Structure-activity relationship, conformational and biological studies of temporin l analogues

Article abstract of DOI:10.1021/jm1012853

Temporins are naturally occurring peptides with promising features, which could lead to the development of new drugs. Temporin-1Tl (TL) is the strongest antimicrobial peptide, but it is toxic on human erythrocytes and this fact makes the design of synthetic analogues with a higher therapeutic index vital.We studied the structure-activity relationships of a library of TL derivatives focusing on the correlation between the α-helix content of the peptides, the nature of their cationic residues, and their antibacterial/antiyeast/ hemolytic activities. We found that the percentage of helicity of TL analogues is directly correlated to their hemolytic activity but not to their antimicrobial activity. In addition, we found that the nature of positively charged residues can affect the biological properties of TL without changing the peptide's helicity. It is noteworthy that a single amino acid substitution can prevent the antimicrobial activity of TL, making it a lytic peptide presumably due to its self-association. Last, we identified a novel analogue with properties that make it an attractive topic for future research.

Full text of DOI:10.1021/jm1012853

ARTICLE
pubs.acs.org/jmc
Structure-Activity Relationship, Conformational and Biological
Studies of Temporin L Analogues
Maria Luisa Mangoni,||,§ Alfonso Carotenuto,||,† Luigia Auriemma,† Maria Rosaria Saviello,†
Pietro Campiglia,‡ Isabel Gomez-Monterrey,† Stefania Malfi,† Ludovica Marcellini,§ Donatella Barra,§
Ettore Novellino,† and Paolo Grieco*,†
†Department of Pharmaceutical and Toxicological Chemistry, University of Naples “Federico II”, Via D. Montesano 49, I-80131 Naples,
Italy
‡Department of Pharmaceutical Science, University of Salerno, I-84084 Fisciano, Salerno, Italy
§Istituto Pasteur—Fondazione Cenci Bolognetti, Department of Biochemical Sciences, “A. Rossi Fanelli”, University of Rome “La
Sapienza”, I-00185 Rome, Italy
S Supporting Information
b
ABSTRACT: Temporins are naturally occurring peptides with promising
features, which could lead to the development of new drugs. Temporin-
1Tl (TL) is the strongest antimicrobial peptide, but it is toxic on human
erythrocytes and this fact makes the design of synthetic analogues with a
higher therapeutic index vital.We studied the structure-activity relation-
ships of a library of TL derivatives focusing on the correlation between the
R-helix content of the peptides, the nature of their cationic residues, and
their antibacterial/antiyeast/hemolytic activities. We found that the per-
centage of helicity of TL analogues is directly correlated to their hemolytic
activity but not to their antimicrobial activity. In addition, we found that
the nature of positively charged residues can affect the biological properties
of TL without changing the peptide’s helicity. It is noteworthy that a single
amino acid substitution can prevent the antimicrobial activity of TL, making it a lytic peptide presumably due to its self-
association. Last, we identified a novel analogue with properties that make it an attractive topic for future research.
’ INTRODUCTION
because, in order to become resistant to these AMPs, microbes
have to change the composition of their membrane, which is a
very expensive solution and not conducive to their survival.7
Although development of resistance to AMPs has been
demonstrated experimentally in vitro, it occurs at rates that are
many times lower than those observed for conventional
antibiotics.7-10 In addition, besides direct antimicrobial activity,
AMPs display other biological effects including neutralization
of endotoxins, chemokine-like activities, immunomodulating
properties, and induction of both angiogenesis and wound
repair.11-15 Taken together, these properties make AMPs pro-
mising candidates for development as new anti-infective agents
and food preservatives.
There are several sources for peptide antibiotics, but amphi-
bian skin of the Rana genus is one of the richest.16,17 Nowadays,
temporins (first isolated from the skin secretions of specimens of
the European red frog Rana temporaria)18 are the largest family
of AMPs, with more than 100 isoforms.19 Temporins are among
the smallest AMPs (10-14 residues long) found in nature to
date.20,21 They have an amidated C-terminus and a low net
In recent decades, the widespread use and abuse of conven-
tional antibiotics has led to the emergence of multidrug-resistant
bacterial and fungal strains. The World Health Organization
states that, worldwide, 95% of the Staphylococcus aureus isolates
are now resistant to penicillin and up to 60% are resistant to
methicillin.1 As a consequence, infections due to resistant
microbes have become the third most common cause of human
death worldwide.2
In light of this, the search for new antibiotic agents with a new
mode of action is extremely important. Gene-encoded cationic
antimicrobial peptides (AMPs) that are produced in bacteria,
insects, plants, and vertebrates including humans3,4 are interest-
ing compounds which could give rise to novel therapeutic
approaches.
