Dissociation of Antimicrobial and Hemolytic Activities in
Cyclic Peptide Diastereomers by Systematic Alterations in
Amphipathicity*
Leslie H.
Kondejewski
,
Masood
Jelokhani-Niaraki
,
Susan W.
Farmer§,
Bruce
Lix
,
Cyril M.
Kay
¶,
Brian D.
Sykes
¶,
Robert E. W.
Hancock§, and
Robert S.
Hodges
¶
From the
Protein Engineering Network of Centres of
Excellence and ¶ Department of Biochemistry, University of
Alberta, Edmonton, Alberta T6G 2S2, Canada and the
§ Department of Microbiology and the Canadian Bacterial
Diseases Network, University of British Columbia,
Vancouver, British Columbia V6T 1Z3, Canada
 |
ABSTRACT |
We have investigated the role of
amphipathicity in a homologous series of head-to-tail cyclic
antimicrobial peptides in efforts to delineate features resulting in
high antimicrobial activity coupled with low hemolytic activity
(i.e. a high therapeutic index). The peptide GS14,
cyclo(VKLKVd-YPLKVKLd-YP), designed on the
basis of gramicidin S (GS), exists in a preformed highly amphipathic
-sheet conformation and was used as the base compound for this study. Fourteen diastereomers of GS14 were synthesized; each contained a different single enantiomeric substitution within the framework of
GS14. The
-sheet structure of all GS14 diastereomers was disrupted as determined by CD and NMR spectroscopy under aqueous conditions; however, all diastereomers exhibited differential structure
inducibility in hydrophobic environments. Because the diastereomers all
have the same composition, sequence, and intrinsic hydrophobicity, the
amphipathicity of the diastereomers could be ranked based upon
retention time from reversed-phase high performance liquid chromatography. There was a clear correlation showing that high amphipathicity resulted in high hemolytic activity and low
antimicrobial activity in the diastereomers. The latter may be the
result of increased affinity of highly amphipathic peptides to outer
membrane components of Gram-negative microorganisms. The diastereomers possessing the most favorable therapeutic indices possessed some of the
lowest amphipathicities, although there was a threshold value below
which antimicrobial activity decreased. The best diastereomer exhibited
130-fold less hemolytic activity compared with GS14, as well as greatly
increased antimicrobial activities, resulting in improvement in
therapeutic indices of between 1,000- and 10,000-fold for a number of
microorganisms. The therapeutic indices of this peptide were between
16- and 32-fold greater than GS for Gram-negative microorganisms and
represents a significant improvement in specificity over GS. Our
findings show that a highly amphipathic nature is not desirable in the
design of constrained cyclic antimicrobial peptides and that an optimum
amphipathicity can be defined by systematic enantiomeric substitutions.
 |
INTRODUCTION |
The ever-increasing development of bacterial resistance to
traditional antibiotics has reached alarming levels, making it essential that new antibiotics be developed (1). Ideally, these new
antibiotics should possess both novel modes of action as well as
different cellular targets compared with existing antibiotics to
decrease the likelihood of development of cross-resistance. Antimicrobial peptides may represent such a new class of antibiotics, and their design and structure-activity relationships have become an
area of active research in recent years (see Refs. 2 and 3 and
references therein). Although their exact mode of action has not been
established, it has been proposed that the cytoplasmic membrane is the
main target of these peptides, where their accumulation results in
increased permeability and loss of barrier function. The development of
resistance to these membrane active peptides is not expected because
this would require substantial changes in the lipid composition of cell
membranes. Indeed, the induction of resistance to such peptides has not
been seen for a number of the antimicrobial peptides (2). Because both
their mode of action and cellular targets are different from those of
the traditional antibiotics, antimicrobial peptides represent a truly new class of antibiotics and are therefore attractive candidates for
development as such.
Two major classes of the cationic antimicrobial peptides are the
-helical and the
-sheet peptides. The
-helical class (for example cecropins, magainins, and melittin) are linear peptides that
exist as disordered structures in aqueous media and become helical upon
interaction with hydrophobic solvents or phospholipid vesicles. Unlike
the
-helical peptides,
-sheet peptides are cyclic peptides
constrained in this conformation either by disulfide bonds
(tachyplesins, protegrins, and polyphemeusins) or by cyclization of the
backbone (gramicidin S and tyrocidines). Although the
-sheet conformations of these peptides may be further stabilized in the presence of a hydrophobic or lipid environment, they exist largely in a
"preformed"
-sheet conformation in aqueous environments due to
their structural constraints. From numerous structure-activity studies
on both natural and synthetic antimicrobial peptides, a number of
factors believed to be important for antimicrobial activitiy have been
identified. These include the presence of both hydrophobic and basic
residues, as well as a defined secondary structure (
-helical or
-sheet), either preformed or inducible, and an amphipathic nature
that segregates basic and hydrophobic residues to opposite sides of the
molecule in lipid or lipid-mimicking environments (2-5).
Many of the antimicrobial peptides show poor selectivity for bacteria
in that they are also toxic to higher eukaryotic cells. To make the
antimicrobial peptides useful as therapeutics therefore requires
delineation of the features responsible for antimicrobial activity from
those responsible for toxicity to higher eukaryotic cells (typically
measured as hemolytic activity). The obvious goal is to design peptides
that have high antimicrobial activity coupled with low toxicity,
i.e. a high specificity or high therapeutic index. Recent
studies on a number of linear peptides have attempted to delineate
features responsible for these activities and found that high
amphipathicity (6, 7), high hydrophobicity (6, 8), as well as high
helicity (9, 10) were correlated with increased hemolytic activity.
