Effect of ring size on conformation and biological activity of cyclic cationic antimicrobial peptides

Article abstract of DOI:10.1021/jm801648n

In a series of cyclic peptides based on GS10, an analogue of gramicidin S (GS), the ring size was varied from 10 to 16 amino acids. Alternative addition of basic and hydrophobic amino acids to the original GS10 construct generated a variety of even-number

Full text of DOI:10.1021/jm801648n

2090
J. Med. Chem. 2009, 52, 2090–2097
Effect of Ring Size on Conformation and Biological Activity of Cyclic Cationic Antimicrobial
Peptides
Masoud Jelokhani-Niaraki,*,† Leslie H. Kondejewski,‡,| Laura C. Wheaton,† and Robert S. Hodges§
Department of Chemistry, Wilfrid Laurier UniVersity, Waterloo, Ontario N2L 3C5, Canada, Protein Engineering Network of Centres of
Excellence, UniVersity of Alberta, Edmonton, Alberta T6G 2S2, Canada, and Department of Biochemistry and Molecular Genetics, UniVersity
of Colorado DenVer, School of Medicine, Anschutz Medical Campus, Aurora, Colorado 80045
ReceiVed December 28, 2008
In a series of cyclic peptides based on GS10, an analogue of gramicidin S (GS), the ring size was varied
from 10 to 16 amino acids. Alternative addition of basic and hydrophobic amino acids to the original GS10
construct generated a variety of even-numbered rings, i.e., GS10 [cyclo-(VKLdYPVKLdYP)], GS12 [cyclo-
(VKLKdYPKVKLdYP)], GS14 [cyclo-(VKLKVdYPLKVKLdYP), and GS16 [cyclo-(VKLKVKdYP-
KLKVKLdYP)] (d stands for D-enantiomers). The odd-numbered analogues (11-, 13-, and 15-mers) were
derived from these four peptides by either addition or deletion of single basic (Lys) or hydrophobic (Leu or
Val) amino acids. The resulting peptides, divided into three groups on the basis of peptide ring size (10- to
12-meric, 13- and 14-meric, and 15- and 16-meric), illustrated a diverse spectrum of biological activity
correlated to their ring size, degree of ꢀ-structure disruption, charge, hydrophobicity, amphipathicity, and
affinity for lipid membranes. Two of these peptides with potent antimicrobial activities and high therapeutic
indexes (4.5- to 10-fold compared with GS) are promising candidates for development of broad-spectrum
antibiotics.
Introduction
potent activity against a broad range of microbes, which include
Gram-positive and Gram-negative bacteria and fungi, GS is
highly hemolytic against mammalian erythrocytes.13 It has been
shown that GS and GS-like cationic peptide antibiotics interact
with both mammalian and microbial cell membranes to change
or destroy the membrane integrity causing lysis, which could
lead to cell death. Interaction with the microbial membranes
may not be the only mechanism for the biological activity of
GS or GS-like antimicrobial peptides. Generally, the mechanism
of biological activity of antimicrobial peptides can include
inhibitory interactions with the components of metabolic
pathways and immune systems of organisms.14 Specifically, it
has been reported that GS interacts with other biomolecules,
such as carbohydrates15 and enzyme components of metabolic
pathways.16
Resurgence of once extinguished microbes, their resistance
against existing drugs, and reappearance of lethal diseases
caused by these “modern microbes” have caused serious concern
worldwide.1 Development of potent broad-spectrum antimicro-
bial peptides with low toxicity against mammalian cells has been
a challenge and an important component of novel drug design.2,3
The main barrier in development of novel antimicrobial peptide
drugs is the complex mechanism of their biological activity.
Mammalian and bacterial cell membranes have been considered
as the primary, if not the only, target of antimicrobial peptides.
Strong and destructive interaction of peptides with mammalian
membranes, such as erythrocytes, is toxic and therefore should
be avoided in the design of effective antimicrobial peptides.
In our previous studies on cyclic cationic antimicrobial
peptides, we have chosen a naturally found decameric peptide,
gramicidin S (GSa), as the principal peptide construct in the
systematic design of antimicrobial peptides with enhanced
antimicrobial and reduced hemolytic activities.4-8 GS, a cyclic
cationic peptide, is biosynthesized in the Bacillus breVis and
has been widely used as a topical antibiotic.9 GS structure is
amphipathic and composed of a double-stranded antiparallel
ꢀ-sheet connected by a pair of type II′ ꢀ-turns.10-12 Despite its
We have previously reported that amphipaticity of cyclic
cationic peptides could be utilized in dissociating their antimi-
crobial and hemolytic activities5 and have also studied the
detailed biophysical properties and mechanism of interaction
of these peptides with biological membranes.17-20 In this
comprehensive study, we investigate the effect of ring size,
amphipathicity, and interaction with mammalian and bacterial
model membranes on the biological activity of 10- to16-meric
cyclic cationic peptides (13 peptides in total, Table 1). The
original construct for the peptide series in Table 1 is GS10, a
D-Tyr- and Lys-containing analogue of GS (Figure 1 and Table
1). Among these peptides, GS10 and GS14 have positively
charged residues on one face (2 and 4 charges, respectively)
and hydrophobic residues on the opposite face of the molecule
(amphipathic structure) and possess the highest ꢀ-sheet-ꢀ-turn
(ꢀ-structure) content (Figure 1). On the other hand, GS12 and
GS16 have evenly distributed charged residues on both faces
of the molecule (4 and 6 charges in total, with 2 and 3 charges
on each side, respectively) (nonamphipathic structure) and
possess the lowest ꢀ-structure content (Figure 1). Odd-numbered
cyclic peptides were derived from these four peptides by either
addition or deletion of single basic (Lys) or hydrophobic (Leu
* To whom the correspondence should be addressed. Phone: +1 (519)
884-0710 (extension 2284). Fax: +1 (519) 746-0677. E-mail: mjelokhani@
wlu.ca.
†
Wilfrid Laurier University.
University of Alberta.
Present Address: Caprion Pharmaceuticals Inc., 7150 Alexander-
‡
|
Fleming, St. Laurent, Quebec H4S 2C8, Canada.
