Cationic amphipathic peptide analogs of cathelicidin LL-37 as a probe in the development of antimicrobial/anticancer agents

Article abstract of DOI:10.1002/psc.3254

Cathelicidin LL-37 belongs to the class of human defense peptides and is overexpressed in many cancers. Segments of LL-37 derived through biochemical processes have a wide range of activities. In this study, novel analogs of the 13-amino acid cathelicidin 17-29 amide segment F¹⁷KRIV²¹QR²³IK²⁵DF²⁷LR-NH₂ were prepared and examined for their antimicrobial and hemolytic activities, as well as for their cytotoxicity on cancer bronchial epithelial cells. Selected substitutions were performed on residues R²³ and K²⁵ in the hydrophilic side, V²¹and F²⁷ in the hydrophobic side of the interphase, and F¹⁷ that interacts with cell membranes. Specific motifs IIKK and LLKKL with anticancer and antimicrobial activities isolated from animals were also inserted into the 17-29 fragment to investigate how they affect activity. Substitution of the amino-terminal positive charge by acetylation and replacement of lysine by the aliphatic leucine in the peptide analog Ac-FKRIVQRIL²⁵DFLR-NH₂ resulted in significant cytotoxicity against A549 cancer cells with an IC₅₀ value 3.90 μg/mL, with no cytotoxicity to human erythrocytes. The peptide Ac-FKRIVQI²³IKK²⁶FLR-NH₂, which incorporates the IIKK motif and the peptides FKRIVQL²³L²⁴KK²⁶L²⁷LR-NH₂ and Ac-FKRIVQL²³L²⁴KK²⁶L²⁷LR-NH₂, which incorporate the LLKKL motif, displayed potent antimicrobial activity against gram-negative bacteria (MIC 3–7.5 μg/mL) and substantial cytotoxicity against bronchial epithelial cancer cells, (IC₅₀ 12.9–9.8 μg/mL), with no cytotoxic activity for human erythrocytes. The helical conformation of the synthetic peptides was confirmed by circular dichroism. Our study shows that appropriate substitutions, mainly in positions of the interphase, as well as the insertion of the motifs IIKK and LLKKL in the cathelicidin 17-29 segment, may lead to the preparation of effective biological compounds.

Full text of DOI:10.1002/psc.3254

Received: 18 December 2019
DOI: 10.1002/psc.3254
Revised: 4 May 2020
Accepted: 6 May 2020
R E S E A R C H A R T I C L E
Cationic amphipathic peptide analogs of cathelicidin LL-37 as a
probe in the development of antimicrobial/anticancer agents
Athanasios Tzitzilis | Anastasia Boura-Theodorou | Vassilios Michail |
Stylianos Papadopoulos | Dimitrios Krikorian | Marilena E. Lekka |
Anna-Irini Koukkou | Maria Sakarellos-Daitsiotis | Eugenia Panou-Pomonis
Department of Chemistry, University of
Ioannina, Ioannina, Greece
Cathelicidin LL-37 belongs to the class of human defense peptides and is over-
expressed in many cancers. Segments of LL-37 derived through biochemical pro-
cesses have a wide range of activities. In this study, novel analogs of the 13-amino
acid cathelicidin 17-29 amide segment F17KRIV21QR23IK25DF27LR-NH2 were pre-
pared and examined for their antimicrobial and hemolytic activities, as well as for
their cytotoxicity on cancer bronchial epithelial cells. Selected substitutions were per-
formed on residues R23 and K25 in the hydrophilic side, V21and F27 in the hydropho-
bic side of the interphase, and F17 that interacts with cell membranes. Specific motifs
IIKK and LLKKL with anticancer and antimicrobial activities isolated from animals
were also inserted into the 17-29 fragment to investigate how they affect activity.