Antimicrobial activity on the other hand was found to be less dependent
on peptide helicity (9, 10). Furthermore, decreases in either
hydrophobicity or amphipathicity were either found to increase (7, 8)
or to decrease antimicrobial activity (6, 11), depending on the
peptides studied. In both cases, however, specificity for bacteria over
erythrocytes could be increased either by increasing activity coupled
with decreased hemolysis (7, 8) or because the hemolytic activity was
decreased more readily than antimicrobial activity (6, 11).
Relatively few studies have investigated structural features
responsible for the hemolytic and antimicrobial properties of the
cyclic
-sheet peptides. The fact that these peptides are constrained
and therefore have less conformational freedom compared with the linear
-helical peptides suggests that the properties of these peptides may
be different. We have utilized the 10 residue head-to-tail cyclic
peptide gramicidin S (GS)1
(12) as the basis of our design for novel antimicrobial agents. GS has
the sequence cyclo(Val-Orn-Leu-d-Phe-Pro)2 and
exists in an antiparallel
-sheet conformation with the strands fixed
in place by two type II'
-turns (5, 13, 14). The
-sheet structure
gives the molecule a preformed amphipathic nature with four hydrophobic
residues (Val and Leu) making up one face of the molecule and two basic
Orn residues making up the other face. This amphipathicity, along with
high hydrophobicity, has long been thought to be important for the
antimicrobial properties of GS-like peptides (5, 15-18). A previous
study with cyclic
-sheet antimicrobial peptides based on GS
indicated that it is possible to dissociate antimicrobial and hemolytic
activities through gross manipulation of
-sheet structure and
amphipathicity (19). In this manuscript, we report on the effect of
small incremental changes in amphipathicity (directed hydrophobicity
and positive charge) on the antimicrobial and hemolytic properties of
cyclic 14 residue peptides. We have utilized the cyclic
tetradecapeptide, GS14,
cyclo(VKLKVd-YPLKVKLd-YP), as the model peptide
in this study. This peptide has been shown to exist in a highly
amphipathic
-sheet structure, with six hydrophobic residues on one
face of the molecule and four basic residues on the opposite face (19, 20). Unlike GS, which exhibits broad spectrum antimicrobial activity as
well as hemolytic activity, GS14 was found to possess limited
antimicrobial activity and very high hemolytic activity (19). Because
amphipathicity is intimately linked to
-sheet structure in GS14, any
change in
-sheet structure was predicted to directly affect the
amphipathicity of the molecule. We have created a series of GS14
diastereomers in which each contains a different single residue
enantiomeric substitution within the framework of GS14 resulting in a
series of cyclic peptides possessing gradated disruption of
-sheet
structure and amphipathicity. The present method of enantiomeric
substitutions within a constrained backbone system has the advantage
that all peptides retain the same sequence, intrinsic hydrophobicity,
and basicity but differ only in structure. We show that the
amphipathicity of these peptides has a large effect on their biological
properties and that by defining the optimum amphipathicity in the
framework of these cyclic peptides, the balance between hemolytic and
antimicrobial activities can be optimized.
 |
MATERIALS AND METHODS |
Peptide Synthesis, Purification, and Cyclization--
All
peptides were synthesized by solid phase peptide synthesis using
standard t-butyloxycarbonyl chemistry, cleaved from the resin, and purified by preparative RP-HPLC as reported previously (19).
For all peptides proline was the C terminus because racemization can
occur during the cyclization reaction with other
residues.2 Purity of linear
peptides was verified by analytical RP-HPLC, and correct peptide masses
were verified by electrospray mass spectrometry on a Fisons VG Quattro
triple quadrupole mass spectrometer (Manchester, UK). Pure linear side
chain protected peptides were cyclized, deprotected, and purified by
preparative RP-HPLC as described (19). Purified cyclic peptides were
homogeneous by analytical RP-HPLC and gave correct primary ion
molecular weights by mass spectrometry as well as appropriate amino
acid analysis ratios. Peptide concentrations of stock solutions were
determined by amino acid analysis for subsequent use in biological assays.
Analytical Reversed-phase Analysis of
Diastereomers--
Peptides were analyzed by RP-HPLC on a Zorbax SB-C8
column (150 × 2.1-mm inner diameter, 5 µm particle size, 300 Å pore size; Rockland Technologies, Wilmington, DE) using a Hewlett
Packard 1100 chromatograph at 70 °C with a linear AB gradient of 1%
B/min (where solvent A was 0.5% aqueous trifluoroacetic acid and
solvent B was 0.5% trifluoroacetic acid in acetonitrile) at a flow
rate of 0.25 ml/min.
Circular Dichroism Measurements--
CD spectra were recorded on
a Jasco J-500C spectropolarimeter (Jasco, Easton, MD) as described
(19). Spectra were recorded in either 5 mM sodium acetate
buffer, pH 5.5, or 5 mM sodium acetate buffer, pH 5.5, containing 50% trifluoroethanol (TFE).
NMR Spectroscopy--
NMR spectroscopy was carried out under
aqueous conditions on a Varian Unity 300 MHz spectrometer equipped with
a 5-mm inverse detection probe. Each peptide was dissolved in 500 µl
of 90% H2O/10% D2O (or 100% D2O)
giving a sample conentration of 1-2 mM, and the pH was
adjusted to 5.5. 1H double quantum filtered two-dimensional
correlated spectroscopy, rotating frame Overhauser effect spectroscopy,
and total correlation spectroscopy spectra were collected at 25 °C
and processed as described (21). The chemical shift index was
calculated for selected peptides as described by Wishart et
al. (22).