AMPs are evolutionarily conserved molecules of the innate
immune system of a wide range of organisms and protect the host
from the constant interactions with invading microbes before the
adaptive immunity is activated.5 In contrast with commonly used
drugs which operate on specific intracellular targets,6 many
AMPs physically permeate and destroy the cell membrane,
making it difficult for the bacteria to develop resistance. This is
Received: October 6, 2010
Published: February 14, 2011
r
2011 American Chemical Society
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Table 1. Sequences of TL Analogues
compd
sequencesa
TL
H-Phe1-Val2-Gln3-Trp4-Phe5-Ser6-Lys7-Phe8-Leu9-Gly10-Arg11-Ile12-Leu13-NH2
H-Leu1-Leu2-Gln3-Trp4-Leu5-Ser6-Lys7-Leu8-Leu9-Gly10-Arg11-Leu12-Leu13-NH2
H-Leu1-Leu2-Gln3-Trp4-Leu5-Ser6-Lys7-Leu8-Leu9-Gly10-Arg11-Trp12-Leu13-NH2
H-Phe1-Val2-Gln3-Trp4-Phe5-Ser6-Lys7-Phe8-Leu9-Leu10-Arg11-Ile12-Leu13-NH2
H-Phe1-Val2-Pro3-Trp4-Phe5-Ser6-Lys7-Phe8-Leu9-Gly10-Arg11-Ile12-Leu13-NH2
H-Phe1-Leu2-Pro3-Trp4-Phe5-Ser6-Lys7-Phe8-Leu9-Gly10-Arg11-Ile12-Leu13-NH2
H-Phe1-Val2-Pro3-Trp4-Phe5-Ser6-Lys7-Phe8-Leu9-Pro10-Arg11-Ile12-Leu13-NH2
H-Phe1-Val2-Pro3-Trp4-Phe5-Ser6-Lys7-Phe8-Leu9-DPro10-Arg11-Ile12-Leu13-NH2
H-Phe1-Val2-Gln3-Trp4-Phe5-Ser6-Lys7-Phe8-Leu9- Gly10-Lys11-Ile12-Leu13-NH2
H-Phe1-Val2-Gln3-Trp4-Phe5-Ser6-Lys7-Phe8-Leu9- Gly10-Orn11-Ile12-Leu13-NH2
H-Phe1-Val2-Gln3-Trp4-Phe5-Ser6-Arg7-Phe8-Leu9- Gly10-Arg11-Ile12-Leu13-NH2
H-Phe1-Val2-Arg3-Trp4-Phe5-Ser6-Lys7-Phe8-Leu9- Gly10-Arg11-Ile12-Leu13-NH2
H-Phe1-Val2-Arg3-Trp4-Phe5-Ser6-Arg7-Phe8-Leu9- Gly10-Arg11-Ile12-Leu13-NH2
H-Phe1-Val2-Pro3-Trp4-Phe5-Ser6-Lys7-Phe8-Leu9- Gly10-Orn11-Ile12-Leu13-NH2
H-phe1-val2-gln3-trp4-phe5-ser6-lys7-phe8-leu9-Gly10-arg11-ile12-leu13-NH2
H-leu1-ile2-arg3-Gly4-leu5-phe6-lys7-ser8-phe9-trp10-gln11-val12-phe13-NH2
TL-1
TL-2
[Leu10]TL
[Pro3]TLb
[Leu2, Pro3]TL
[Pro3, Pro10]TL
[Pro3, DPro10]TL
[Lys11]TL
[Orn11]TL
[Arg7]TL
[Arg3]TL
[Arg3, Arg7]TL
[Pro3, Orn11]TL
D-TL
RI-TL
a Residue variations compared to TL are highlighted in bold. b Already described in refs 29 and 30. Lower case indicates D-amino acids.
positive charge at neutral pH (2 to þ3), due to the presence of
only 1 or 2 basic residues in their sequence. Most of these
peptides adopt an amphipathic R-helical-like preferential con-
formation in a hydrophobic environment. They act mainly on
Gram-positive bacteria, including methicillin-resistant clinical
isolates,22-26 with minimal inhibitory concentrations ranging from
2.5 to 20 μM.23 Interestingly, temporin-1Tl (TL, H-Phe1-
Val2-Gln3-Trp4-Phe5-Ser6-Lys7-Phe8-Leu9-Gly10-Arg11-Ile12Leu13-
NH2) has a higher and broader spectrum of activity than the other
isoforms also being active against fungi and Gram-negative bacteria
such as Pseudomonas aeruginosa and Escherichia coli.27,28 In addition,
TL is cytotoxic to three different human tumor cell lines (Hut-78,
K-562, and U-937) but it also kills human erythrocytes at micro-
bicidal concentrations. Otherwise, most of the temporins are
practically nonhemolytic.26
By combining spectroscopic and computational analyses, we
had previously obtained a molecular-level resolution of the
interactions between TL and membrane mimicking micelles29,30
and, on the basis of these results, we designed a novel analogue,
[Pro3]TL, which showed an increased antimicrobial activity
toward Gram-positive bacteria and yeast cells and a decreased
hemolytic activity compared to the native TL.29
However, to have a clearer understanding of the structural
elements that are responsible for the cell selectivity of TL, we
decided to perform an extensive SAR study of this peptide. We
designed and synthesized a new library of TL derivatives and
investigated the correlation between the R-helical content of the
compounds (by CD analysis) and their antibacterial/antiyeast/
hemolytic activities. Furthermore, we also analyzed the influence
of positively charged residues of the TL template on its biological
activity. The results of these experiments will assist in engineer-
ing analogues of TL with a better therapeutic index.
chosen because of its high helical propensity.31 Starting from TL-1,
we replaced Leu12 with Trp, thereby obtaining TL-2. Trypto-
phan membrane anchoring properties could significantly modu-
late the peptide antimicrobial activity and toxicity.32 Then, to
increase the helical character of the C-terminal region, we
replaced the Gly10 residue of TL with a Leu, thus obtaining the
compound [Leu10]TL.