Substitution of the amino-terminal positive charge by acetylation and replacement of
lysine by the aliphatic leucine in the peptide analog Ac-FKRIVQRIL25DFLR-NH2
resulted in significant cytotoxicity against A549 cancer cells with an IC50 value
3.90 μg/mL, with no cytotoxicity to human erythrocytes. The peptide Ac-
FKRIVQI23IKK26FLR-NH2, which incorporates the IIKK motif and the peptides
FKRIVQL23L24KK26L27LR-NH2 and Ac-FKRIVQL23L24KK26L27LR-NH2, which incor-
porate the LLKKL motif, displayed potent antimicrobial activity against gram-negative
bacteria (MIC 3–7.5 μg/mL) and substantial cytotoxicity against bronchial epithelial
cancer cells, (IC50 12.9–9.8 μg/mL), with no cytotoxic activity for human erythro-
cytes. The helical conformation of the synthetic peptides was confirmed by circular
dichroism. Our study shows that appropriate substitutions, mainly in positions of the
interphase, as well as the insertion of the motifs IIKK and LLKKL in the cathelicidin
17-29 segment, may lead to the preparation of effective biological compounds.
Correspondence
Eugenia Panou-Pomonis, Department of
Chemistry, University of Ioannina, 45110
Ioannina, Greece.
Email: epanou@uoi.gr
K E Y W O R D S
amphipathic α-helical peptides, antimicrobial activity, cathelicidin LL-37 analogs, cytotoxicity,
hemolytic assay
Athanasios Tzitzilis, Anastasia Boura-Theodorou, and Vassilios Michail contributed equally.
J Pep Sci. 2020;e3254.
wileyonlinelibrary.com/journal/psc
© 2020 European Peptide Society and John Wiley & Sons, Ltd.
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https://doi.org/10.1002/psc.3254

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TZITZILIS ET AL.
1
|
INTRODUCTION
dodecylphosphocholine and sodium dodecyl sulfate (SDS) has rev-
ealed that aromatic residues F5, F6, F17, and F27 on the hydrophobic
nonpolar side of the helical 2-31segment of LL-37 are important sites
for the interaction of the protein with cell membranes. The same is
also true for residues R7, R19, R23, R29, and R34 on the polar hydro-
philic side of the helix.23
According to the Schiffer-Edmunson projection, amphipathic helical
cationic peptides isolated over the last few decades from a variety of
plant and animal organisms are characterized as natural antimicro-
bials.1,2 They represent the first line of defense for organisms and tar-
get a wide variety of microbes that include bacteria, viruses, parasites,
and fungi. Antimicrobial peptides that also inactivate a wide range of
cancer cell types and act as anticancer agents have been isolated. The
inappropriate and irrational use of antibiotics has induced the world-
wide emergence and spreading of resistant microorganisms. As well,
radiation and chemotherapy are conventional treatments for cancer,
which is one of the main causes of disease-related death worldwide.
These treatments suffer from low performance and serious side
effects.3–6 The development of new antimicrobial/anticancer thera-
peutics is of urgent priority. Amphipathic peptides or their analogs
may be a potential alternative.
With the aim of further contributing to the development of new
effective antimicrobial/anticancer compounds, we report the design,
synthesis, and study of cationic amphipathic peptide analogs of the
cathelicidin 17-29 segment. Selected substitutions were made on resi-
dues R23, K25, V21, and F27 of both sides of the interface, as well as on
residue F17, which is related to cell membrane interactions.8,23
Sequences containing two consecutive lysines and the hydrophobic
residues of leucine and isoleucine (e.g., IIKK, IIKKI, IKK, and LLKKL)
with anticancer and antimicrobial activities have been isolated from
animals.4,5 The IIKK and LLKKL motifs were inserted into the
17-29 segment and studied.8,35–37 Peptide-based factors as
polarity/hydrophobicity, aromaticity, total charge, and side chain ste-
reochemical volume are discussed in relation to their biological
properties.
Many relatively small-size peptides have a number of advantages.
These include favorable pharmacokinetic profiles and bioavailability,
good solubility, lessened potential for development of tolerance
because of the short interaction time with cells, low toxicity, and
selective recognition by microbial and cancer cells.7 Cationic antimi-
crobial amphipathic peptides rupture the cell membrane through
membrane-disrupting mechanisms (carpet, barrel-stave, and toroidal
models), do not act via receptors, and decrease tolerance. Antimicro-
bial peptides can be divided into those that cause cell death by direct
cell lysis and those that penetrate the cell membranes and kill the cell
without lysis by interacting with critical intracellular targets. Interest-
ingly, peptides that are active in microbial and cancer cells but inactive
in normal cells have also been reported.8 Peptides that contribute to
the defense of an organism are expected to show strong toxicity and
selectivity to cancer cells compared with normal cells.