Molecular Modelling--
A model of GS14 was constructed using
Insight II (Biosym Technologies Inc., San Diego, CA) on a Silicon
Graphics workstation starting with the linear peptide
LKVd-YPLKVKLd-YPVK. The model was constructed by
specifying standard antiparallel
-sheet
,
values of
139 ° and +135 °, respectively (23). Two type II'
-turns
were incorporated into the model designating D-Tyr and Pro
residues as residues i + 1 and i + 2 of the turns. Dihedral angles
(
,
) used for the turns were 60 °,
120 ° and
80 °, 0 ° for i + 1 and i + 2 residues, respectively (23). The formation of the turns brought the N and C termini into close proximity, and an
amide bond was formed between the termini. The model was subjected to
energy minimization using the consistent valence force field (24) with
a distance-dependent dielectric constant of 4 at pH 7 with
no cross-terms. The potential energy of the model was minimized in two
steps, first using the steepest descent algorithm for 100 iterations
followed by 100 iterations using the VA09A algorithm.
Calculation of Peptide Hydrophobic Moment and
Hydrophobicity--
The mean residue hydrophobic moment (µ) of GS14
and a comparable linear
-helical peptide (7) was calculated using
the consensus hydrophobicity scale of Eisenberg et al. (25)
to facilitate comparison with other antimicrobial peptides. Due to the
cyclic nature of GS14, calculation of µ was carried out for each half of the molecule, and the value reported represented the average of
these values. This procedure was necessary to account for the two
-turns in the molecule and appears to be a reasonable assumption based on the molecular model. The two segments used for the
calculations were: VKLKVYP and LKVKLYP; a value of
= 180 ° was
used for the angle at which successive side chains emerge from the
backbone of the
-sheet.
Measurement of Antibacterial, Antifungal, and Hemolytic
Activity--
Minimal inhibitory concentrations (MICs) were measured
using a standard microtitre dilution method in Luria Broth no salt medium utilizing the same bacterial strains as reported (19). MICs were
determined as the lowest peptide concentration that inhibited growth
after 24 h at 37 °C. The hemolytic activity of peptides was
measured in saline utilizing human erythrocytes as described (16, 19).
The concentration of peptide required for complete hemolysis was
determined visually after 24 h at 37 °C.
Measurement of Peptide Outer Membrane
Interactions--
Dansyl-polymyxin B displacement from
Pseudomonas aeruginosa lipopolysaccharide (LPS) was measured
to determine the binding affinity of the peptides to LPS (19).
Permeabilization of bacterial outer membranes was measured by
monitoring peptide mediated 1-N-phenylnaphthalamine (NPN)
fluorescence increases utilizing Escherichia coli UB1005 cells (16, 19).
 |
RESULTS |
Design of Cyclic Peptides
In this study we systematically replaced each residue in the
sequence of the highly amphipathic
-sheet peptide, GS14, with its
enantiomer (Table I). The rationale for
these substitutions was based on the following observations:
(a) In a previous study (19) we found that with certain
cyclic peptides the lack of
-sheet structure and amphipathicity
under aqueous conditions was the key to achieving a high specificity
for microbes over human erythrocytes (i.e. a high
therapeutic index). In that study, the disruption of
-sheet
structure and amphipathicity was accomplished by utilizing peptides
that could not form
-sheet structures due to the number of residues
in the ring (19, 20). (b) During the initial synthesis and
cyclization of GS14 we found that racemization of the C terminus could
occur during the cyclization reaction when the C terminus was a non-Pro
residue and that this racemization led to loss of
-sheet
structure.2 Together, these observations led us to
hypothesize that GS14 could be transformed into a peptide possessing a
high therapeutic index by disrupting the
-sheet character, and hence
the amphipathicity, of the molecule. To accomplish this, we synthesized
all 14 possible diastereomers of GS14; each contains a different single
amino acid enantiomeric substitution. We also synthesized two
diastereomers with two and four enantiomeric substitutions,
respectively (Table I). The peptides were characterized with respect to
their structural and biological properties.
Structure of GS14 Diastereomers
Circular Dichroism Spectroscopy--
The CD spectra of GS14 and
representative single residue substitution diastereomers under aqueous
conditions are shown in Fig.
1A. It has been shown by NMR
spectroscopy that both GS and GS14 possess a similar
-sheet
conformation and that they also exhibit similar CD spectra with large
negative ellipticities at 206 and 223 nm (19, 20), reminiscent of a
combination of
-sheet structure and type II
-turns (26). All of
the single replacement diastereomers as well as those containing two or
four enantiomeric substitutions exhibited CD spectra more typical of
disordered structures, indicating that enantiomeric substitutions
within the framework of GS14 resulted in the disruption of
-sheet
structure. The CD spectra of GS14 and representative diastereomers
recorded in the presence of the lipid-mimicking solvent, TFE, are shown in Fig. 1B. TFE is generally thought of as a helix-inducing
solvent (see Ref. 27 and references therein); however, recent studies have shown that aqueous solutions containing TFE can also stabilize
-hairpin and
-turn structures (28, 29). In 50% TFE the molar ellipticities around 206 and 223 nm were significantly enhanced for
GS14 as well as all the diastereomers, suggesting an enhancement or
stabilization of
-sheet structure in the peptides. However, because
the shape of the observed CD spectra of these cyclic peptides is a
combination of contributions by
-turns, aromatic residues, and
-sheet structure, it is difficult to assign particular secondary structural elements to these spectra (26). One striking difference in
the CD spectra of all diastereomers in the presence of TFE was
displayed by GS14K4 (and GS14K11; not shown), with a large blue-shifted
negative ellipticity in the vicinity of 200 nm when compared with all
other diastereomers (Fig. 1B). This large increase in molar
ellipticity at 205 nm for GS14K4 and GS14K11 is also evident from Fig.