§
University of Colorado Denver, School of Medicine.
a Abbreviations: Boc, tert-butyloxycarbonyl; BOP, benzotriazole-1-yloxy-
tris-(dimethylamino)phosphonium hexafluorophosphate; CD, circular dichro-
ism; DIEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; GS,
gramicidin S; HBTU, N-[(1H-benzotriazol-1yl)(dimethylamino)methylene]-
N-methylmethanaminium hexafluorophosphate N-oxide; HOBt, 1-hydroxy-
benzotriazole; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG,
phosphatidylglycerol; Tris, tris(hydroxymethyl)aminomethane hydrochloride.
10.1021/jm801648n CCC: $40.75
2009 American Chemical Society
Published on Web 03/11/2009

Ring Size and ActiVity of Cyclic Cationic Peptides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2091
Table 1. Peptide Sequences,a Molecular Mass,b Retention Time,c Relative Hydrophobicity,d and Hemolytic Activitye
peptide (no of AA) Ch/Hypf
sequence
MW (Da), c /m RT (min) [∆GWO
]
[∆GWIF
]
[∆∆GIFO
]
hemolytic activity (%)
GS (10)
GS10 (10)
GS12 (12)
2/4
2/4
4/4
3/4
3/4
4/3
4/6
4/5
3/6
4/5
6/6
5/6
6/5
5/6
cyclo-(VOLFP5VOLFP10)
1140.7/1140.8
1200.7/1200.8
1456.9/1457.2
1328.8/1330.0
1328.8/1328.9
1343.8/1345.0
1669.1/1670.3
1570.0/1570.3
1541.0/1540.3
1556.0/1556.2
33.5
30.3
22.9
24.4
25.7
19.1
30.9
25.8
28.7
26.1
22.8
25.9
21.6
22.7
-0.096 -0.036 -0.060
100 (1)
100 (1)
cyclo-(VKLYP5VKLYP10)
0.104
0.55
0.35
0.35
0.72
0.35
0.41
0.16
0.48
0.66
0.52
0.73
0.52
0.002
0.17
0.092
0.092
0.23
0.11
0.11
0.04
0.16
0.22
0.17
0.23
0.17
0.102
0.39
0.26
0.26
0.48
0.24
0.30
0.12
0.32
0.44
0.35
0.50
0.35
cyclo-(VKLKYP6KVKLYP12)
cyclo-(VKLKYP6*VKLYP11)
cyclo-(VKL*YP5KVKLYP11)
cyclo-(VK*KYP5KVKLYP11)
cyclo-(VKLKVYP7LKVKLYP14)
cyclo-(VKLK*YP6LKVKLYP13)
cyclo-(VKL*VYP6LKVKLYP13)
cyclo-(VK*KVYP6LKVKLYP14)
5.1 (19.6)
4.9 (20.4)
8.6 (11.4)
3.1 (32.2)
100 (1)
20.2 (4.9)
78.2 (1.3)
15.7 (6.4)
12.7 (7.9)
82 (1.2)
GS12-K7 (11)
GS12-K4 (11)
GS12-L3 (11)
GS14 (14)
GS14-V5 (13)
GS14-K4 (13)
GS14-L3 (13)
GS16 (16)
cyclo-(VKLKVKYP8KLKVKLYP16) 1925.3/1925.6
cyclo-(VKLKV*YP7KLKVKLYP15) 1797.2/1797.5
cyclo-(VKLK*KYP7KLKVKLYP15) 1826.2/1826.5
cyclo-(VKL*VKYP7KLKVKLYP15) 1797.2/1797.5
GS16-K6 (15)
GS16-V5 (15)
GS16-K4 (15)
7.3 (13.7)
9.6 (10.4)
a O represents ornithine. Underlined amino acids (AAs) represent the D-enantiomers. The “*” symbol shows the missing amino acids compared with the
parent peptide in bold letters. b Calculated (c) and measured (m) molecular masses (MW) of peptides, using electrospray mass spectrometry. Molecular
masses were measured as [M + H]+. c Retention times (RT) on the RP-HPLC Zorbax 300 SD C8 (150 mm × 2.1 mm i.d., 5 µm particle size, 300 Å pore
size, Rockland Technologies, Wilmington, DE) column. Solvents A (water + 0.05%TFA) and B (acetonitrile + 0.05%TFA) were used at a flow rate of 0.2
mL/min with a gradient from 100% A f 100% B in 40 min. d Relative free energy of transfer of the peptides from water to octanol (∆GWO) and water to
POPC bilayer interface (∆GWIF), and the difference between the two (∆∆GIFO) were calculated on the basis of whole-residue hydrophobicity scales.26 The
free energy of transfer of peptide bonds to octanol and the effect of the cyclic peptide backbone structure were not considered in these calculations. “W”
stands for water; “O” stands for octanol; “IF” stands for interfacial; “IFO” stands for interfacial to octanol. All reported values in brackets are total free
energy of transfer per residue. Free energies are in kcal/(mol ·nr), where “nr” is the “number of residues” in peptides. e Percentage of the hemolysis of human
red blood cells at 100 µg/mL peptide concentration in 24 h at 37 °C. Numbers in parentheses represent the fold reduction (improvement) of peptide hemolytic
activity relative to 100% hemolysis (1 in parentheses). These numbers are calculated as 100/% hemolytic activity. f “Ch/Hyp” is the ratio of the number of
charged residues (Ch) (Lys or Orn) to the number of hydrophobic residues (Hyp) (Leu and Val) in peptides.
40 °C. Peptides were purified by RP-HPLC and their molecular
masses were determined by electrospray mass spectrometry (Table
1). All peptides were of g95% purity as verified by analytical RP-
HPLC. Concentrations of the peptides in aqueous stock solutions
were determined by amino acid analysis ((5% error). Phospho-
lipids, 1-palmitoyl-2-oleoyol-sn-glycero-3-phosphocholine (POPC),
1-palmitoyl-2-oleoyol-sn-glycero-3-phosphoethanolamine (POPE),
and 1-palmitoyl-2-oleoyol-sn-glycero-3-[phosphor-rac-1-glycerol]
(sodium salt) (POPG) were from Avanti Polar (Alabaster, AL). All
other chemicals were of reagent grade and used as received without
further purification.