The anticancer activity of the synthesized peptide analogs was
investigated by measuring the cytotoxicity of the peptides on
the cancer obronchial epithelial cell line A549, using the
(3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) photo-
metric assay, and concentration that causes 50% inhibition (IC50
)
was determined.8 The antimicrobial properties of the synthesized
peptides were studied in gram-negative and gram-positive bacteria,
the minimum inhibitory concentration (MIC) was estimated, and the
concentration of the peptide that causes lysis of 50% of erythro-
cytes (EC50) was established.38–40
The data concerning the correlation of the anticancer and antimi-
crobial properties of the peptide analogs will help to understand the
prerequisites for the development of effective pharmaceuticals.
The detailed mechanisms of action of anticancer peptides are not
fully understood, but it is accepted that in most cases, they cause
necrosis and/or apoptosis as they affect the cell or the
mitochondria.3,9–14 The net negative charge of cancer and microbial
cells, due to the presence of anionic compounds in the cell membrane,
such as phospholipids, phosphatidylserine, glycosyl, and sialyl com-
pounds, is an important difference from the zwitterion-neutral form
of normal mammalian cells. Other important aspect is cholesterol,
which modulates membrane fluidity and facilitates the destabilization
of cancer cells by anticancer peptides.15–22
2
| MATERIALS AND METHODS
2.1 | Peptide synthesis
The synthesis of the peptides was performed by the stepwise
solid-phase synthesis procedure on
a Rink-Amide (AM) resin
(0.37 mmol/g resin) using the 9-fluorenylmethyloxycarbonyl
(Fmoc) methodology. Arginine was used as Fmoc-Arg (Pbf)-OH
(Pbf: 2,2,4,6,7-pentamethyl-dihydrobenzofurane-5-sulfonyl), lysine
as Fmoc-Lys (Boc)-OH (Boc: tert-butyloxycarbonyl), aspartic acid as
Fmoc-Asp (OtBu)-OH (OtBu: Otert-butyloxy), and glutamine as
Fmoc-Gln (Trt)-OH. Fmoc-groups were removed using 20%
piperidine in dimethylformamide. The coupling reactions were per-
formed using an Fmoc–amino acid/HBTU/HOBt/DIEA/resin with
a molar ratio of 3/3/3/6/1 (HBTU: O-benzotriazol-1-yl-N,N,N0,N0
tetramethyluronium hexafluorophosphate, HOBt: hydroxybenzo-
1.2,3-triazole, and DIEA: N, N-diisopropylethylamine). Completion
of coupling reactions was ensured by the use of the ninhydrin-
Cathelicidin is the 18 kDa precursor human protein of the active
37 amino acid form, LL-37, that results following proteolysis. LL-37
belongs to the class of human defense peptides and is secreted from
bone marrow, leukocytes, epithelial tissues, and the gastrointestinal
system. LL-37 is overexpressed in many cancers, including ovarian,
breast, and lung cancer.23–28 Segments of LL-37 derived from bio-
chemical processes have diverse actions that include antiseptic,
antibacterial, antimicrobial, apoptotic, metastatic, and immunological
activities.29–34
Two- and three-dimensional nuclear magnetic resonance spec-
troscopy conducted in a membrane-like environment comprised of

TZITZILIS ET AL.
3 of 13
based Kaiser test. The peptides were cleaved from the resin
by treatment with trifluoroacetic acid/triisopropylsilane/water
(TFA/TIS/H2O, 95/2.5/2.5). The resin was removed by filtration,
the filtrate was evaporated under reduced pressure, and the prod-
uct was precipitated with cold diethyl ether. The crude products
were purified by semipreparative reverse phase-high performance
liquid chromatography (RP-HPLC) using a C18 column. Appropriate
programs were applied using eluent A (H2O/0.1% TFA) and B (ace-
tonitrile [CH3CN]/0.1% TFA). Yields ranged from 40% to 55%. The
purity of the peptides was checked by analytical HPLC, and the
correct molecular masses were confirmed by electrospray
ionization-mass spectrometry (ESI-MS) (Table 1).
