of pseudin-2, while maintain-
ing the amphipathic character of the molecule, by sub-
103 stitution of the amino acid residues Leu5, Val8, Ile12, Ile16,
102 and Ile19 on the hydrophobic face by the more hydrophobic
residue, phenylalanine is shown in Table 1. Effects on
0
30
60
90
120
180
240
300

24 hrs Time
antimicrobial potency of increasing hydrophobicity were
(min)
much less than the corresponding effects produced by
Fig. 2. Survival of (A) S. aureus and (B) E. coli in Mueller–Hinton broth increasing cationicity. Only the analog containing the
after addition of 1MIC of [Lys3, Lys10, Lys14, Lys21]pseudin-2 Symbols: substitution (Ile19YPhe) showed increased potency com-
.
(o) incubations in the presence and ( ) in the absence of peptide. Data
pared with the native peptide (2-fold against E. coli). The
points show meanFS.D. for three independent experiments.
HC50 value for all analogs was N200 AM but the hemolytic

din-2 displayed the same 4- to 8-fold increase in potency

against E. coli and S. aureus compared with the native Table 2
peptide but the hemolytic activity of the tetra-substituted Minimum inhibitory concentrations (AM) of lysine-substituted analogs of
pseudin-2 against reference strains of microorganisms
analogue increased 2-fold compared with pseudin-2
(HC50=180 AM vs 360 AM for pseudin-2). The cytolytic Native [K3,14] [K3,10,14] d-[K3,10,14] [K3,10,14,21]
activity against L929 mouse fibroblasts of the tetra- Escherichia coli 20 5 5 5 5
ATCC 25922
substituted analog was approximately 7-fold greater than
Enterobacter cloacae 80 10 5 40 2.5
that of the native peptide (LC50=10 AM vs 75 AM for HNTCC 53001
pseudin-2). Substitution by an additional l-lysyl residue in Klebsiella pneumoniae 40 5 5 40 5
the penta-substituted analog [Lys3, Lys10, Lys14, Lys21, KK3 9904
Lys24] pseudin-2) increased hemolytic activity further Pseudomonas aeruginosa 80 10 5 40 5
ATCC 27853
(HC50=95 AM without increasing antimicrobial potency.
Proteus mirabilis N80 N80 N80 N80 N80
In a time-kill assay using E. coli, 99.9% of the bacteria ATCC 25933
were killed within 96 min by [Lys 3, Lys 10, Lys 14, Staphylococcus aureus N80 40 20 N80 20
Lys21]pseudin-2 at a concentration of 1MIC (Fig. 2). In NCTC 8325
contrast, the effect of peptide on S. aureus was bacterio- Staphylococcus 80 20 10 N80 10
epidermidis RP62A
static. Even at a concentration of 2MIC, the analog did not
Streptococcus Group B 80 10 10 N80 10
produce 100% cell-death, although a 3–4 log decrease in the HNTCC 80130
CFU was observed after a 24 h incubation (data not shown). Enterococcus faecalis N80 N80 N80 N80 N80
The microorganisms surviving this exposure remained ATCC 29212
sensitive to the peptide as evidenced by an unchanged Candida albicans N80 N80 N80 80 N80
ATCC 90028
MIC value.
 T. Pál et al. / Regulatory Peptides 129 (2005) 85–91
Table 3
MIC of [d-Lys3, d-Lys10, d-Lys14]pseudin-2 against clinical isolates of antibiotic resistant Escherichia coli
Strains MIC (AM) AMP AMC CXM CAZ CTX
16858/02 5 R R R R R
533/03 5 R R R R R
1990/03 5 R R R R R
4660/2 5 R R R R R
4493 5 R R R R R
AMP: Ampicillin; AMC: Amoxi-Clav; CXM: Cefuroxime; CAZ: Ceftazidime; CTX: Cefotaxime; CRO: Ceftriaxone; FEP: Cefepim; ATM: Aztreonam; IPM:
Imipenem; GM: Gentamicin; AK: Amikacin; TMP/SMX: Trimethoprim–Sulphamethoxasole; NA: Nalidixix acid; CIP: Ciprofloxaxin; R: Resistant; I:
Intermediate, S: Susceptible.
activity of the Phe8-, and Phe16-substituted peptides were
significantly greater than that of pseudin-2.
4. Discussion
A major obstacle to the development of antimicrobial
peptides as useful anti-infectives, particularly if they are
to be administered systemically, is their varying degrees
of toxicity towards mammalian cells. This study describes
the design of analogs of a naturally occurring peptide,
pseudin-2 that show increased potency against micro-
organisms but appreciably less cytolytic activity against
erythrocytes and fibroblasts. The composition of the
bacterial cytoplasmic cell membrane is rich in acidic
phospholipids whereas the plasma membrane of mamma-
lian cells, such as human erythrocytes, contains a much
higher proportion of zwitterionic phosphatidylcholine and
sphingomyelin phospholipids [20]. Consequently, an
increase in peptide cationicity should promote interaction
with the more negatively charged bacterial cell membrane
and increase antimicrobial potency. The positive charge on
the peptide is also believed to facilitate interaction with,
and passage across, the bacterial cell wall, both in the
case of gram-negative bacteria that contain negatively
charged lipopolysaccharides and of gram-positive bacteria
that contain negatively charged teichoic and teichuronic
acids [21]. The present study has confirmed this pre-
diction. By selective substitution of neutral and acidic
amino acids by l-lysine, it has been possible to design
analogs of the naturally occurring antimicrobial peptide,
pseudin-2 that show increased potency (up to 32-fold)
against a range of pathogenic microorganisms yet retain
low hemolytic activity. Consistent with previous studies
with analogs of magainin-2 [22,23], increasing positive
charge on pseudin-2 resulted in an increase in antimicro-
bial activity until a limit was reached whereupon further
increases in cationicity did not result in any further
increase in activity.
As well as molecular charge, antimicrobial potency is
determined by conformation (degree of a-helicity) and
amphipathicity [13,20]. Several studies have also demon-
strated a direct correlation between degree of amphipa-