Preparation and Characteristics of Large Unilamellar
Vesicles. Large unilamellar vesicles (LUVs) were prepared as
reported previously.6,18,19 In brief, chloroform solutions of the
lipids, POPC, and POPE/POPG (7/3 molar ratio) were dried under
a mild nitrogen flush to form a thin film, which was further dried
overnight under reduced pressure. The lipid film was then hydrated
with a buffer solution containing 10 mM Tris, 150 mM NaF, and
0.1 mM EDTA at pH 7.4. Obtained multilamellar vesicle disper-
sions were then freeze-thawed a few times and extruded through
a 100 nm filter in a LiposoFast apparatus (Avestin Inc., Ottawa,
Canada).21 Prepared unilamellar vesicles (average diameter of ∼80
Figure 1. Molecular models for even-numbered ring cyclic cationic
nm) were stable in the dark at room temperature and for several
peptides. GS10 and GS14 are ꢀ-sheet-ꢀ-turn amphipathic peptides,
days at 4 °C. Lipid concentrations were measured as described
and GS12 and GS16 are nonamphipathic peptides with unordered
previously.6
disrupted ꢀ-sheet-ꢀ-turn structures.
Two phospholipid vesicle systems were utilized for this study.
The first system consisted of POPC vesicles that can be considered
as a model for mammalian red blood cell membranes. The second
system was composed of POPE and POPG phospholipids ([PE]/
[PG]) 7/3) to model the negatively charged bacterial membranes.
These two lipid bilayer systems were in the liquid crystalline state,
which is compatible with biological membranes.
Instrumentation. Peptides were analyzed on a Hewlett-Packard
1100 chromatograph (Agilent Technologies, Santa Clara, CA) at
70 °C. Retention times were measured on a RP-HPLC Zorbax 300
SD C8 (150 mm × 2.1 mm i.d., 5 µm particle size, 300 Å pore
size, Rockland Technologies, Wilmington, DE) column. Solvents
A (water + 0.05% TFA) and B (acetonitrile + 0.05% TFA) were
used at a flow rate of 0.2 mL/min with a gradient from 100% A f
100% B in 40 min.
or Val) amino acids and had a variety of conformations, positive
charges, and amphipathicities.
Experimental Section
Materials. Gramicidin S was purchased from Sigma (St. Louis,
MO) and further purified by reversed-phase high performance liquid
chromatography (RP-HPLC). As previously described, all other
peptides were synthesized by solid-phase procedures using Boc
chemistry and Boc-Pro-PAM resin to initiate the synthesis.5,6
Briefly, HBTU-HOBT coupling reagents were used to activate the
C-terminus of the amino acids. Lys side chains were formyl-
protected throughout the syntheses. Linear peptides were cleaved
from the resin and purified by RP-HPLC. Purified linear peptides
were then cyclized in a head-to-tail manner (Pro at the C-terminus)
using BOP, HOBt, and DIEA in DMF. Finally, Lys residues of
the cyclized peptides were deprotected in dilute methanolic HCl at
Far-UV CD spectra were obtained on a Jasco 720 spectropola-
rimeter (Tokyo, Japan). Ellipticities are reported as mean residue
ellipticity, [θ]MRE. Spectra were measured in buffer solutions and

2092 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Jelokhani-Niaraki et al.
phospholipid vesicle dispersions in buffer. The buffer solution was
composed of Tris (10 mM), NaF (150 mM), and EDTA (0.1 mM)
at pH 7.4. All measurements were in quartz cells with 0.1 cm path
length at 25 °C. Sodium fluoride was used in buffer to reduce the
high noise-to-signal levels of chloride ions at wavelengths below
200 nm.
Molecular modeling of peptides was based on the global
minimization of peptide structures, using the Insight II software
(Accelrys, San Diego, CA) on a Silicon Graphics workstation.5,6
Antimicrobial and Hemolytic Activity Assays. Microbial
strains, both bacteria and fungi, were prepared as described
previously.5-7 Briefly, minimal inhibitory concentrations (MICs)
were measured using a standard microtiter dilution method in Luria
broth no salt medium utilizing the same bacterial strains. MICs
were determined as the lowest peptide concentration that inhibited
growth after 24 h at 37 °C.
Hemolytic activity of peptides was measured in saline in the
presence of human erythrocytes as described5-7 and is reported as
the percentage of hemolysis at 100 µg/mL peptide concentrations
in 24 h at 37 °C.
Results
For a systematic evaluation of the relation between structure
and biological activity, the 10- to 16-meric cyclic peptides
exhibited in Table 1 are divided into three groups on the basis
of their ring size and amino acid composition (number of
charged and hydrophobic residues). The first group includes
GS10, GS12, and its three 11-meric analogues (GS {10-12}
group). GS is also included in this group for comparison. The
second group includes GS14 and its three 13-meric analogues
(GS {13,14} group). The third group is composed of GS16 and
its three 15-meric analogues (GS {15,16} group). Structures of
the peptides with even-numbered rings are shown in Figure 1.
These structures are based on an NMR study of the periodicity
of the ꢀ-sheet content of cyclic gramicidin S analogues.22
Stereochemical considerations23 and experimental results22 both
confirm that cyclic peptides with 2(2n + 1) residues (n ) 1, 2,
3,...) (GS, GS10, and GS14 in Table 1 and Figure 1) can form
amphipathic ꢀ-sheet ring structures, with hydrophobic residues
(Val, Leu) on one face and hydrophilic residues (Lys or Orn)
on the other face of the ring. On the other hand, cyclic peptides
composed of 4n residues (n ) 2, 3,...) (GS12 and GS16 in table
and Figure 1) can only form unordered nonamphipathic
conformations (distorted ꢀ-sheets), with a distribution of
hydrophobic and hydrophilic residues on both faces of the ring.
Odd-numbered cyclic peptides of Table 1 (GS11, GS13, and
GS15 peptides) contain intermediate conformations, between
ꢀ-sheet and unordered, with different degrees of ꢀ-structure
(ꢀ-sheet-ꢀ-turn) content and amphipathicity. A detailed study
of structure-function relationships considering the ꢀ-structure
content, amphipathicity, and biological activity in the 10- to
16-meric cyclic peptides will be described in the following
experimental results.