TABLE 1
Parameters and characterization of the synthesized peptides
Peptides
RP-HPLC gradient elutiona
tR (min)b
ESI-MS
Net charge
1 F17KRIV21QR23 24
I
K25D26F27LR-NH2
A:B 90:10
A:B 30:70
A:B 90:10
A:B 30:70
A:B 90:10
A:B 30:70
A:B 80:20
A:B 20:80
A:B 90:10
A:B 0:100
A:B 90:10
A:B 30:70
A:B 90:10
12.5
Calculated [M + 3H]3+: 573.7
Found [M + 3H]3+: 573.96
Calculated [M + 3H]3+: 562.36
Found [M + 3H]3+: 562.68
Calculated [M + 3H]3+: 548.33
Found [M + 3H]3+: 548.97
Calculated [M + 3H]3+: 582.7
Found [M + 3H]3+: 581.61
Calculated [M + 3H]3+: 704.14
Found [M + 3H]3+: 703.36
Calculated [M + 3H]3+: 583.00
Found [M + 3H]3+: 582.04
+5
2 L17KRIVQRIKDFLR-NH2
3 A17KRIVQRIKDFLR-NH2
4 Ac-FκRIVQRIL25DFLR-NH2
5 Ac-FK (SA)RIVQRIL25DFLR-NH2
6 FKRIVQRIR25DFLR-NH2
7 Ac-FKRIVQRIR25DFLR-NH2
12.5
13.0
17.0
17.0
13.5
17.5
+5
+5
+3
+2
+5
+4
Calculated [M + 3H]3+: 59
5.05
A:B 30:70
A:B 90:10
A:B 30:70
A:B 90:10
A:B 30:70
A:B 90:10
A:B 30:70
A:B 90:10
A:B 30:70
A:B 90:10
A:B 30:70
A:B 90:10
A:B 30:70
A:B 90:10
A:B 30:70
A:B 90:10
A:B 30:70
A:B 90:10
A:B 30:70
A:B 90:10
A:B 30:70
A:B 90:10
A:B 30:70
Found [M + 3H]3+: 595.94
Calculated [M + 3H]3+: 564.37
Found [M + 3H]3+: 563.26
Calculated [M + 3H]3+: 559.03
Found [M + 3H]3+: 558.12
Calculated [M + 3H]3+: 573.25
Found [M + 3H]3+: 572,21
Calculated [M + 3H]3+: 562.37
Found [M + 3H]3+: 561.09
Calculated [M + 3H]3+: 576.38
Found [M + 3H]3+: 575.10
Calculated [M + 2H]2+: 845.60
Found [M + 2H]2+: 843.69
Calculated [M + 2H]2+: 866.62
Found [M + 2H]2+: 864.72
Calculated [M + 3H]3+: 552.34
Found [M + 3H]3+: 551.10
Calculated [M + 3H]3+: 566.40
Found [M + 3H]3+: 565.12
Calculated [M + 3H]3+: 578.05
Found [M + 3H]3+: 577.09
Calculated [M + 2H]2+: 887.59
Found [M + 2H]2+: 886.79
8 FKRIVQκ23IKDFLR-NH2
12.5
10.5
14.5
10.2
13.8
17.5
22.5
14.5
18.0
15.5
20.5
+5
+4
+3
+5
+4
+5
+4
+5
+4
+5
+4
9 FKRIVQL23IKDFLR-NH2
10 Ac-FKRIVQL23IKDFLR-NH2
11 FKRIVQRIKDL27LR-NH2
12 Ac-FKRIVQRIKDL27LR-NH2
13 FKRIVQI23IKK26FLR-NH2
14 Ac-FKRIVQI23IKK26FLR-NH2
15 FKRIVQL23L24KK26L27LR-NH2
16 Ac-FKRIVQL23L24KK26L27LR-NH2
17 FKRIL21QRIKDFLR-NH2
18 Ac-FKRIL21QRIKDFLR-NH2
Abbreviations: ESI-MS, electrospray ionization-mass spectrometry; RP-HPLC, reverse phase-high performance liquid chromatography.
aA: H2O/0.1%TFA, B: CH3CN/0.1%TFA.
btR: retention time; SA: sialic acid.

4 of 13
TZITZILIS ET AL.
2.2 | Circular dichroism
hemolysis and 100% hemolysis were determined by using Tris buffer
and 0.5% NH4OH, respectively. The experiments were performed in
triplicate. Concentrations causing 50% hemolysis (EC50) were derived
from the dose-response curves.