89
Antibiotics GM AK TMP/SMX NA CIP
CRO FEP ATM IPM
R R R S I R R R R
R R R S R R R R R
R R R S R S S R R
R R R S S S R R R
R R R S S I R R R
thicity and hemolytic activity in peptides that form an a-
helix [24–26]. It has been suggested that increasing
amphipathicity has a relatively modest effect on the ability
to permeabilize the negatively charged cell membrane of
microorganisms but a marked effect on the more zwitter-
ionic phospholipid membrane of the erythrocyte. It is
probable, therefore, that the increase in amphipathicity
produced by the substitutions Asn21YLys and Gln24YLys
in the tetra- and penta-substituted analogs is responsible
for their enhanced hemolytic activity.
Previous studies with model amphipathic a-helical
peptides have shown that selective substitutions by d-
amino acids may produce analogs that retain antimicrobial
activity but show reduced hemolytic activity and increased
resistance to proteolytic enzymes [27–29]. The analog [d-
Lys3, d-Lys10, d-Lys14]pseudin-2 is active not only
against reference strains of gram-negative bacteria but
also against highly antibiotic-resistant strains of E. coli yet
displays almost undetectable hemolytic activity. However,
the peptide has lost the ability to inhibit growth of the
gram-positive bacteria tested. It has been shown using
model peptides that a stabilized amphipathic a-helical
conformation is an absolute requirement for cidal activity
against gram-positive bacteria whereas the structural
requirements for activity against gram-negative bacteria
are less stringent [13]. Provided that cationicity and
hydrophobicity are maintained, peptides with impaired
helicity were still active. It is suggested, therefore, that
substitution by d-lysine residues in the pseudin-2 analog
is responsible for destabilization of the a-helix and the
consequent loss of activity against gram-positive bacteria
and the reduction in cytolytic activity against L929
fibroblasts.
Time-kill studies with the tetralysine-substituted analog
of pseudin-2 indicated that the interaction of the peptide
with gram-negative bacteria (E. coli) was different from the
interaction with gram-positive bacteria (S. aureus). The
peptide was clearly bacteriocidal against E. coli and the time
required to produce cell death was similar to that measured
for the frog skin peptide, ranatuerin-1 [16]. However,
ranatuerin-1 was also bacteriocidal against S. aureus
whereas [Lys3, Lys10, Lys14, Lys21]pseudin-2, at a concen-
tration of 1MIC, was clearly bacteriostatic (Fig. 2). There
90 T. Pál et al. / Regulatory Peptides 129 (2005) 85–91
is no universal agreement regarding the precise mechanism
by which bacterial cell death occurs and it is clear that no
single mechanism is applicable to all peptides and bacteria
but the dcarpetT model, proposed by Shai and co-workers
[5,30] is gaining broad acceptance. In this model, the
antimicrobial peptides coat the surface of the cell membrane
like a carpet with the positive charges on the peptide binding
to the phospholipid head groups. As concentration increases
and approaches the MIC value, the peptides oligomerize and
reorientate so that the hydrophobic residues insert into the
hydrophobic core of the lipid bilayer leading to membrane
permeabilization and eventually disintegration into peptide-
coated vesicles. Membrane collapse may be facilitated by
the formation of transient transmembrane holes or pores that
will allow passage of low molecular mass molecules
generating osmotic imbalance. In addition, peptides, for
example the thrombin-induced platelet microbicidal pro-
teins, may exercise a growth-inhibitory action independent
of membrane destruction by interacting with intracellular
targets to inhibit protein and DNA synthesis [31]. It
suggested, as one possibility, that the pseudin-2 analog
has only a limited ability to permeabilize the cytoplasmic
membrane of S. aureus and inhibits growth by suppressing
pathways of macromolecular synthesis in the cell. In
contrast, interaction with E. coli results in large-scale
permeabilization of the membrane due to micellization
and leads to cell death.
Studies using a series of analogs related to magainin-2
[24,25] and using model peptides [32] have shown that
increasing overall hydrophobicity had relatively little
influence on activity against gram-negative bacteria but
enhanced activity against gram-positive bacteria while, at
the same time, increasing hemolytic activity. Similarly,
studies using the model peptide K 2 A 3 XA 5 XA 2 -
WA2XA3K4-amide, where X represents a range of amino
acids, demonstrated that the analog with X=Phe had the
greatest hydrophobicity as measured by retention times on
reverse-phase HPLC and highest potency against both
gram-negative and gram-positive bacteria [33]. However,
in the case of pseudin-2, increasing the hydrophobicity of
the peptide by replacement of non-polar residues on the
hydrophobic face of the peptide by phenylalanine residue
did not enhance antimicrobial activity against S. aureus.
The substitutions Val8YPhe, and Ile16YPhe did produce
analogs with the predicted increased hemolytic activity
(Table 1).
Acknowledgments
This work was supported by an Interdisciplinary Grant
(03/12-8-03-01) from the United Arab Emirates University
and a Faculty Support Grant (NP/02/04) from the Faculty of
Medicine and Health Sciences, U.A.E. University. The
authors thank Mahendra Patel, Anne John, and Bency
Varghese for technical assistance.
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