Biological Activities and Conformation of GS {10-12}
Group. Peptides of this group, their retention times (RTs) and
their charged-to-hydrophobic residue ratios (ch/hyp) are listed
in Table 1. Odd-numbered ring 11-meric peptides of this group
were generated by deleting charged (GS12-K4 and GS12-K7)
or hydrophobic (GS12-L3) residues from GS12.
The direct relation between the hemolytic activity and RT
for GS {10-12} peptides is exhibited in Figure 2A and Table
1. GS data are also shown for comparison. Retention times
represent the degree of interaction of the hydrophobic surface
of the peptides and the stationary phase of the reversed-phase
HPLC column: the longer the RT, the higher is the affinity of
the peptide for the stationary phase.24 As seen in this figure,
Figure 2. Relation between retention times with hemolytic (A) and
antimicrobial (B) activities of the peptides in the GS {10-12} group.
GS data are shown for comparison.
decrease in RT is accompanied by decrease in hemolytic activity.
The ꢀ-sheet containing peptide GS10 (Figure 1), as well as GS,
had the strongest hemolytic activity and highest RT, and the
less amphipathic or nonamphipathic peptides of this group had
lower retention times and weaker hemolytic activites.
As can be observed in Figure 2B and Tables 2 and 3, there
is also a direct relation between the antibacterial activities of
the peptides in GS12 {10-12} group and their RTs: the higher
the RT, the stronger is the activity (lower MIC values) against
both Gram-positive and Gram-negative bacteria. On average,
activities against Gram-positive bacteria were much higher than
Gram-negative bacteria. Similar to antibacterial activities,
antifungal activities of the peptides of this group decreased with
a decrease in their retention times (Figure 2B and Table 4); an
exception was GS12, which had a lower RT but slightly stronger
antifungal activity compared with GS12-K7.
To investigate possible relations between the biological
activity of peptides and their conformation, CD spectra of the
peptides were measured in three different milieus: buffer, PC
vesicles (erythrocyte model), and PE/PG vesicles (bacterial
membrane model).
CD spectra for the GS12 {10-12} group are exhibited in
Figure 3. Figure 3A shows a ꢀ-sheet-ꢀ-turn spectrum for GS10,
with two negative maxima at 205 and 222 nm. The positive
maximum of the spectra (185-188 nm) is not shown. In
comparison with the GS10 spectrum, a substantial decrease in

Ring Size and ActiVity of Cyclic Cationic Peptides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2093
Table 2. Activity of Peptides against Gram-Positive Bacteriaa
E. faecalis
S. aureus
S. epidermis
S. mitis
S. pneumoniae
S. pyrogenes
C. jeikeium
mean activityb
peptide
GS
GS10
(µg/ mL)
(µg/ mL)
(µg/ mL)
(µg/ mL)
(µg/ mL)
(µg/ mL)
(µg/ mL)
(µg/ mL)
2 (50.0)
2 (50.0)
64 (31.0)
32 (64.0)
8 (145)
256 (12.0)
32 (3.0)
4 (124)
4 (32.0)
8 (80.0)
4 (197)
2 (61.0)
8 (171)
2 (50.0)
2 (50.0)
64 (31.0)
64 (32.0)
16 (73.0)
256 (12.0)
32 (3.0)
4 (124)
4 (32.0)
4 (159)
8 (98.0)
2 (61.0)
64 (21.0)
32 (32.0)
2 (50.0)
2 (50.0)
64 (31.0)
16 (128)
16 (73.0)
256 (12.0)
16 (6.0)
4 (124)
4 (32.0)
2 (318)
2 (394)
2 (61.0)
8 (171)
16 (65.0)
0.5 (200)
1 (100)
2 (980)
2 (1020)
1 (1160)
64 (50.0)
2 (50.0)
0.5 (990)
1 (128)
0.5 (1270)
2 (394)
0.5 (244)
8 (171)
1 (100)
1 (100)
16 (122)
8 (255)
2 (581)
256 (12.0)
4 (25.0)
0.5 (990)
0.5 (256)
0.5 (1270)
1 (787)
0.5 (244)
2 (685)
1 (1040)
0.5 (200)
0.5 (200)
2 (980)
1 (2040)
0.5 (2330)
8 (403)
4 (25.0)
1 (100)
16 (122)
2 (1020)
2 (581)
128 (25.0)
8 (12.0)
2 (248)
4 (32.0)
2 (318)
1 (787)
2 (61.0)
8 (171)
1 (1040)
1.4 (74.0)
1.2 (82.0)
16.0 (122)
7.2 (281)
3.3 (354)
115.9 (28.0)
7.2 (14.0)
1.2 (406)
1.8 (71.0)
1.5 (429)
1.8 (435)
1.1 (110)
5.9 (230)
3.0 (350)
GS12
GS12-K7
GS12-K4
GS12-L3
GS14
GS14-V5
GS14-K4
GS14-L3
GS16
1 (10)
0.125 (3960)
0.5 (256)
0.5 (1270)
0.5 (1570)
0.5 (244)
0.5 (2740)
0.25 (4170)
GS16-K6
GS16-V5
GS16-K4
4 (260)
4 (260)
a Numbers in parentheses are the “therapeutic indices (TIs)”, calculated as 100 × (100/% hemolytic activity)/(antimicrobial activity). Best TI values for
different bacterial strains and the peptide with best overall TI are highlighted. b Geometric mean of MIC values (after 24 h) against Gram-positive bacteria.
Geometric mean for “n” numbers: aˆ ) (∏ni)1ai)1/n ) (a1a2...an)1/n
Table 3. Activity of Peptides against Gram-Negative Bacteriaa
.