Circular dichroism (CD) spectra were recorded from 260 to 180 nm at
a scan speed of 50 nm/min. The average of three scans was deter-
mined. The procedure was performed using a Jasco J-710 spectro-
polarimeter equipped with a 0.1-cm path-length quartz cell and
peptide concentration 10−4 M at 25ꢀC. Phosphate-buffered saline
(PBS, p 7.4), trifluoroethanol (TFE) in PBS, and sodium dodecylsulfate
(SDS) were used in all experiments. The spectra were smoothed after
subtraction of the solvent contribution. The α-helical content was
2.5 | Cytotoxicity test
The possible anticancer activity of the peptide analogs of cathelicidin
LL 17-29 was assessed using A549 cancer bronchial epithelial cells,
with comparison against DLF lung fibroblasts as normal control cells.
We investigated the cytotoxicity of the peptides for cancer and con-
trol cells using the MTT photometric viability assay as previously
described.43,44 The results were used to determine the IC50. A549 or
DLF cells (18,000 cells/well) were precultured in a sterile 96-well
plate. After 24 h of incubation, cells were treated with 100 μL of pep-
tide solutions (10-200 μg/mL) and incubated for a further 24 h. Then,
10 μL of MTT solution (5 mg/mL) was added to each well and incu-
bated for 4 h. The supernatant was removed from each well and the
formazan crystals that had formed in each well were dissolved in
100 μL dimethyl sulfoxide (DMSO). After 2 h, the absorbance inten-
sity was measured at 570 nm using a microplate reader and compared
with a standard curve to determine the number of viable cells in each
sample. Wells without peptides were used as a positive control and
wells without cells as a negative control (blank) of the assay. The
experiments were carried out three times independently
estimated on the basis of [θ]222 nm values ([θ] in deg.cm2.dmol−1
according to the Chen equation41 as follows:
)
%α−helical content = ½θꢁ222=½θꢁ∞ð1−k=nÞ,
where n is the number of residues of the peptide and [θ]222 the molar
ellipticity at 222 nm. The estimated value [θ]∞, which corresponds to a
100% helical content at 222 nm, is 39,500 and the constant k = 2.57.
2.3 | Antimicrobial activity
The peptides were tested for their antimicrobial activity against gram-
negative bacteria (Escherichia coli DH5a, Pseudomonas aeruginosa
PAO, and Zymomonas mobilis ATCC 10988), gram-positive bacteria
(Bacillus subtilis DELTA and Mycobacterium smegmatis mc2 155), and
the fungus Candida parapsilosis. E. coli is a representative of potent
pathogens, and P. aeruginosa PAO and Z. mobilis ATCC 10988 possess
natural multidrug resistance. The selection of the gram-positive bacte-
ria was based on differences of their membrane lipid components. Ali-
quots (100 μL) containing 2 × 106 logarithmic phase bacteria per
milliliter obtained from microorganisms grown in the appropriate cul-
ture medium were dispensed into wells of 96-well microplates. Pep-
tide stock solution was obtained by solubilizing the peptides in water
followed by filter sterilization using a 0.22-μm pore size filter. After
serial dilutions of the stock solution, 100 μL was added to each well to
obtain a final concentration of 5 to 500 μg/mL. Control wells con-
tained filter-sterilized water instead of peptide solution. The experi-
ments were performed in triplicate. Inhibition of the growth was
determined by measuring the absorbance at 620 nm after incubation
for 24 h at the appropriate temperature for each microorganism. The
antimicrobial activities were expressed as the minimum concentration
at which 100% inhibition of growth was observed (the MIC).
3
| RESULTS AND DISCUSSION
3.1 | Design of the peptides
The design of the peptides was based on the Schiffer-Edmunson heli-
cal wheel projection of the cathelicidin 17-29 amide segment. The aim
of the design was to preserve and improve the amphipathic character
of the peptides Figure 1. Generally, the amides are more proteolytic
2.4 | Hemolytic activity assay
The hemolytic activity of the peptides was monitored by determining
hemoglobin release from a suspension of erythrocytes suspension as
previously described.42 In brief, fresh human red blood cells were
washed three times in 10 mM Tris buffer containing 150 mM NaCl,
pH 7.4. Cell suspensions containing 1.9 × 108 cells/mL were incu-
bated with varying amounts of peptides solubilized in Tris buffer. Zero
FIGURE 1 Schiffer-Edmundson α-helical wheel projection of the
sequence 17-29 of LL-37, FKRIVQRIKDFLR-NH2

TZITZILIS ET AL.