Replacement of Gln3 by Pro residue in TL led to [Pro3]TL
analogue, which was previously found to exhibit an increased
antimicrobial activity and a decreased hemolytic activity.29 This
reduction was ascribed to the break of the N-terminal helical
structure. Starting from [Pro3]TL (Table 1), we changed Val2
with Leu ([Leu2, Pro3]TL) to get the Phe-Leu-Pro N-terminal
sequence that is characteristic of many temporins including
temporin-1Ta
(H-Phe1-Leu2-Pro3-Leu4-Ile5-Gly6-Arg7-Val8-
Leu9-Ser10-Gly11-Ile12-Leu13-NH2). Furthermore, to break the
helical structure at the C-terminus of [Pro3]TL too, we replaced
Gly10 with a Pro ([Pro3, Pro10]TL) or with a DPro ([Pro3,
DPro10]TL) residue. Subsequently, we focused our attention on
charged residues of TL. Different modes of interaction were
recently found for Arg11 and Lys7 with membrane mimicking
micelles.30 More specifically, Arg11 was found to interact mainly
with DPC phosphate groups because of the very favorable
electrostatic interaction between the protonated guanidinium
group and the negatively charged phosphate groups. In contrast,
the Lys7 side chain interacted mainly with water molecules. To
obtain an insight into these different interaction propensities and
their biological significance, we exchanged Arg11 for a Lys
([Lys11]TL) or for an Orn ([Orn11]TL), whereas Lys7 was
replaced by an Arg ([Arg7]TL). Because Gln3 also had previously
shown itself to be highly capable of interacting with water
molecules,30 we substituted Gln3 for an Arg residue ([Arg3]TL).
Moreover, both the water interacting Gln3 and Lys7 were
simultaneously replaced by Arg ([Arg3, Arg7]TL). Finally, be-
cause of the interesting antimicrobial and toxicity profiles of
[Orn11]TL (see below), we combined Arg11/Orn with Gln3/Pro
substitutions, obtaining the analogue [Pro3, Orn11]TL. All D-
isomer (D-TL) and retro-inverse (RI-TL) classic analyses were
also performed (Table 1) to verify the hypothesis that the
’ RESULTS
Design. Several TL analogues (Table 1) were synthesized in
order to evaluate the impact of helical content and charge
interactions on their biological activity. In compound TL-1, we
replaced all the hydrophobic amino acids Phe1, Val2, Phe5, Phe8,
and Ile12 of the TL template with leucine residues. Leucine was
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Table 3. Hemolysis (%) of TL Analogues
peptides
(μM) TL TL-1 TL-2
[Leu10
TL
]
[Pro3] [Leu2,Pro3] [Pro3,Pro10
]
[Pro3,DPro10
TL
]
[Lys11
TL
]
[Orn11
TL
] ]
[Arg7] [Arg3] [Arg3,Arg7] [Pro3,Orn11
TL
TL
TL
TL
TL
TL
TL
D-TL RI-TL
24
12
6
3
1.5
94 100
100
100
78
13
8
83
80
80
78
69
92
42
10
6
93
64
19
9
5
5
4.5
2
2
2.5
2
0.6
1.5
0.4
100
59
14
5
89
62
22
7
98
90
29
9
100
100
52
20
7
100
92
43
14
6
72
12
2
1
0.7
90
88
46
13
5
81
49
15
6
92
48
13
3
73
25
9
4
4.5
4
2.6
3
4
1
Figure 1. CD spectra of TL and some selected analogues in (a) DPC, (b) SDS (TL, pink line; [Leu10]TL, black line; [Leu2, Pro3]TL, red line; [Pro3,
Pro10]TL, green line; D-TL, blue line; RI-TL, yellow line). CD spectra of the other analyzed analogues are reported in Figure A of the Supporting
Information.
Figure 2. CD spectra of TL (red line) and [Leu10]TL (green line) in LPS.
molecular mechanism underlying the antimicrobial activity of TL
is not stereospecific or mediated by receptor recognition.
Biological Activity. All the synthesized compounds were
evaluated for their biological activity against yeasts, Gram-
positive and Gram-negative bacteria (Table 2), and human
erythrocytes (Table 3).
almost all the microorganisms tested but endowed with a potent
hemolytic activity (69% hemolysis at 1.5 μM). The antimicrobial
activity of [Leu2, Pro3]TL was found to be slightly lower than
that of TL (about 2-fold higher MICs), whereas a marked
reduction in the hemolytic activity was observed when compared
with the parent peptide TL although it was more hemolytic than
[Pro3]TL. The analogue [Pro3, Pro10]TL showed a significantly
lower antibacterial activity (2-8-fold higher MIC values with
respect to the natural TL) and had a negligible lytic effect on red
blood cells. Interestingly, the activity of [Pro3, Pro10]TL on
Candida albicans was preserved. Likewise, the analogue [Pro3,
DPro10]TL appeared to be nonhemolytic and with an antimi-
crobial activity even weaker than that of [Pro3, Pro10]TL.
Analogues [Lys11]TL and [Orn11]TL displayed an antimicro-
bial activity similar to that of TL being [Orn11]TL generally more
active than [Lys11]TL. Moreover, both [Lys11]TL and
Analogue TL-1 (Leu1, Leu2, Leu5, Leu8, Leu12 TL) had, in
general, a higher antimicrobial activity and a lower hemolytic
capacity (at a peptide concentration range from 3 to 12 μM)
compared with the parent peptide TL. Differently, TL-2 analo-
gue (Trp12 TL-1) showed a pronounced reduction in the
antimicrobial activity against the majority of the microbial strains
when compared to TL and turned out to be much more
hemolytic (Table 3).