2, where molar ellipticity changes in the
different environments at both 205 and 220 nm are summarized
graphically. The majority of peptides exhibited greater negative molar
ellipticities at both these wavelengths in the presence of TFE. It is
noteworthy, however, that although all diastereomers exhibited
essentially identical spectra under aqueous conditions, all displayed
considerably different CD spectra in TFE, indicating the induction of
different backbone conformations in a hydrophobic environment,
depending on the position of the enantiomeric substitution. The
diastereomers also displayed similar structural changes in the presence
of either SDS micelles or phospholipid vesicles (data not shown).

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Fig. 1.
CD spectra of GS14 and representative GS14
diastereomers. A, spectra were recorded in 5 mM sodium acetate buffer, pH 5.5, at 20 °C at a peptide
concentration of approximately 0.3 mM. Samples were GS14
( ), GS14V1 ( ), GS14K2 ( ), GS14L3 ( ), GS14K4 ( ), and
GS14V5 ( ). B, spectra were recorded in 5 mM
sodium acetate buffer, pH 5.5, containing 50% TFE, at 20 °C.
Samples were as in A.
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Fig. 2.
Environment-dependent CD spectral
changes in GS14 diastereomers. Molar ellipticities at 205 (A) and 220 nm (B) are shown for the
diastereomers under aqueous conditions (black bars) and in
the presence of 50% TFE (gray bars).
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NMR Spectroscopy--
The extent of disruption of the
-sheet
structure in the GS14 diastereomers was confirmed/demonstrated by NMR
spectroscopy for selected
analogs.3 The regions of the
1H NMR spectrum containing the amide and aromatic proton
resonances are shown in Fig. 3 for GS14
(top spectrum) and one diastereomer, GS14K2 (bottom
spectrum). The GS14 spectrum showed 12 well resolved amide
resonances (each is a doublet corresponding to the amide resonances of
Val1 to Tyr6 and Leu8 to
Tyr13), and a single pair of doublets corresponding to the
meta (2,6) and ortho (3,5) protons of both Tyr6 and
Tyr13. The diverse chemical shifts for the amide resonances
indicate a well ordered structure, and the downfield chemical shifts
indicate significant
-sheet structure. The single pair of doublets
for Tyr6 and Tyr13 indicates that both are in
equivalent environments. By comparison, the amide resonances observed
for GS14K2 were much less diverse and centered more around the random
coil value, indicating disruption of
-sheet structure. In addition,
two pairs of doublets were observed for Tyr6 and
Tyr13, indicating that they are not equivalent in the
structure. The H
chemical shift deviations from random coil values
for GS14 and three diastereomers are shown in Fig.
4. Positive chemical shift deviations
greater than 0.1 ppm indicate downfield shifted resonances relative to
random coil values, and a cluster of three or more continuous positive
chemical shifts is indicative of
-sheet structure (22). It is
apparent that the H
chemical shifts for GS14 (Fig. 4A)
were all
-sheet-like within the
-strands of the molecule (see
molecular model below) and non-
-sheet chemical shifts in the turns
defined by the D-Tyr-Pro sequence. It is clear from the
chemical shift analysis of representative diastereomers (Fig. 4,
B, C, and D) that the strands
contained less
-sheet structure and the turns became more disrupted
compared with GS14. It is also noteworthy that the three diastereomers
differed in both the extent and location of disruption, indicating that
they were structurally different under aqueous conditions.

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Fig. 3.
One-dimensional 1H NMR spectra of
the amide regions for GS14 and GS14K2. Spectra were recorded in
H2O, pH 5.5, at 20 °C.
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Fig. 4.
Chemical shift analysis of GS14 and
representative GS14 diastereomers. The chemical shift deviations
of -proton resonances relative to random coil values are shown for
GS14 (A), GS14V1 (B), GS14K2 (C), and
GS14V10 (D). Three or more continuous positive chemical
shift deviations greater than 0.1 ppm (dashed line) are
indicative of -sheet structure.
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Molecular Model of GS14--
Both CD and NMR spectroscopy
indicated that GS14 possesses a
-sheet structure similar to GS. A
model of GS14 was constructed to contain the essential features as
present in GS, namely, an antiparallel
-sheet structure with two
type II'
-turns defined by the D-Tyr-Pro sequence. As
shown in Fig. 5, the incorporation of
both the cyclic constraint (due to backbone cyclization of the peptide)
as well as secondary structural constraints (
-sheet and turns)
results in a highly amphipathic molecule where Val and Leu residues
make up the hydrophobic face and Lys residues make up the basic face of
the molecule (Fig. 5, lower panel). There are potentially
six hydrogen bonds that could be formed to stabilize the
-sheet
structure of GS14 located between all Val and Leu residues. The
non-H-bonded sites are all occupied by Lys residues (Fig. 5,
upper panel).

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Fig. 5.
Molecular model of GS14. The model was
constructed to contain -sheet dihedral angles for residues in the
strands and two type II' -turns defined by the
Xaa-D-Tyr-Pro-Xaa sequence. An amide bond was formed
between the N and C termini, and energy minimization of the structure
carried out as described under "Materials and Methods." Upper
panel, a top view of the backbone of GS14 indicating the positions
of potential interstrand hydrogen bonds. Lower panel, a side
view of GS14 indicating the relative positioning of hydrophobic and
basic residues.