A. caloaceticus
(µg/ mL)
E. cloacae
(µg/ mL)
E. coli
(µg/ mL)
K. pneumoniae
(µg/ mL)
P. aeruginosa
(µg/ mL)
S. maltophilia
(µg/ mL)
S. marcescens
(µg/ mL)
mean activityb
(µg/mL)
peptide
GS
GS10
8 (12.0)
4 (25.0)
16 (122)
8 (255)
8 (145)
64 (50.0)
16 (6.0)
2 (248)
4 (32.0)
2 (318)
2 (394)
2 (61.0)
2 (685)
2 (521)
32 (3.0)
64 (2.0)
16 (6.0)
32 (3.0)
64 (31.0)
64 (32.0)
64 (18.0)
256 (13.0)
128 (1.0)
16 (31.0)
64 (2.0)
16 (4.0)
4 (197)
16 (6.0)
32 (3.0)
64 (31.0)
64 (32.0)
32 (36.0)
256 (13.0)
128 (1.0)
8 (62.0)
64 (2.0)
8 (80.0)
4 (197)
32 (3.0)
8 (12.0)
16 (6.0)
256 (0.3)
256 (0.3)
256 (8.0)
256 (8.0)
256 (4.0)
256 (13.0)
256 (0.4)
256 (2.0)
256 (0.5)
256 (2.5)
256 (3.0)
256 (0.5)
256 (5.0)
256 (4.0)
23.8 (4.0)
39.0 (2.0)
128 (1.0)
256 (8.0)
256 (8.0)
256 (5.0)
256 (13.0)
64 (2.0)
GS12
256 (8.0)
256 (8.0)
256 (4.0)
256 (13.0)
256 (0.4)
128 (4.0)
256 (0.5)
128 (5.0)
64 (12.0)
64 (2.0)
128 (15.0)
64 (31.0)
32 (36.0)
256 (13.0)
64 (2.0)
16 (31.0)
32 (4.0)
16 (40.0)
32 (25.0)
4 (30.0)
105.0 (19.0)
86.1 (24.0)
70.7 (16.0)
210.0 (15.0)
95.1 (1.0)
26.3 (19.0)
70.7 (2.0)
29.0 (22.0)
19.5 (40.0)
19.5 (6.0)
GS12-K7
GS12-K4
GS12-L3
GS14
GS14-V5
GS14-K4
GS14-L3
GS16
64 (8.0)
256 (0.5)
128 (5.0)
64 (12.0)
128 (1.0)
128 (11.0)
256 (4.0)
GS16-K6
GS16-V5
GS16-K4
8 (15.0)
32 (43.0)
16 (65.0)
8 (15.0)
8 (171)
8 (130)
256 (5.0)
256 (4.0)
64 (21.0)
8 (130)
43.1 (32.0)
32.0 (32.0)
a Numbers in parentheses are the “therapeutic indices (TIs)”, calculated as 100 × (100/% hemolytic activity)/(antimicrobial activity). Best TI values for
different bacterial strains and the peptide with best overall TI are highlighted. b Geometric mean of MIC values (after 24 h). For the definition of geometric
mean, please see footnote “b” in Table 2.
Table 4. Activity of Peptides against Fungia
C. albicans
C. neoformans
mean activityb
peptide
GS
GS10
(µg/ mL)
(µg/ mL)
(µg/ mL)
8 (12.0)
16 (6.0)
1 (100)
1 (100)
2.8 (35.0)
4.0 (25.0)
45.2 (43.0)
64.0 (32.0)
11.3 (103)
181.0 (18.0)
11.3 (9.0)
5.7 (88.0)
5.7 (23.0)
4.0 (159)
GS12
128 (15.0)
128 (16.0)
64 (18.0)
256 (13.0)
64 (2.0)
32 (15.0)
32 (4.0)
16 (40.0)
64 (12.0)
32 (4.0)
16 (122)
32 (12.0)
2 (581)
128 (25.0)
2 (50.0)
1 (500)
1 (128)
1 (637)
64 (12.0)
1 (122)
64 (21.0)
8 (130)
GS12-K7
GS12-K4
GS12-L3
GS14
GS14-V5
GS14-K4
GS14-L3
GS16
64.0 (12.0)
5.7 (22.0)
64.0 (21.0)
22.6 (46.0)
GS16-K6
GS16-V5
GS16-K4
64 (21.0)
64 (16.0)
a Numbers in parentheses are the “therapeutic indices (TIs)”, calculated
as 100 × (100/% hemolytic activity)/(antimicrobial activity). Best TI values
for different fungal strains and the peptide with best overall TI are
highlighted. b Geometric mean of MIC values (after 24 h). For the definition
of geometric mean, please see the footnote “b” in Table 2.
Figure 3. CD spectra of the GS {10-12} group peptides in buffer
(A), POPC (B), and POPE/POPG (C) vesicles. Relation between
ellipticities sensitive to changes in ꢀ-structure (205, 215, and 225 nm)
are shown.
the negative maxima at 222 nm (45-75%) and blue shift of
the lower wavelength negative maxima are indicative of
conformational change and decrease of the ꢀ-structure
(ꢀ-sheet-ꢀ-turn structure) content of GS12 and 11-meric
peptides in buffer. Negative ellipticities of peptides were
generally enhanced in both zwitterionic PC (Figure 3B) and
negatively charged PE/PG (Figure 3C) lipid systems, which
imply that these positively charged cyclic peptides interact with
lipid membranes with different affinities and change or enhance
their conformation. Detailed mechanistic studies on interaction
of cyclic cationic peptides with lipid membrane systems have
been reported previously.18,19
Ellipticities at three wavelengths (205, 215, and 225 nm) can
indicate the changes in the ꢀ-sheet (212-218 nm) and ꢀ-turn
(205-225 nm) content of the peptide conformations.25 As

2094 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Jelokhani-Niaraki et al.
GS14 (Figure 1) had the most potent hemolytic activity and
highest RT, and the less-amphipathic peptides of this group had
lower RTs and weaker hemolytic activities. GS14-L3 and GS14-
V5 peptides had comparable RTs, hemolytic activities, and ch/
hyp ratios (4/5) (Table 1).
In contrast to the 10- to 12-meric peptides, there is an inverse
relation between the antibacterial activities of the peptides in
GS {13,14} group and their RTs; the higher the RT, the lower
is the activity against both Gram-positive and Gram-negative
bacteria (Tables 2 and 3 and Figure 4B). Similar to their
hemolytic activities, GS14-L3 and GS14-V5 had comparable
antibacterial activities. Antifungal activities of the peptides of
this group also decreased with an increase in their retention times
(Figure 4B and Table 4).