Surprisingly, the substitution of the Gly10 with Leu in
[Leu10]TL produced a compound devoid of activity against
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[Orn11]TL showed a little (∼2-fold) decrease in the hemolytic
activity in comparison with TL, up to 12 μM.
Peptides [Arg7]TL, [Arg3]TL, and [Arg3, Arg7]TL had a
reduced antimicrobial activity (approximately, 2-fold higher
MICs than TL). The only exception was Pseudomonas aeruginosa
ATCC 27853, toward which they all showed a higher activity
than the native peptide. The hemolytic capacity of [Arg7]TL was
reduced by half with respect to TL at a peptide concentration of 6
μM, while [Arg3]TL and [Arg3, Arg7]TL analogues had a toxic
effect on human erythrocytes similar to that of TL at all
concentrations used.
Finally, the antimicrobial activity of [Pro3, Orn11]TL was
slightly lower than that of TL. However, of the most active
antimicrobial compounds, [Pro3, Orn11]TL manifested the low-
est hemolytic activity.
As expected, D-TL (all D-isomer) had very similar antimicro-
bial and hemolytic activities to those of the natural TL, whereas a
lower biological activity was displayed by the retro-inverso
analogue RI-TL.
Figure 3. Calcein leakage from PE/PG (triangles), PG/CL (squares),
and PC/Cho (circles) LUVs after addition of TL (filled symbols) or
[Leu10]TL (empty symbols) at different concentrations. Fluorescence
recovery was measured for 10 min after the peptides were mixed with the
vesicles, and its maxima was reported. Leakage was calculated as
indicated in the Experimental Section. Values are means of three
independent measurements with (SD.
Conformational Studies. To explore the conformational
behavior of TL analogues, we performed a CD study of these
peptides in water, SDS/water, and DPC/water solutions. CD
spectra in water (pH 7.4) revealed the presence of disordered
conformers for all compounds with a minimum close to 198 nm
(data not shown). In contrast, in SDS and DPC micelles solution,
the shape of CD spectra suggested the propensity to form
defined secondary structures. In particular, CD spectra showed
two minima around 209 and 222 nm, characteristic of R-helix
structures (Figure 1 and Supporting Information).
Helical content was predicted from CD spectra using the
SOMCD method33 (Table 4). In the same Table 4, the hydro-
phobicity (H) and the mean hydrophobic moment (amphipathicity,
M) are also reported. Two consensus scales were used: the Eisenberg
consensus scale,34 which is the most used in the scientific literature,
and the combined consensus scale (CCS),35 which is extended to
nonproteinogenic amino acids and which allowed the calculation of
H and M for ornitine-containing peptides.
Permeabilization of Large Unilamellar Vesicles
(LUV). Among the synthesized analogues, [Leu10]TL revealed
itself to be the peptide with the lowest antimicrobial activity
especially against Gram-negative bacteria and with the highest
toxic effect on human erythrocytes (Table 2), which is in
agreement with its high content of R-helix (see Table 4). To
check whether this drastic decrease in the antimicrobial activity
was due to the peptide’s inability to permeate bacterial mem-
branes, we used calcein-loaded large unilamellar vesicles (LUV)
of different lipid composition. More precisely, to mimic the lipid
composition of the cytoplasmic membrane of a representative
Gram-negative bacterium (e.g., E. coli), we used LUV made of
phosphatidylethanolamine (PE)/phosphatidylglycerol (PG)
(7:3, w-w). The membrane of a representative Gram-positive
bacterium (e.g., S. aureus) was instead mimicked by PG/cardi-
olipin (CL) (6:4, w/w) LUV.37 Liposomes made of phosphati-
dylcholine (PC)/cholesterol (Cho) (9:1, w/w), to mimic the
composition of the outer leaflet of mammalian erythrocytes,38
were also included for comparison. Different concentrations of
peptide were added to the LUV suspension, and membrane
permeability was measured by following fluorescence recovery
due to calcein release from the vesicles.39 As indicated in Figure 3,
calcein leakage induced by [Leu10]TL (empty symbols) from
both PE/PG (triangles) and PG/CL (squares) liposomes was
similar to that caused by the native peptide TL (filled symbols)
and increased in a dose-dependent manner with a slightly higher
leakage from PG/CL LUV. Overall, these results indicate that,
despite lacking antimicrobial activity, [Leu10]TL is able to bind
and destabilize the two representative bacterial model mem-
branes to a similar extent to that of TL. In contrast, when
zwitterionic (PC/Cho) LUV were employed, the effect of
[Leu10]TL (empty circles) was double that of the native peptide
(filled circles) at the lowest peptide/lipid molar ratio used
(0.0025). This latter finding is in line with the stronger hemolytic
activity of [Leu10]TL compared to TL when tested at low
concentrations (up to 6 μM) (Table 3).
Generally, helical contents observed in SDS and DPC micelle
solutions were similar with a reduction in SDS of about 10%
compared to DPC. The highest helical content was observed in
TL-2 and in [Leu10]TL (75% and 76%, respectively, in DPC). In
the latter, Gly10 was replaced by a Leu residue, and this substitu-
tion probably enhanced the helical content of the C-terminal
end. In contrast, peptides bearing one ([Pro3]TL, [Leu2,
Pro3]TL, and [Pro3, Orn11]TL) or two ([Pro3, Pro10]TL and
[Pro3, DPro10]TL) proline residues showed the lowest helical
content (about 30% and 5%, respectively, in DPC) in accordance
with the design strategy. Substitutions carried out on peptides
[Lys11]TL/[Orn11]TL did not change the helical content of the
peptide (about 50-60% in DPC). D-TL showed a higher
percentage of left-handed helix structure (60% in DPC) while
only 30% R helix was observed in RI-TL analogue.