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Reversed-phase HPLC Analysis--
Retention time on reversed-phase
HPLC can be used as a measure of peptide hydrophobicity (30, 31). It is
well known, however, that the formation of a hydrophobic binding domain
due to peptide secondary structure can affect peptide interactions with
reversed-phase matrices, this effect having been observed both with
-helical peptides (32, 33) and
-sheet peptides (21, 34). GS14 and
the GS14 diastereomers have exactly the same composition and sequences,
and therefore all have the same intrinsic hydrophobicity. Any
differences in retention times would be due to differences in their
effective hydrophobicity, which is related to the ability of the
peptide to form a hydrophobic preferred binding domain for interaction
with the hydrophobic surface of the HPLC matrix. Conversely, the
positioning of positive charges in the vicinity of the hydrophobic
binding domain would also serve to destabilize the hydrophobic
interactions and substantially decrease the overall hydrophobicity of
the preferred hydrophobic binding domain (7, 35, 36). Because retention
time of the GS14 diastereomers on RP-HPLC is a measure of the ability
to form a large hydrophobic binding domain and at the same time
segregate hydrophobic and hydrophilic side chains to opposite sides of
the molecule, it is therefore also a measure of amphipathicity in these
peptides. The RP-HPLC separation of a mixture of GS14 and the 14 diastereomers is shown in Fig. 6. There
was a wide range of retention times observed for the analogs, and all
had a lower retention time than GS14. As seen from the model of GS14
(Fig. 5) the parent molecule exists in a highly amphipathic
-sheet
conformation with a large hydrophobic preferred binding domain formed
by six hydrophobic residues on one face of the molecule, as well as the
basic residues sequestered on the opposite face of the molecule. All
diastereomers had lower retention times compared with GS14 due to
decreased amphipathicity caused by either a decreased size of the
hydrophobic preferred binding domain or the relative positioning of
hydrophobic and basic residues. Measurement of retention times of the
diastereomers on RP-HPLC is therefore a convenient means for ranking
and comparing peptide amphipathicity in this homologous series of
peptides. The extent of disruption of amphipathicity was dependent on
the position of the enantiomeric substitution, with diasteromers
containing substitutions in the non-H-bonded sites (Lys in this case)
being much more disruptive than those with substitutions in the
H-bonded sites (Val and Leu). Enantiomeric substitutions in the center of the
-sheet (Leu3 and Val10) as well as
Pro residues in the turns were least disruptive.

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Fig. 6.
RP-HPLC separation of GS14 and single residue
substitution diastereomers. A mixture of GS14 and the 14 diastereomers was separated by RP-HPLC as described under "Materials
and Methods." The position of the enantiomeric substitution is shown
for each peak on the chromatogram.
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Membrane Interactions of GS14 Diastereomers
Interaction with Bacterial Lipopolysaccharide--
Cationic
peptides interact with Gram-negative bacteria by initially binding to
LPS prior to self-promoted uptake across the outer membrane (37, 38).
We investigated the interaction between GS14 diastereomers and the
bacterial outer membranes by monitoring the displacement of LPS-bound
dansyl-polymyxin B by the peptides (19, 39). The LPS binding affinity
of GS14 was extremely strong, approaching that of polymyxin B itself
(19), whereas all the diastereomers had lower affinity (Table I). There
is a good correlation between binding affinity and retention time on
RP-HPLC (Fig. 7), with those peptides
having a longer retention time exhibiting higher affinity for LPS. This
indicates that the peptides that are more amphipathic (a greater
hydrophobicity of the preferred binding domain and therefore also a
larger oriented basic face) also bind tighter to LPS. The binding
affinity of GS was lower than all the diastereomers, although GS has
the greatest retention time on RP-HPLC (Table I), showing that the
increased number of basic residues and size of the molecule (compared
with GS) also play a role in binding the outer membranes. These
findings are in line with those of a previous study (19) where it was found that both the number of hydrophobic residues and the number of
positive charges, as well as their relative positioning, were important
in LPS binding.

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Fig. 7.
Correlation between amphipathicity and LPS
binding affinity in GS14 diastereomers. Retention times on RP-HPLC
were determined for each diasteromer as shown in Fig. 6, and LPS
binding affinities as listed in Table I were measured as described
under "Materials and Methods." The position of the enantiomeric
substitution is shown for each point. The line is drawn to
guide the eye.
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Permeabilization of Outer Membranes to NPN--
Permeabilization
of E. coli outer membranes by GS14 diastereomers was
monitored using the hydrophobic fluorescent probe NPN. NPN fluorescence
is substantially increased when it is incorporated into the hydrophobic
bacterial cell membrane (after permeabilization) compared with its
fluorescence in the presence of bacterial cells under non
permeabilizing conditions (40). Outer membrane destabilization by
representative GS14 diastereomers is shown in Fig.
8. All the diasteromers were found to
exhibit essentially similar capacities to permeabilize the outer
membrane to the hydrophobic probe at high concentrations; however, at
lower concentrations all were more effective than GS14.

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Fig. 8.
Permeabilization of E. coli
UB1005 cells to NPN. Peptide-mediated outer membrane
destabilization was monitored by fluorescence increase due to NPN
partitioning into the hydrophobic membrane interior. Samples were GS14
( ), GS14V1 ( ), GS14K2 ( ), GS14K4 ( ), and GS14V5
( ).
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Structure-Activity Relationships in GS14 Diastereomers
Hemolytic Activity--
GS14 exhibits extremely high hemolytic
activity against human erythrocytes as shown in Table I. The
diastereomers exhibited a wide range of activities, ranging from
activity similar to GS14 to greatly reduced hemolytic activity. As
shown in Fig. 9, there was a clear
relationship between amphipathicity and hemolytic activity. Those
analogs that were more amphipathic were also more hemolytic. Most
significantly, however, in the least hemolytic single substitution
diastereomer, GS14K4, hemolytic activity was reduced more than 130-fold
compared with the parent GS14. In keeping with this trend, hemolytic
activity was further reduced in those diastereomers containing either
two or four enantiomeric substitutions due to further decreases in
amphipathicity (Table I).