As with the peptides in group GS {10-12}, biological
activities of GS {13,14} peptides were related to their confor-
mation. CD spectra of the peptides in buffer and the two lipid
vesicle systems are exhibited in the Supporting Information
(Figure I). Similar to GS10, GS14 has a typical ꢀ-sheet-ꢀ-
turn conformation.18 Compared with GS14, the CD spectra of
13-meric peptides imply a decrease in the ꢀ-structure content
of these peptides in buffer and lipid vesicles. Enhancement of
negative ellipticities of peptides in both PC and PE/PG lipid
systems indicates interaction of peptides with lipid membranes
as well as their conformational changes (see above).18,19 Similar
to the peptides of the GS {10-12} group, there is a direct
relation between the ꢀ-structure content of the peptides in lipid
vesicles and their RTs and hemolytic activities; the lower the
content of the ꢀ-structure, the lower the RTs and the weaker
the hemolytic activities are. However, antibacterial activities
of the peptides of the GS {13,14} group are inversely related
to their ꢀ-structure content in the PE/PG lipid system; the lower
the ꢀ-structure content of the peptides, the more potent their
antimicrobial activities (Figure 4 and Supporting Information).
Among the peptides of the GS {13,14} group, GS14-L3 and
GS14 had the highest and lowest overall TIs against Gram-
positive and Gram-negative bacteria as well as fungi, respec-
tively (Tables 2-4).
Biological Activities and Conformation of GS {15,16}
Group. Odd-numbered ring 15-meric peptides of this group
were generated by deleting charged (GS16-K4 and GS16-K6)
or hydrophobic (GS16-V5) residues from GS16. The sequences,
RTs, and ch/hyp ratios of the GS {15,16} group are listed in
Table 1.
Similar to GS {10-12} and GS {13,14} peptides, the
hemolytic activities and RT values for GS {15,16} peptides are
directly related (Figure 5A and Table 1). As observed in Figure
5A, a decrease in RTs is accompanied by a decrease in
hemolytic activity. The nonamphipathic peptide of this group,
GS16 (Figure 1), had a weak hemolytic activity and second
highest RT, and the most amphipathic peptide of this group,
GS16-K6, had the highest RTs and strongest hemolytic activities.
Figure 4. Relation between retention times with hemolytic (A) and
antimicrobial (B) activities of the peptides in the GS {13,14} group.
mentioned above, since there is also a direct correspondence
between RTs and the biological activity (hemolytic and anti-
bacterial) of the peptides in the GS {10-12} series, the
ꢀ-structure content in PC (erythrocyte membrane) and PE/PG
(bacterial membrane) lipid systems can be directly related to
the biological activity; the higher is the ꢀ-structure content of
the peptides (higher negative ellipticities), the more potent are
their biological activities. Among the peptides of the GS
{10-12} group, GS10 and GS12-L3 had the highest and lowest
overall biological activities, RT and ꢀ-sheet content, respec-
tively. As observed in Tables 2-4, the therapeutic index (TI)
parameters, calculated as the ratio of hemolytic to antimicrobial
activities, is used to define the degree of dissociation of
hemolytic (toxic, lethal) activity from antimicrobial activities.
In the GS {10-12} group, GS12-K4 had the highest overall TI
against Gram-positive bacteria and fungi; however, despite better
average antibacterial activity against Gram-negative bacteria,
its TI was slightly lower than GS12-K7 (Tables 2-4).
Biological Activities and Conformation of GS {13,14}
Group. Odd-numbered ring 13-meric peptides of this group
were generated by deleting charged (GS14-K4) or hydrophobic
(GS14-L3 and GS14-V5) residues from GS14, and their
sequences, RTs, and ch/hyp ratios are listed in Table 1.
Hemolytic activity and RTs for GS {13,14} peptides are
directly related (Figure 4A and Table 1). As observed in Figure
4A, a decrease in RTs is accompanied by a decrease in
hemolytic activity. The amphipathic ꢀ-sheet containing peptide
Similar to the 10- to 12-meric peptides, and in contrast to
GS {13,14} series, there is a direct relation between the
antibacterial activities of the peptides in the GS {15,16} group
and their RTs; the higher the RT, the higher is the activity
against both Gram-positive and Gram-negative bacteria (Tables
2 and 3 and Figure 5B). Like the two other groups, activities
of the 15- and 16-meric peptides against Gram-positive bacteria
were much higher than Gram-negative bacteria. Antifungal
activities of the peptides of this group also increased with an
increase in their retention times (Figure 5B and Table 4); an
exception was GS16, which had a higher RT but lower
antifungal activity compared with GS16-K4.

Ring Size and ActiVity of Cyclic Cationic Peptides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2095
calculated on the basis of whole-residue hydrophobicity scale
by calculating the sum of the free energy of transfer of individual
amino acids from water to octanol (∆GWO), representing the
lipid bilayer hydrophobic core.26 To compare the hydrophobicity
of peptides with different ring sizes, free energy of transfer
values are shown as [∆GWO] ) ∆GWO per residue. As seen in
this table, peptides with comparable RT values can have
different overall hydrophobicity (for example, GS14-L3, GS16-
K6, GS14-V5, and GS12-K4) and vice versa (for example,
GS12-K4 and GS12-K7). For a relation between the amphipa-
ticity (RTs) and ꢀ-sheet content of all three peptide groups in
buffer and lipid membranes, see Figure III in the Supporting
Information.