CD spectra of TL and [Leu10]TL were also acquired in 100
μM LPS (lipopolysaccharide) solution (Figure 2). Note that LPS
is the major component of the outer membrane of Gram-
negative bacteria.36 According to Rosenfeld et al.,28 TL adopted
an R-helix structure when added to LPS micelles in solution (two
minima at 209 and 224 nm). In contrast, CD spectrum of
[Leu10]TL showed only one minimum at 205 nm. Using the
SOMCD method, the secondary structure of [Leu10]TL in LPS
solution could be estimated as random coil (41%) and β-strand
(49%) conformations.
’ DISCUSSION
The purpose of this study was to develop analogues of TL with
a higher therapeutic index than the native peptide. Generally,
activities of AMPs against bacteria, fungi, and mammalian cells
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Table 4. Peptide Sequences, Percentage of r-Helix Character, Percentage of Hemolysis, Hydrophobicity (H), and
Amphipathicity (M)
peptide
%helixa
%helixb
%hemolysisc
H Eisenberge
M Eisenberge
H CCSf
M CCSf
TL
59
42
75
76
25
35
10
3
52
37
68
69
22
32
6
48
25
78
80
10
19
4.5
0.6
14
22
29
52
43
2
0.06461
0.03
0.4188
0.3949
0.3851
0.4029
0.3804
0.3798
0.3899
3.0231
3.46
3.46
3.95
3.46
3.9
4.844
5.07
5.07
4.13
4.42
4.63
4.29
TL-1
TL-2
[Leu10]TL
0.01769
0.09308
0.1123
0.1115
0.09461
[Pro3]TL
[Leu2, Pro3]TL
[Pro3, Pro10]TL
[Pro3, DPro10]TL
[Lys11]TL
[Orn11]TL
[Arg7]TL
3.64
1
54
52
55
59
52
34
60d
30d
47
45
48
50
45
29
50d
24d
0.1185
0.3832
3.03
3.1
4.84
4.81
4.85
5.14
5.15
4.38
0.01077
-0.02077
-0.07462
0.4724
0.4917
0.5446
3.01
2.71
2.71
3.54
[Arg3]TL
[Arg3, Arg7]TL
[Pro3, Orn11]TL
D-TL
46
15
RI-TL
a Measured in DPC micelle solution. b Measured in SDS micelle solution. At peptide concentration of 6 μM. d Left-handed helices. According to
c
e
Eisenberg consensus scale.34 f According to combined consensus scale (CCS).35
are determined by a complex interaction between cationicity,
hydrophobicity, R-helicity, and amphipathicity.6,40,41 Hence, we
designed a first group of analogues with the aim of increasing
(TL-1, TL-2, and [Leu10]TL) or decreasing ([Leu2, Pro3]TL,
[Pro3, Pro10]TL, [Pro3, DPro10]TL) the percentage of helicity.
Actually, TL-1 turned out to have less helical content than TL. In
a second group of analogues ([Lys11]TL, [Orn11]TL, [Arg7]TL,
[Arg3]TL, [Arg3, Arg7]TL), we investigated the importance
of the nature of the positively charged side chains by exchang-
ing guanidinium groups with amine groups and vice versa
([Lys11]TL, [Orn11]TL, [Arg7]TL) or by inserting a further
positive charge in the peptide sequence ([Arg3]TL, [Arg3,
Arg7]TL).
antibacterial activity on TL. Nevertheless, disruption of the
C-terminal helix does not affect the anti-Candida activity of the
peptide (Table 2). Taken together, these findings suggest a
“carpet-like” or a “dynamic peptide-lipid supramolecular pore”
mechanism underlying the antimicrobial activities of temporin L
analogues.29 Indeed, unlike the “barrel-stave” model, a helical
structure encompassing the entire peptide sequence is not
required for these two types of mechanisms.
Interestingly, [Leu10]TL (76% helical content in DPC) was
the analogue with the highest hemolytic capacity and the lowest
antimicrobial activity. This could be due to the peptide’s oligo-
meric state, probably induced by contact with LPS in Gram-
negative bacteria or with teichoic acids in Gram-positive bacteria.
The large size of the oligomers would make it more difficult for
the peptide to diffuse through the cell wall into the target
cytoplasmic membrane.42 This would explain the very low
antimicrobial activity of [Leu10]TL despite its ability to permeate
bacterial membranes to a similar extent to that found with TL
(Figure 3). It is worthwhile noting that our CD spectra in LPS
solution (Figure 2) revealed a contribution of ∼50% of β-sheet
structures for [Leu10]TL, whereas the native peptide TL adopted
an R-helix structure in LPS solution. Such β-sheet structures
might be associated with amyloid-like aggregates. In fact, small
β-sheets are unstable in aqueous solution without aggregation
unless they are stabilized by, for example, disulfide bridges.43
In the second group of peptides ([Lys11]TL, [Orn11]TL,
[Arg7]TL, [Arg3]TL, [Arg3, Arg7]TL), the helical content, in
both DPC or SDS, was almost constant and similar to that of the
parent TL although the hemolytic activity was not preserved.