5 of 13
stable and enhance the α-helical conformation inducing better activ-
8 and by leucine in analogs 9 and 10. Leucine replaced phenylala-
nine in position 27 of the hydrophobic side in analogs 11 and 12.
Leucine also replaced valine in position 21 of the hydrophobic side
in analogs 17 and 18. The IIKK segment (23-26) substituted for
aspartic acid 26 and arginine 23 of the hydrophilic face in analogs
13 and 14,while the LLKKL segment (23-27) substituted for argi-
nine 23 and aspartic acid 26 of the hydrophilic face as well as F27
and I24 of the hydrophobic face in analogs 15 and 16. Finally, sialic
acid, which is successfully incorporated in microbial pathogens
where it assists in evasion of host immunity,45 was inserted into
the ε- amino group of lysine in position 18.
ity. Selected substitutions were performed on residues R23, K25, V21
,
and F27 of both sides of the interface, as well as on residue F17. The
specific motifs IIKK and LLKKL, which are found in animal peptides,
were inserted into the 17-29 segment and their influence on antimi-
crobial and anticancer activities was evaluated.
Table 1 shows the sequence 17-29 amide of LL-37(1) and the
synthesized analogs (2-18). The aromatic phenylalanine residue in
position 17 of the hydrophobic side of the amphipathic helix was
substituted by the aliphatic leucine and alanine residues in com-
pounds 2 and 3, respectively. The positively charged lysine in posi-
tion 25 of the interface was replaced by the aliphatic leucine
residue, in analogs 4 and 5 and by arginine, another positively
charged residue, in analogs 6 and 7. Arginine in position 23, which
is also located in the interface, was substituted by lysine in analog
The location of the replaced residues within the amphipathic
helix, their charges, and hydrophilic/hydrophobic properties, as well
as their stereochemical volume, were investigated in relation to their
biological activities.
FIGURE 2 Circular dichroism (CD) spectra
of the peptides 1(
15( ), and 16(
), 4(
), 14(
),
) (100 μM). A,
Phosphate-buffered saline (PBS); B, sodium
dodecyl sulfate (SDS) 8 mM; C,
trifluoroethanol (TFE)/PBS (50/50, v/v)

6 of 13
TZITZILIS ET AL.
3.2 | CD analysis
side of the amphipathic helix, by aliphatic residues leucine and alanine
in peptides 2 and 3, respectively, did not substantially affect the activ-
ity of the natural sequence (peptide 1), indicating that phenylalanine
may not interact with the cell membranes in this analog. Substitution
of the positively charged lysine in position 25 of the interface by
leucine and acetylation of the amino-terminal group increased the
activity in peptide 4. Analog 5, which incorporates sialic acid into the
ε-amino group of lysine in position 18, displayed reactivity only
against B. subtilis DELTA, although the native sequence (peptide 1)
was inactive to this microorganism. Substitution of lysine 25 by
arginine, another positively charged residue, did not substantially
change the reactivity in analog 6. The same finding was also found for
the acetylated N-terminus peptide 7.
All the CD experiments of the peptides in this study were carried out
in PBS (pH 7.4), TFE in PBS (50/50, v/v), and in the SDS membrane
mimic at the critical micelle concentration of 8 mM38–40 Figure 2. The
percent helicity of the peptides was calculated on the basis of [θ]222
nm values (Table 2). The hydrophobic moment of the peptides was
also calculated Table 2.46,47 The native sequence 17-29 of LL-37 (pep-
tide 1) indicated a percent helicity of 51% in SDS and 83% in
TFE/PBS. All the synthesized analogs preserved or increased, in some
cases, their alpha helical content indicating that the selected substitu-
tions did not affect the overall helical conformation, which might be
critical for their activity. The calculated hydrophobic moments of all
free peptides were almost the same.
Replacement of arginine in position 23, which is also located in
the interface, by lysine in peptide 8 did not enhance the activity of
the peptide compared with the peptide 1. Arginine in position 23 was
also substituted by leucine in peptides 9 and 10. This substitution
increased the activity against E. coli DH5a (MIC 30 μg/mL and
100 μg/mL for peptide 9 and 10, respectively), whereas these
peptides showed low reactivity (MIC 20 and 100 μg/mL, same respec-
tive order) against P. aeruginosa PAO. On the contrary, peptides 9 and
10 were not effective against the tested gram-positive bacteria and
the fungus. These findings highlighted the role of position 23 in the
interphase. Replacement of the aromatic phenylalanine in position
27 of the hydrophobic side by leucine produced comparable reactiv-
ities against all tested bacteria and fungus, for peptides 11 and 12.