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Fig. 9.
Correlation between amphipathicity and
hemolytic activity in GS14 diastereomers. Retention times on
RP-HPLC were determined for each diasteromer as shown in Fig. 6, and
hemolytic activity as listed in Table I was measured as described under
"Materials and Methods." The line is drawn to guide the
eye.
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Gram-negative Antibacterial Activity--
The antimicrobial
activity of the diastereomers against a range of Gram-negative
microorganisms is shown in Table II. GS14 itself had no activity against any of the microorganisms tested. The
majority of diastereomers exhibited at least some activity against most
of these microorganisms, with some analogs displaying very strong
activity. The therapeutic index of the diastereomers was calculated as
a measure of specificity of the peptide for the microorganism over
human erythrocytes (Table II). It is apparent that GS14 has an
extremely low therapeutic index, a value of less than 0.01, indicating
that it has much greater activity against erythrocytes than
Gram-negative microorganisms. The majority of diastereomers exhibited
an increase in therapeutic index, with a number of these showing a
substantial improvement in specificity (indices greater than 10)
representing a greater than 1,000-fold improvement over GS14. The
therapeutic indices for GS14 and the two best diastereomers, GS14K4 and
GS14K11, are boxed in Table II to highlight the large differences in
specificities between these peptides. The best diastereomer, GS14K4,
demonstrated a greater than 6,500-fold increase in specificity for two
separate microorganisms (P. aeruginosa H188 and E. coli DC2). The improved therapeutic index reflects both the
enhanced antimicrobial activity and the decreased hemolytic activities
of the diastereomers. The activity and specificity of GS is also shown
in Table II for comparison. The best GS14 diastereomer, GS14K4,
exhibited substantially greater specificity than GS for all the
Gram-negative microorganisms tested, with therapeutic indices in the
range of 13-35-fold greater than GS itself. There was a relationship
between both Gram-negative antimicrobial activity and specificity with
amphipathicity as shown in Fig. 10. For
all microorganisms tested, the antimicrobial activity increased with
decreasing retention time on RP-HPLC (decreasing amphipathicity). This,
coupled with decreased hemolytic activity in those diastereomers with
lower amphipathicity (Fig. 9), resulted in substantial increases in the
therapeutic indices compared with GS14. Diastereomers containing either
two or four enantiomeric substitutions were generally less active
against Gram-negative microorganisms than the best single substitution
analogs, but in the case of GS14K2K4, due to decreases in both
antimicrobial and hemolytic activities, this analog exhibited a
therapeutic index against E. coli DC2 similar to those of
the two best diastereomers (Table II).

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|
Fig. 10.
Antimicrobial activity and microbial
specificity of GS14 diastereomers against Gram-negative
microorganisms. The minimal inhibitory concentration ( ) and
therapeutic index ( ) of the single residue enantiomeric substitution
analogs shown in Table II are plotted as a function of retention time
on RP-HPLC. Plots are for P. aeruginosa H188 (A),
E. coli UB1005 (B), and E. coli DC2
(C). MIC values of >200 µg/ml are plotted as 400 µg/ml.
The lines are drawn to guide the eye.
|
|
Gram-positive Antibacterial and Antifungal Activity--
The
activity of the diastereomers against Gram-positive microorganisms and
yeast is shown in Table III. GS14 was
inactive against four of the Gram-positive microorganisms tested but
exhibited strong activity against both Enterococcus faecalis
(MIC 2.3 µg/ml) and Corynebacterium xerosis (MIC 6.2 µg/ml). All of the single residue substitution diastereomers also
exhibited strong activity against both E. faecalis and
C. xerosis. However, they also displayed strong activity
against Staphylococcus epidermidis and moderate activity
against the remainder of the Gram-positive microorganisms tested. GS14
was essentially inactive against the yeast Candida albicans,
but all of the diastereomers exhibited antifungal activity ranging from
moderate to strong. Coupled with the decreased hemolytic activity of
the diastereomers, there was again a large increase in therapeutic
indices relative to GS14 for all of these microorganisms. Increases in
selectivity over GS14 were in the order of 200-fold for E. faecalis, 1,000-fold for C. xerosis, 3,000-fold for
C. albicans, and 10,000-fold for S. epidermidis
for the best diastereomers (GS14K4 and GS14K11). The therapeutic
indices for GS14 and the best diastereomers are boxed in Table III to
highlight these differences. As for Gram-negative microorganisms, there
was a relationship between amphipathicity and Gram-positive activity,
antifungal activity, and specificity (Fig.
11). For those microorganisms against which GS14 exhibited activity (E. faecalis and C. xerosis), the activities remained relatively constant in all the
analogs, but the therapeutic indices were increased due to a reduction
in hemolytic activity. For the remainder of the microorganisms against
which GS14 was inactive, there was an increase in both activity and therapeutic index with decreasing amphipathicity. The most active diastereomers displayed activities that were essentially equal to GS
itself for three of the Gram-positive microorganisms and yeast but with
therapeutic indices in the order of 10-fold higher than GS, due
primarily to reduced hemolytic activity. However, for three other
microorganisms, which included two Staphylococcus aureus
strains and Bacillus subtilis, the best diastereomers were approximately 10-fold less active than GS, although the therapeutic indices were comparable.

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Fig. 11.