More precisely, replacement of Arg11 by Lys ([Lys11]TL) or by
Orn ([Orn11]TL) gave rise to an approximately 2-fold reduction
in hemolytic activity. This means that structural features of
arginine (e.g., the bifurcated structure or high hydrogen-bonding
potential of the guanidinium group), rather than cationicity
per se, do favor the interaction between TL and the eukaryotic
cell membrane (as already demonstrated for cathepsin G
derivatives).44
Helical contents of the peptides were evaluated by CD
spectroscopy in water, SDS, and DPC micelle solutions
(Figure 1 and Table 4). SDS and DPC micelles are considered
to be good models of negatively charged (microbial) and
zwitterionic (eukaryotic) membranes respectively.29,30
All peptides exhibited a helical content ranging from 3% to
76% in DPC (Table 4). Considering the first group of peptides
(TL-1, TL-2, [Leu10]TL, [Leu2, Pro3]TL, [Pro3, Pro10]TL,
[Pro3, DPro10]TL) and the lead compounds TL and
[Pro3]TL, a clear and direct correlation between the helical
content and the hemolytic activity could be detected at 6 μM
(Table 4). This is consistent with the “barrel stave” mechanism
proposed for the hemolytic activity of TL and its derivatives.29,30
In contrast, the antimicrobial activity was not dependent on
the helicity of the peptide; for instance, TL-1 (37% helix in SDS)
exhibited a higher antimicrobial activity than TL-2 (68% helix in
SDS) against the tested microbial strains except for Acinetobacter
spp. (Table 2). However, for the analogues [Pro3, Pro10]TL or
[Pro3, DPro10]TL bearing a Pro or a DPro residue at position 10
(in addition to Pro3), to also break the helical structure at the
C-terminus, the antibacterial activity was significantly reduced
compared to that of [Pro3]TL (Table 2). These results indicate
that a helical structure at the C-terminus of the peptide sequence,
rather than at its N-terminal end, is crucial in order to confer
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Replacement of Lys7 by Arg ([Arg7]TL) also decreased
hemolytic activity by half (Table 3). This indicates different
functions of Lys vs Arg charged residues in the peptide interac-
tion with zwitterionic membranes depending on their position
along the peptide sequence, which is in agreement with our
previous MD simulations results.30
’ CONCLUSION
Overall, our studies have indicated that the R-helical content
and the nature of positively charged residues in the sequence of
TL induce different effects on the hemolytic and the antimicro-
bial activity of the peptide. Regarding the hemolytic activity, the
helical content of the peptide does play an important role as
proved by the existence of a direct correlation between the
percentage of helicity and the lytic effect of the peptide on human
erythrocytes. On the contrary, no direct correlation has been
detected between the helicity of the peptide and its antimicrobial
activity despite the finding that disruption of the C-terminal R-
helix drastically reduces the toxic effect of the peptide on different
species of bacteria but not on Candida. This finding can address
the design of further selective antifungal agents starting from the
TL template by breaking its helical structure both at the N- and at
the C-terminal regions.
We have also demonstrated that the nature of positively
charged residues can differently affect the antimicrobial/hemo-
lytic activity of TL (depending on their position along the
peptide’s sequence) without modifying its helical content. More
particularly, the presence of an amine instead of a guanidinium
group at position 11 reduces the lytic effect of the peptide on red
blood cells but does not significantly alter its antimicrobial
activity. Note, too, that insertion of a further positively charged
residue (i.e., Arg) in position 3 does not improve the antimicro-
bial potency of the peptide despite its higher cationicity and
amphiphilicity nor does it induce changes in the helical content/
hemolytic activity of TL.
Remarkably, our studies have also indicated that replacement
of Gly10 with a leucine almost abolishes the antimicrobial activity
of TL and causes a dramatic increase in hemolytic activity, which
might be due to an oligomerization of the peptide.
Finally, it is important to underline that when the breakage of
the N-terminal helical structure of TL (due to Pro3) is combined
with the replacement Arg11/Orn ([Pro3, Orn11]TL), the hemo-
lytic activity of the peptide is suppressed by up to 12 μM without
significant changes in the antimicrobial activity of the native
peptide. These findings, together with the unique properties of
temporins (short size and low positive charge), have contributed
to making [Pro3, Orn11]TL an interesting template for the future
development of temporin-based anti-infective agents with a
better therapeutic index.
Analogously, the exchange of guanidinium with amine groups
at position 11 ([Lys11]TL and [Orn11]TL) or the substitution of
Lys with Arg in position 7 ([Arg7]TL) were found to be
responsible for a slightly reduced antimicrobial activity. Bearing
in mind what was stated above, these results underline the fact
that side chains of basic residues also have a different role in the
interaction of TL with anionic membranes depending on their
position along the peptide sequence.
Note also that [Orn11]TL resulted more active than TL
against the Gram positive S. aureus Cowan I, the Gram-negative
P. aeruginosa ATCC 27853, and the yeast C. albicans ATCC
10231 (Table 3).
Replacement of Gln3 by Arg ([Arg3]TL) increased the net
positive charge of the peptide to þ4. Helical percentage
and hemolytic and antimicrobial activity of this analogue results
(Table 2 and 4) results were very similar to those of TL,
suggesting that a positive charge at this position does not affect
the helicity or the biological activity of the peptide. Probably,
residue in position 3 is not involved in the interaction with a
negative counterpart but rather it is confined in a hydrophilic
environment.