Discussion
Systematic disruption of the ꢀ-sheet-ꢀ-turn structure of
amphipathic ꢀ-sheet peptides GS10 and GS14 by single
enantiomeric substitution of the amino acid residues of the
peptide rings (D-enantiomer in place of L-amino acids and
L-enantiomer in place of D-Tyr) resulted in significant changes
in the biological activities of these peptides.5-7,27 These results
showed that by systematic disruption of the ꢀ-structure, the
larger and more flexible rings can adopt a diverse array of
conformations with different degrees of amphipathicity, and this
diversity in conformation and amphipathicity could be the key
factor in design of novel peptides with higher TI and antimi-
crobial specificity. Another method to disrupt the structure of
cyclic ꢀ-sheet peptides and their amphipathicity is systematic
addition and deletion of amino acids in the peptide ring. By
adoption of the latter method in this study, odd- and even-
numbered rings were generated and divided into three groups
on the basis of their ring size: rings with limited conformational
flexibility, GS {10-12} group; rings with moderate conforma-
tional flexibility, GS {13,14}; and conformationally flexible
rings, GS {15,16}. Interestingly, variation in the ring size and
charge of the cationic cyclic peptides resulted in diversity and
specificity of biological function, dissociation between hemolytic
and antimicrobial activities, and improvement in TIs.
Among the peptides of Table 1, less amphipathic or nonam-
phipathic peptides of group GS {10-12}, with disrupted
ꢀ-sheet, had the weakest hemolytic activities. After GS10, GS12-
K4 (+3) (more amphipathic than GS12 and less amphipathic
than GS10 (+2)) had the second best overall biological activity
as well as the best overall TI in this group (Tables 2-4). Since
there is a direct correlation between RTs, ꢀ-structure content,
and hemolytic and average antibacterial activities in this group,
an optimal ꢀ-structure content and amphipathicity can be achieved
for the best dissociation between the hemolytic and antimicrobial
activities (best TI). GS12-K4, the lead peptide of this group,
had an RT of 25.7 min, as well as ∼45% less ꢀ-structure content
and weaker overall antimicrobial activity compared with GS10.
In contrast to the GS {10-12} group, in the GS {13,14}
group amphipathicity was reduced by deleting amino acid
residues from the ꢀ-sheet-ꢀ-turn peptide GS14 (+4). Hemolytic
activities were also reduced with reduction in ꢀ-structure content
but were stronger than 11-meric peptides and GS12. GS14 was
the most hemolytic peptide of this group but had the lowest
TIs. The low antimicrobial activity of GS14 can be related to
its strong interaction with and entrapment in the nonlipid
components of the bacterial cell walls. In this group there is an
inverse correlation between RTs and ꢀ-structure content, and
average antibacterial activities in this group. GS14-L3, with the
highest TI, had an RT of 26.1 min and 55% and 45-50% less
ꢀ-structure content in buffer and the two lipid systems,
respectively, compared with GS14.
Figure 5. Relation between retention times with hemolytic (A), and
antimicrobial (B) activities of the peptides in the GS {15,16} group.
As with the peptides of the two previous groups, biological
activities of GS {15,16} peptides were related to their confor-
mation. CD spectra of the peptides in buffer and the two lipid
vesicle systems are exhibited in the Supporting Information
(Figure II). Compared with GS10 and GS14 (ꢀ-sheet-ꢀ-turn
peptides), all peptides of this group have low ꢀ-structure
contents. The spectra in the PC lipid system were different from
those in PE/PG system, which implies different conformations
and peptide-lipid interactions in this milieu. Overall the CD
spectra in lipid systems, especially PE/PG, indicate that the
cyclic peptides interact with lipid membranes and change their
conformation. As in the cases of GS {10-12} and GS {13,14}
groups, there is a direct relation between the ꢀ-structure content
of the peptides and their RTs and hemolytic activities; the lower
the content of ꢀ-structure in lipid vesicles, the lower the RTs
and the weaker the hemolytic activities. However, the relation
between antimicrobial activity and the ꢀ-structure content in
PE/PG lipid system is not as straightforward. GS16 with the
second best overall antibacterial activity had the lowest ꢀ-struc-
ture content in the GS {15,16} group. In this group of peptides,
GS16 had the highest overall TI against both Gram-positive
and Gram-negative bacteria, whereas GS16-K4 had the highest
TI against fungi (Tables 2-4).
Retention Time, Hydrophobicity, Amphipathicity, and
Conformation. Table 1 exhibits the relation between the
apparent amphipathicity, as measured by RTs, and hydrophobic-
ity of the cyclic peptides. Hydrophobicities of peptides were

2096 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Jelokhani-Niaraki et al.
Table 5. Summary of Therapeutic Index, Mean Antimicrobial Activity,
and Fold Improvement of the Lead Peptides
All peptides of the GS {15,16}group had disrupted ꢀ-sheet
(CD spectra and RT values), and two of these peptides, GS16-
K4 (+5) and GS16-V5 (+6), with comparable conformations
in PC vesicles, had very weak hemolytic activities. GS16-K6
(+5) was the most amphipathic peptide of this group (highest
RT and ꢀ-structure content) and had the strongest hemolytic
and average antimicrobial activities. However, because of its
potent hemolytic activity, this peptide had the lowest TIs of
the GS {15,16} group. GS16 had the best overall TIs of this
group for antibacterial activity, and GS16-K4 had the best TI
for antifungal activity. Similar to the GS {10-12} group, there
is a direct correlation between RTs, ꢀ-structure content, and
hemolytic and average antibacterial activities in the GS
{15,16}group.
GS GS10 GS12-K4 GS14-L3 GS14-V5 GS16 GS16-K4
Microbial Strain: Gram-Positive
TI
74
1.4 1.2
1.1
82
354
3.3
4.8
429
1.5
5.8
406
1.2
5.5
435
1.8
5.9
350
3.0
4.7
activity (µg/mL)a
fold improvementb
Microbial Strain: Gram-Negative
TI
4
2
16
70.7
4
22
29.0
5.5
19
26.3
4.8
40
19.5
10
32
32.0
8
activity (µg/mL)a 23.8 39.0
fold omprovementb
0.5
Microbial Strain: Fungi
25 103 159
11.3 4.0
2.9 4.5
TI
35
2.8 4.0
0.7
88
5.7
2.5
12
64.0
0.3
46
22.6
1.3
activity (µg/mL)a
fold improvementb
a Geometric mean of antimicrobial activity. For the definition of
geometric mean, please see the footnote “b” in Table 2. b Fold improvement
of the TI values of peptide analogues relative to GS. Highlighted parameters
denote the analogues with best improvement in their TI relative to GS.
Average antimicrobial activities of these analogues are comparable to or
better than GS.