Antimicrobial activity and microbial
specificity of GS14 diastereomers against Gram-positive microorganisms
and yeast. The minimal inhibitory concentration ( ) and
therapeutic index ( ) of the single residue enantiomeric substitution
analogs shown in Table III are plotted as a function of retention time
on RP-HPLC. Plots are for S. epidermidis (A),
E. faecalis (B), and C. albicans
(C). The lines are drawn to guide the
eye.
|
|
 |
DISCUSSION |
The role of amphipathicity (polarization of hydrophobicity and
positive charge) in antimicrobial and hemolytic activities of a series
of homologous cyclic peptides was investigated in this study. All the
GS14 analogs had exactly the same composition and sequence and
therefore shared similar intrinsic physicochemical properties (basicity
and hydrophobicity). Our findings show that enantiomeric substitutions
within the framework of GS14 were responsible for the disruption of
both the
-sheet structure and amphipathicity in the diastereomers.
However, all sites were not equivalent in that each substitution
resulted in a unique amount of perturbation of amphipathicity,
depending on the substitution position. Enantiomeric substitutions in
the non-H-bonded sites of GS14 (Lys residues) resulted in the greatest
disruption of amphipathicity. Molecular modelling studies utilizing the
-sheet backbone structure of GS14 have shown that enantiomeric
substitutions at positions corresponding to the Lys residues result in
the positioning of the positively charged side chains closer to the
hydrophobic face of the molecule, whereas substitutions in the H-bonded
sites result in repositioning of the hydrophobic side chain on the same
face of the molecule (data not shown). Similar effects have been noted
with amphipathic helices where substitution of basic residues on the
hydrophobic face of the helix had a greater effect on amphipathicity
than hydrophobic substitutions on the hydrophilic face, which have little effect on the hydrophobicity of the preferred hydrophobic binding domain (7, 35, 36, 41). The GS14 diastereomers were found to
undergo changes in backbone conformation in hydrophobic lipid-mimicking
environments, similar to that seen with linear nonconstrained peptides.
However, due to the constrained nature of the cyclic peptides, these
conformational changes would be expected to be relatively small
compared with linear peptides.
The observed amphipathicities of the diastereomers correlated well with
their hemolytic properties, showing that hemolytic activity is driven
by high amphipathicity, and more likely, the presence of a large
hydrophobic face. Analogous findings were reported in a previous study
on GS-like cyclic 10 residue peptides in which
-sheet structure and
the basic face were retained, but only hydrophobic residues were
altered (16). The fact that GS itself has high hemolytic activity,
although approximately 10-fold less that GS14, again supports this
conclusion, because GS also has an amphipathic nature with a large
hydrophobic face, albeit smaller than GS14. We assume that the
presentation of a large hydrophobic face promotes the partitioning into
membranes of any cell, whereas partitioning into bacterial membranes,
which tend to carry strong negative charges and have a large (internal
negative) electrical potential gradient, is further promoted by the
cationic charge of these GS peptides. Our findings indicate that in the context of GS-like cyclic peptides, hemolytic activity can be reduced
either through reduction of amphipathicity (to reduce the "directed
hydrophobicity" of the hydrophobic domain) as well as by reduction of
overall peptide hydrophobicity. Both these design elements can be
incorporated into peptides either through sequence or structural
(e.g. enantiomeric substitutions and ring size)
manipulation. Similar findings associating increased hydrophobicity or
amphipathicity with increased hemolytic activity have also been
reported for nonconstrained linear peptides (6-8, 42). Modulation of
amphipathicity and hydrophobicity therefore appears to be a generally
applicable method of regulating hemolytic activity in antimicrobial
peptides of different structural classes.
The antimicrobial activity of the GS14 diastereomers against
Gram-negative microorganisms was found to increase with decreasing amphipathicity. This is likely a reflection of the higher binding affinity for outer membrane components by analogs with high
amphipathicity (Fig. 7). These high affinity interactions would be
expected to decrease the ability of the peptides to penetrate to and
accumulate at their site of action on the inner membrane. All
diastereomers were found to have similar capacities to destabilize the
outer membrane following binding. It would therefore appear that
binding to outer membrane components, regardless of the affinity, is
sufficient to destabilize the outer membrane, but only those peptides
with appropriate binding affinity can easily penetrate to the inner membrane. There was a similar trend of increasing Gram-positive antimicrobial activity with decreasing amphipathicity in the
diastereomers. Although these microorganisms have no outer membrane,
they possess a peptidoglycan layer that contains negatively charged
groups from teichoic acid as well as from the amino acids composing the peptidoglycan layer. It is possible that a similar mechanism may be
responsible for the decreased antimicrobial activity by highly amphipathic diastereomers whereby binding to peptidoglycan components results in decreased accumulation of the peptides in the cytoplasmic membrane. Unlike GS14 and the diastereomers, GS exhibited strong antimicrobial activity against all the microorganisms tested. These
differences may again be related to outer membrane binding because GS
was found to have approximately 100-fold lower affinity for LPS
compared with GS14.