This is in line with our previous data showing that Gln3 of
TL mainly interacts with water molecules when the peptide is
bound to a DPC micelle.30 The results obtained with
[Arg3]TL have also indicated that, in contrast to what is
observed in magainins and other host defense peptides,45,46
a direct relationship between cationicity and antimicrobial
activity is not a general rule. This is also supported by the
results found with [Arg3, Arg7]TL (net charge þ4), a peptide
with a similar hemolytic but a slightly reduced antimicrobial
activity compared to that of TL. Moreover, [Arg3, Arg7]TL
showed the highest hydrophobic moment (M) and the lowest
hydrophobicity (H) among the peptides reported in this study
(Table 4). Hydrophobic moment, which measures the amphi-
philicity of the peptide in an R-helix, is considered to be one of
the most important factors governing antimicrobial acti-
vity.46,47 However, the results found with [Arg3, Arg7]TL
and other analogues have clearly shown that the hydrophobic
moment does not control the antimicrobial activity of tem-
porins. In addition, data reported in Table 4 do not show any
correlation between hydrophobicity and hemolytic (or anti-
microbial) activity in temporin L analogues. Note that [Pro3,
Orn11]TL, which combines [Pro3]TL and [Orn11]TL sub-
stitutions, showed the lowest hemolytic effect among the most
active antimicrobial analogues (Table 3). These results con-
firm that both the breakage of the N-terminal helical structure
of TL and the absence of a guanidinium group at position 11
can almost abolish the hemolytic activity of the peptide
without causing drastic changes in its antimicrobial activity
(Table 2). Finally, we found that the antimicrobial and the
hemolytic activities of the D-isomer were similar to those of
TL, demonstrating that antimicrobial activity of temporins is
not mediated by receptor recognition and that peptide-
membrane interactions are the major step in the mechanism
of action of these peptides.
’ EXPERIMENTAL SECTION
Synthesis. NR-Fmoc-protected amino acids, HBTU, HOBt, and
Rink amide resin, were purchased from GL Biochem Ltd. (Shanghai,
China). Peptide synthesis solvents, reagents, as well as CH3CN for HPLC,
were reagent grade and were acquired from commercial sources and used
without further purification unless otherwise noted. Peptides were synthe-
sized according to the solid phase approach using standard Fmoc metho-
dology in a manual reaction vessel and in a stepwise fashion.48 For example,
for the synthesis of TL, NR-Fmoc-Leu-OH was coupled to Rink amide
resin (0.5 g, 0.7 mmol NH2/g). The following protected amino acids were
then added stepwise NR-Fmoc-Ile-OH, NR-Fmoc-Arg(Pbf)-OH, NR-
Fmoc-Gly-OH, NR-Fmoc-Leu-OH, NR-Fmoc-Phe-OH, NR-Fmoc-Lys-
(Boc)-OH, NR-Fmoc-Ser(tBu)-OH, NR-Fmoc-Phe-OH, NR-Fmoc-Trp-
(Boc)-OH, NR-Fmoc-Gln(Trt)-OH, NR-Fmoc-Val-OH, and NR-Fmoc-
Phe-OH. Each coupling reaction was accomplished using a 3-fold excess of
amino acid with HBTU and HOBt in the presence of DIEA.
The NR-Fmoc protecting groups were removed by treating the
protected peptide resin with a 25% solution of piperidine in DMF
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ARTICLE
(1 ꢀ 5 min and 1 ꢀ 20 min). The peptide resin was washed three times
with DMF, and the next coupling step was initiated in a stepwise manner.
All reactions were performed under an Ar atmosphere. The peptide resin
was washed with DCM (3ꢀ), DMF (3ꢀ), and DCM (4ꢀ), and the
deprotection protocol was repeated after each coupling step. The
N-terminal Fmoc group was removed as described above, and the
peptide was released from the resin with TFA/Et3SiH/H2O (90:5:5)
for 3 h. The resin was removed by filtration, and the crude peptide was
recovered by precipitation with cold anhydrous ethyl ether to give a
white powder which was purified by RP-HPLC on a semipreparative
C18-bonded silica column (Vydac 218TP1010, 1.0 cm ꢀ 25 cm) using a
gradient of CH3CN in 0.1% aqueous TFA (from 10% to 90% in 30 min)
at a flow rate of 1.0 mL/min. The product was obtained by lyophilization
of the appropriate fractions after removal of the CH3CN by rotary
evaporation. Analytical RP-HPLC indicated a purity >98% and the
correct molecular weights were confirmed by LC/ESI-MS (6110
Quadrupole, Agilent Technologies). The peptide concentration was
determined by quantitative amino acid analysis using an Applied
Biosystems model 420A. (Table A and B, Supporting Information).
Microorganisms. The strains used for the antimicrobial assays
were the following: the Gram-negative bacteria Acinetobacter baumannii
ATCC 19606, Acinetobacter junii RT-4, Enterobacter faecalis ATCC
29212, Escherichia coli ATCC 25922, E. coli D21, Pseudomonas aerugi-
nosa ATCC 15692, P. aeruginosa ATCC 27853, Pseudomonas syringae pv
tobaci 1918NCPPB, Yersinia pseudotuberculosis YPIII, the Gram-positive
bacteria Bacillus megaterium Bm11, Staphylococcus aureus ATCC 25923,
Staphylococcus aureus Cowan I, Staphylococcus capitis 1, Staphylococcus
epidermidis ATCC 12228, Streptococcus pyogens ATCC 21547, and the
yeasts Candida albicans ATCC 10231, Saccharomyces cerevisiae, and
Saccharomyces pombe.