Insertion of the IIKK motif (23-26) in peptides 13 and 14 and
the LLKKL motif (23-27) in peptides 15 and 16 found in many defense
proteins improved the antimicrobial activity. The substitutions of R23
3.3 | Antimicrobial activity
The antimicrobial activity of the peptides was studied using gram-
negative bacteria (E. coli DH5a, P. aeruginosa PAO, and Z. mobilis
10,988), gram-positive bacteria (B. subtilis DELTA and M. smegmatis
mc2 155), and the fungus C. parapsilosis. The MIC values of the
peptides are shown in Table 3. Differences in the MIC values could be
attributed to the different composition of the outer envelope of each
microorganism. The native sequence 17-29 of LL-37 (peptide 1)
showed weak antimicrobial activity, with an MIC of 200 μg/mL, for
E. coli DH5a and C. parapsilosis, and an MIC of 50 μg/mL for
M. smegmatis mc2 155. Substitution of the aromatic phenylalanine in
position 17 (which interacts with cell membranes) of the hydrophobic
TABLE 2
Percent helicity on the basis of [θ]222 nm values and hydrophobic moment μH of the peptides
Peptides
μH
PBS
5.8
SDS (8mM)
50.7
51.5
58.3
70.6
68.4
87.0
70.9
53.0
81.6
79.3
55.6
77.4
51.9
70.6
39.7
67.8
44.7
89.8
TFE/PBS (50/50 v/v)
1
FKRIVQRIKDFLR-NH2
8.846
83.1
90.1
46.6
88.6
58.4
55.8
57.4
62.0
60.5
52.0
44.6
76.0
66.4
56.5
53.4
76.5
71.6
99.5
2
L17KRIVQRIKDFLR-NH2
A17KRIVQRIKDFLR-NH2
Ac-FKRIVQRIL25DFLR-NH2
Ac-FK (SA)RIVQRIL25DFLR-NH2
FKRIVQRIR25DFLR-NH2
Ac-FKRIVQRIR25DFLR-NH2
FKRIVQK23IKDFLR-NH2
FKRIVQL23IKDFLR-NH2
Ac-FKRIVQL23IKDFLR-NH2
FKRIVQRIKDL27LR-NH2
Ac-FKRIVQRIKDL27LR-NH2
FKRIVQI23IKK26FLR-NH2
Ac-FKRIVQI23IKK26FLR-NH2
FKRIVQL23L24KK26L27LR-NH2
Ac-FKRIVQL23L24KK26L27LR-NH2
FKRIL21QRIKDFLR-NH2
Ac-FKRIL21QRIKDFLR-NH2
0.840
7.6
3
0.743
8.0
4
-
25.8
46.1
8.4
5
-
6
0.847
7
-
9.2
8
0.846
6.0
9
0.847
15.0
9.8
10
11
12
13
14
15
16
17
18
-
0.843
12.1
7.2
-
0.863
7.7
-
9.9
0.848
5.2
-
14.6
2.8
0.864
-
6.3
Abbreviations: PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; TFE, trifluoroethanol.

TZITZILIS ET AL.
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8 of 13
TZITZILIS ET AL.
by I and D26 by K (peptides 13 and 14) significantly increased
the activity of peptide 13 against P. aeruginosa PAO (MIC 3 μg/mL)
and Z. mobilis 10,988 (MIC 5 μg/mL). Peptide 14 also was effective
against P. aeruginosa PAO (MIC 3 μg/mL) and Z. mobilis 10,988 (MIC
7.5 μg/mL).
The substitutions of R23 and I24 by L, D26 by K ,and F27 by L in
the peptides 15 and 16 also significantly increased the activity against
P. aeruginosa PAO (MIC 3 μg/mL in both peptides) and Z. mobilis
10,988 (MIC 7.5 and 5 μg/mL for peptides 15 and 16, respectively),
and against B. subtilis DELTA (MIC 10 μg/mL for peptide 16).