Our findings relating increased amphipathicity with decreased
antimicrobial activity in the present cyclic peptides are in agreement
with a previous study by Blondelle and Houghten (7), who utilized model
linear
-helical peptides. In contrast, two recent studies by Bienert
and co-workers (6, 11) also utilizing linear
-helical peptides
reported an opposite trend in which antimicrobial activity either
remained relatively constant or decreased with decreasing
amphipathicity. Differences in overall peptide hydrophobic moments
(quantitated amphipathicity, µ), however, may explain these
discrepancies. GS14 has a high hydrophobic moment (µ = 0.531) that is
essentially equivalent to those linear
-helical peptides (7), which
exhibited similar trends in antimicrobial activity (µ = 0.521). In
contrast, the linear
-helical peptides that exhibited the opposite
trends had substantially lower hydrophobic moments (µ = 0.284 to
0.451 and µ = 0.329 to 0.391 (Ref. 6); µ = 0.334 (Ref. 11)). It is
possible that the differences in the observed activities between these
sets of peptides is related to their overall amphipathicity and its
influence on peptide outer membrane interactions. As shown in the
present study, highly amphipathic molecules bind to outer membrane
components with a greater affinity compared with those with decreased
amphipathicity, which apparently results in decreased antimicrobial
activity (presumably by impeding the subsequent movement of the peptide
toward its internal targets or by interfering with co-operativity that
is essential for trans-outer membrane uptake). Thus, increased outer
membrane interactions by the more amphipathic linear
-helical
peptides (7) may explain the similar trend as seen with the highly
amphipathic GS14 peptides. It is possible that the
-helical peptides
with decreased amphipathicity (6, 11) were of low enough amphipathicity
that these outer membrane interactions were reduced. In support of
this, we also found in the present study that decreasing the
amphipathicity past a certain threshold resulted in a trend of
decreasing antimicrobial activity similar to that observed with the
less amphipathic
-helical peptides (6, 11). One may also speculate
that peptide outer membrane interactions between the present cyclic
peptides and linear peptides may also differ. The greater
conformational entropy upon binding of linear peptides would be
expected to result in decreased binding affinity to outer membrane or
peptidoglycan components compared with the present constrained
peptides, thereby facilitating the partitioning of those peptides to
the inner membrane. Linear peptides would also tend to cover a larger
surface area, which may also influence their action on outer membranes.
As mentioned above, we found that there was a threshold amphipathicity
required for antimicrobial activity below which activity decreased as
exemplified by those diastereomers containing either two or four
enantiomeric substitutions. We have also previously found that linear
nonconstrained peptides related to GS are inactive (16). Both these
observations support the concept that there is a minimum amphipathicity
or minimum size of the hydrophobic domain required for antimicrobial
activity in these cyclic peptides. Although the actual mechanism of
lipid membrane disruption by these peptides is not known, there is
evidence suggesting that the formation of nonlamellar phases are a
feature of their mode of action (43). The formation of such phases
would presumably require a certain proportion of the molecule to
partition into the hydrophobic portion of the membrane to result in
such a rearrangement of membrane achitecture. One would therefore
expect that there be a minimum size of a hydrophobic domain required to
allow partitioning of a critical portion of the peptide into the
membrane. Similar to the antimicrobial activity, hemolytic activity
also decreased with decreasing amphipathicity. We were, however, able
to identify an optimum amphipathicity where the therapeutic index was
maximum in the present diastereomers. The fact that complete
specificity was not achieved in the present cyclic peptides by
modulation of amphipathicity may be a reflection that the proposed site
of action of these peptides are biological membranes. Although there are differences between prokaryotic and eukaryotic membrane
composition, these differences may not be large enough to obtain
complete specificity in these antimicrobial peptides, but rather, the
therapeutic indices can be optimized to provide the greatest
discrimination. Similarly, because there are species to species
variations in both cytoplasmic lipid as well as outer membrane
compositions, it is possible that there will be different optimal
peptide properties for different species.
In summary, we have shown that a preformed highly amphipathic nature is
not desirable in the present cyclic peptides because this results in
decreased specificity as well as increased interactions with outer
membrane components. By systematic manipulation of the amphipathicity
of GS14, it was possible to identify peptides possessing optimal
amphipathicities leading to the highest therapeutic indices. These
cyclic peptides and likely other constrained peptides appear to share
similar requirements for activity and specificity as the linear
unconstrained antimicrobial peptides. However, our findings also
suggest that outer membrane interactions may play a significant role in
the observed antimicrobial properties of antimicrobial peptides in
general and further that these interactions may be of greater
importance for conformationally restricted molecules. We are currently
utilizing the best structural framework (amphipathicity) derived from
the present study to investigate the role of intrinsic hydrophobicity
as well as basicity to further optimize the therapeutic indices of
these cyclic peptides. The three-dimensional structures of the present
peptides in aqueous medium and in lipid environments are also being
determined by NMR spectroscopy, and in-depth lipid binding/disruption
studies are currently in progress. Knowledge of both structure and
mechanism will ultimately allow us to design peptides possessing the
greatest activity and specificity.
 |
ACKNOWLEDGEMENTS |
We thank Paul Semchuk, Leonard Daniels, and
Marc Genest for assistance with peptide synthesis and purification and
Bob Luty for CD measurements. We also thank Pierre Lavigne for helpful discussions and Campbell McInnes for preparation of supplementary material.
 |
FOOTNOTES |
*
This study was supported by the Canadian Government through
grants to the Canadian Bacterial Diseases Network (to R. E. W. H.) and the Protein Engineering Network of Centers of
Excellence (to R. S. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
780-492-2758; Fax: 780-492-1473; E-mail:
robert.hodges{at}ualberta.ca.
2
L. H. Kondejewski and R. S. Hodges,
unpublished results.
3
The complete 1H NMR assignments for
these analogs are available at
http://www.pence.ualberta.ca/ftp/kondejewski.html.
 |
ABBREVIATIONS |
The abbreviations used are:
GS, gramicidin S;
LPS, lipopolysaccharide;
MIC, minimal inhibitory concentration;
NPN, 1-N-phenylnaphthylamine;
RP-HPLC, reversed-phase high
performance liquid chromatography;
TFE, trifluoroethanol;
dansyl, 5-dimethylamino- naphthalene-1-sulfonyl.
 |
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