Antimicrobial Assay. Susceptibility testing was performed by
adapting the microbroth dilution method outlined by the Clinical and
Laboratory Standards Institute using sterile 96-well plates (Falcon NJ,
USA). Aliquots (50 μL) of bacteria in midlog phase at a concentration of
2 ꢀ 106 colony-forming units (CFU)/mL in culture medium (Mueller-
Hinton, MH) were added to 50 μL of MH broth containing the peptide
in serial 2-fold dilutions in 20% ethanol. The range of peptide dilutions
used was 0.75-48 μM. The same procedure was followed with yeast
strains but using Winge medium.49 Inhibition of microbial growth was
determined by measuring the absorbance at 600 nm after an incubation
of 18-20 h at 37 °C (30 °C for yeasts), with a 450-Bio-Rad Microplate
Reader. Antimicrobial activities were expressed as the minimal inhibitory
concentration (MIC), the concentration of peptide at which 100%
inhibition of microbial growth is observed after 18-20 h of incubation.
Hemolytic Assay. The hemolytic activity was measured on human
red blood cells as reported previously.50 Briefly, aliquots of a human
erythrocyte suspension in 0.9% (w/v) NaCl were incubated with serial
2-fold dilutions of peptide (dissolved in 20% ethanol prior to use) for 40
min at 37 °C with gentle mixing. The samples were then centrifuged for
5 min at 900g, and the absorbance of the supernatant was measured at
415 nm. Complete lysis was measured by suspending erythrocytes in
distilled water.51
permeation induced by the peptides was monitored at 37 °C for 10
min after peptide addition, by the fluorescence increase (λexcitation = 485
nm; λemission = 535 nm). Complete dye release was obtained by using
0.1% Triton X-100, which caused total destruction of lipid vesicles.52
The percentage of calcein leakage was calculated as 100(F1 - F0)/(Ft -
F0), where F0 represents the fluorescence of intact vesicles, and F1 and Ft
denote the intensities of the fluorescence achieved after peptide and
Triton-X-100 treatment, respectively.
Circolar Dicroism. All CD spectra were recorded using a JASCO
J710 spectropolarimeter at 25 °C with a cell of 1 mm or 10 mm path
length. The CD spectra were acquired by the range from 260 to 190 nm
1 nm bandwidth, 4 accumulations, and 100 nm/min scanning speed.
The CD spectra of the TL and TL derivates, at a concentration of 100
μM, were performed in water (pH = 7.4), in SDS (20 mM) and in DPC
(20 mM) micellar solutions. We acquired these CD spectra also at lower
(10 and 1 μM) peptide concentrations (data not shown), and we did not
observe any significant variation of the spectra, indicating that no self-
assembly phenomena occur within this concentration range. CD spectra
of TL and [Leu10]TL (100 μM) were also acquired in a 100 μM LPS
solution.
’ ASSOCIATED CONTENT
S
Supporting Information. Analytical data of the synthe-
b
sized peptides. CD spectra of TL analogues. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: þ39-081-678620. Fax: þ39-081-678644. E-mail: pagrieco@
unina.it.
Author Contributions
These authors contributed equally to the work.
’ ACKNOWLEDGMENT
This work was supported by grants from Sapienza Universitꢀa
di Roma, Italian Ministero dell’Istruzione, Universitꢀa e Ricerca
(PRIN 2008), and Istituto di Biologia e Patologia Molecolari of
the National Research Council.
’ ABBREVIATIONS USED
SDS, sodium dodecylsulphate; DPC, dodecylphosphocholine;
SAR, structure-activity relationship; CD, circular dichroism;
DCM, dichloromethane; DIPEA, N,N-diisopropylethyl-amine;
DMF, N,N-dimethylformamide; Et3SiH, triethylsilane; Fmoc,
9-fluorenyl-methoxycarbonyl; HOBt, N-hydroxy-benzotriazole;
HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluoro-phosphate; Pbf, 2,2,4,6,7-pentamethyldihydrobenzo-
furan-5-sulfonyl; RP-HPLC, reversed-phase high performance
liquid chromatography; ESI, electrospray ionization; LC-MS,
liquid chromatography-mass spectrometry; TL, temporin-1Tl;
TA, temporin-1Ta; PE, phosphatidyl-ethanolamine; PG, phosp-
hatidylglycerol
Calcein-Loaded LUV and Leakage Assay. Lipid films of PE/
PG (7:3, w/w), PG/CL (6:4, w/w), and PC/Cho (9:1) were prepared
by dissolving dry lipids (2 mg of each lipid mixture) in chloroform/
methanol (2:1, v/v) and evaporating the solvents under a nitrogen
stream. The lipid film was then hydrated with 10 mM Tris and 150 mM
NaCl (pH 7.4) containing 60 mM calcein solution. The liposome
suspension was extruded 10 times through a polycarbonate filter
(pore size, 0.1 μm), and free calcein was removed by gel filtration by
using a Sephadex G-25 column (1.5 cm ꢀ 10 cm; Pharmacia Biotech
AB) at room temperature. Calcein entrapped in the vesicles was highly
concentrated, and the fluorescence was self-quenched. Calcein release
from LUVs (final lipid concentration, 30 μM) due to membrane
’ ADDITIONAL NOTE
Abbreviations used for amino acids and designation of peptides
follow the rules of the IUPAC-IUB Commission of Biochemical
Nomenclature in J. Biol. Chem. 1972, 247, 977-983. Amino acid
symbols denote L-configuration unless indicated otherwise.
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