Overall, peptides with comparable apparent amphipathicity
(as estimated by RT values), but with different ring size and
positive charge, can have different hemolytic or antimicrobial
activities (for example, GS14-L3, GS16-K6 and GS12-K4 or
GS12 and GS16). On the other hand, peptides with comparable
ring size, charge, and RT values can have comparable biological
activities (for example, GS14-L3 and GS14-V5). Hydrophobicity
of peptides ([∆GWO]), exhibited in Table 1, is correlated to the
affinity of peptides for lipid bilayer cores. Interestingly, with
exception of GS, all [∆GWO] values for the peptides are positive.
Apparently, on the basis of the hydrophobicity of their amino
acid sequences, none of the peptides in the three groups have a
tendency to interact with the lipid bilayer hydrophobic core
(negative ∆GWO) or bilayer interface (negative ∆GWIF). How-
ever, the whole-residue hydrophobicity scale does not consider
the structure of peptides. By consideration of the overall
topology, amphipaticity, and hydrophobic surfaces of the
peptides and on the basis of experimental evidence,17-20 cyclic
peptides in the three groups interact with the lipid membrane
surface with different affinities. In a series of isothermal titration
calorimetry (ITC) experiments, the binding affinity of some of
the cyclic peptides (GS10, GS12, GS14, GS14-L3, GS16, and
GS16-K4) for PC and PE/PG systems was measured (ref 19
and unpublished results by L.Wheaton and M. Jelokhani-
Niaraki). On the basis of these measurements, with the exception
of GS14, interactions of peptides with PC vesicles were weak,
endothermic, and entropy-driven. In contrast to all other
peptides, GS14 had an exceptionally strong and exothermic
binding to PC vesicles. Compared with PC vesicles, all peptides
(including GS14) had endothermic binding to PE/PG vesicles.
affinity for lipid membranes are important factors for improving
the TIs while preserving the antimicrobial potency. As exhibited
in Table 5, for average activity against the Gram-positive
bacteria, GS16 and GS14-L3 were as potent as GS and had the
best TI (∼6-fold improvement compared with GS). However,
compared with GS16, GS14-L3 was more specific (higher TI)
for some strains such as S. aureus, S. mitis, and S. pneumoniae
and GS16-K4 was more specific against E. faecalis, S. pyro-
genes, and C. jeikeium (Table 2). Also, both GS14-V5 and
GS12-K4 had good average activities and were specific against
certain bacteria (Tables 2 and 5). Activity against Gram-negative
bacteria was generally weaker than activity against Gram-
positive bacteria. Peptides were practically inactive against S.
marcescens and only weakly active against E. cloacae or P.
aeruginosa (Table 3). GS16 also had the best TI (10-fold
improvement and stronger activity, compared with GS) for
average activity against the Gram-negative bacteria (Table 5)
and was the most specific toward most strains (Table 3). GS16-
V5 and GS16-K4 were more specific against A. caloaceticus
and S. maltophilia, respectively (Table 3). For average activity
against fungi, GS14-L3 had the best TI (4.5-fold improvement
compared with GS) and specificity against the tested strains,
followed by GS12-K4 and GS14-V5 (Tables 4 and 5). In
addition to physicochemical properties of the antimicrobial
peptides, the specificity of peptide activity against different
strains of bacteria could be related to the composition and
structure of different bacterial cell walls and membranes.
Mechanism of interaction of the cyclic cationic peptides with
animal and bacterial cytoplasmic cell membranes has been the
subject of our previous studies.18,19 It is noteworthy to mention
that we have also examined the biological activities and
conformations of linear forms of the even-numbered peptides
(unpublished results of M. Jelokhani-Niaraki and L. H. Konde-
jewski). Compared to their cyclic versions, these linear peptides
had weak biological activities and little ordered secondary
structures; moreover, their conformations were dependent on
the amino acids at their N- and C-termini.
In this study, biophysical properties and biological activities
of the cyclic peptides show a clear relation between their
structure and function. Disruption of the ꢀ-structure in 11-, 13-,
15-, and 16-meric peptides was the decisive step taken in
reducing the hemolytic activity (as much as 32-fold) and
amphipathicity and increasing the selectivity (increase in TI).
Consequently, by systematic modulation of ring size, ꢀ-structure
content, amphipathicity, and affinity for microbial membrane
surfaces, we were able to find lead peptides with significantly
improved TIs compared with GS and its decameric analogue
GS10. These peptides are highlighted in Tables 2-5. In each
of the three groups, peptide conformation and its affinity for
model lipid membranes of erythrocytes and bacteria were closely
related to its biological activity. This finding implies that,
regardless of other possible additional mechanisms for the
biological function of antimicrobial peptides, interaction with
mammalian and bacterial cell membranes in these series of
peptides is an essential component of their biological activity.
From the results of this study it is also clear that more flexible
and larger rings, higher number of positive charges, and lower
Conclusion
Among the peptides in three groups, the 16-meric peptide
GS16, with the largest ring, most flexible and nonamphipathic
structure, and highest number of positive charges, was the lead
compound with best TI for average activity against both Gram-
positive and Gram-negative bacteria (compared with GS, 5.9-

Ring Size and ActiVity of Cyclic Cationic Peptides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2097
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and 10-fold increase, respectively). This peptide also had low
affinity to interact with PC vesicles but had a higher tendency
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With their low toxicity against erythrocytes (6-8 times less
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for optimizing their therapeutic function,7 GS16 and GS14-L3
can therefore serve as promising candidates for developing novel
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Acknowledgment. We thank Marc Genest for peptide
synthesis and purification and amino acid analysis. This study
was supported by grants from the Canada Foundation for
Innovation (CFI, Grant 6786) and the Natural Sciences and
Engineering Research Council of Canada (NSERC, Grant
250119) to M.J.-N. and from the Protein Engineering Network
of Centres of Excellence (PENCE) and National Institutes of
Health (Grant NIHR01-AI-067296) to R.S.H.
Supporting Information Available: CD spectra of GS {13,14}
and GS {15,16} group peptides and relation between retention times
and ꢀ-sheet content of peptides. This material is available free of
charge via the Internet at http://pubs.acs.org.
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