Replacement of valine by leucine in position 21 of the hydropho-
bic side in peptide analogs 17 and 18 increased the activity of peptide
18 against P. aeruginosa PAO (MIC 20 μg/mL) and M. smegmatis mc2
155 (MIC 20 μg/mL).
The overall data of Table 3 demonstrated that the incorpora-
tion of the IIKK and LLKKL motifs into the native sequence 17-29
of the cathelicidin LL-37 significantly improved the antimicrobial
activity against most of the tested microorganisms. Substitution of
the positively charged arginine in position 23 and lysine in position
25, which are located in the interface, by aliphatic residues (leucine
or isoleucine) increased the activity against almost all the tested
bacteria. Replacement of phenylalanine, an aromatic residue, in
positions 17 and 27 of the hydrophobic side of the amphipathic
helix by aliphatic residues leucine and alanine did not enhance the
activity of peptide 1.
FIGURE 4 Hemolytic activity of peptides versus concentration
(μg/mL), A: 12(
), 13(
), 14(
), 15(
), and 16(
);
B: 17(
) and 18(
). The experiments were performed in
triplicate
TABLE 4
Hemolytic activity of the peptides
Peptides
EC50, μg/mL
1 F17KRIV21QR23 24
I
K25D26F27LR-NH2
-
2 L17KRIVQRIKDFLR-NH2
-
3 A17KRIVQRIKDFLR-NH2
-
4 Ac-FκRIVQRIL25DFLR-NH2
5 Ac-FK (SA)RIVQRIL25DFLR-NH2
6 FKRIVQRIR25DFLR-NH2
450
-
-
-
7 Ac-FKRIVQRIR25DFLR-NH2
8 FKRIVQκ23IKDFLR-NH2
-
9 FKRIVQL23IKDFLR-NH2
114
227
-
10 Ac-FKRIVQL23IKDFLR-NH2
11 FKRIVQRIKDL27LR-NH2
12 Ac-FKRIVQRIKDL27LR-NH2
13 FKRIVQI23IKK26FLR-NH2
14.Ac-FKRIVQI23IKK26FLR-NH2
15 FKRIVQL23L24KK26L27LR-NH2
16 Ac-FKRIVQL23L24KK26L27LR-NH2
17 FKRIL21QRIKDFLR-NH2
18 Ac-FKRIL21QRIKDFLR-NH2
-
361
292
411
185
-
FIGURE 3 Hemolytic activity of peptides versus concentration
(μg/mL), A: 1(
7( ), 9(
in triplicate
), 2(
), 3(
), and 4(
); B: 6(
),
-
), and 10(
). The experiments were performed
Note. (−) not active.

TZITZILIS ET AL.
9 of 13
3.4 | Hemolytic assay
The cathelicidin 17-29 segment and all its analogs were also tested
for their toxicity against human erythrocytes. Peptides 1-3, 5-8,
11, 12, 17, and 18 did not exhibit any toxicity against erythrocytes,
while peptides 4, 9, 10, and 13-16 displayed low toxicity at concentra-
tions much higher than their MIC values Figures 3 and 4 and Table 4.
The lack of hemolytic activity can be attributed to the different lipid
composition of the bacteria and erythrocyte membranes. The cationic
AMPs interact with the anionic surface of bacterial membranes, which
lead to the lysis of the bacteria, whereas they cannot interact with the
neutral surface of the erythrocytes.
3.5 | Cytotoxicity test
FIGURE 5 Cytotoxicity of peptide
1, F17KRIV21QR23 24K25D26F27LR-NH2; IC50 (μg/mL), 20.13. The
experiments were carried out three times independently
I
The cytotoxicity of the peptides against A549 bronchial epithelial
cancer cells and the control DLF normal lung fibroblasts was investi-
gated using the MTT photometric assay. The IC50 values of the pep-
tides are shown inTable 5.
The native sequence 17-29 of LL-37 (peptide 1) was cytotoxic to
A549 cells, with an IC50 of 20.13 μg/mL Figure 5. Substitution of phe-
nylalanine in position 17 by the aliphatic residues leucine and alanine
in compounds 2 and 3, respectively, and replacement of phenylalanine
in position 27 of the interphase by leucine in peptides 11 and 12 did
not enhance their cytotoxicity compared to peptide 1. Substitution of
the positively charged lysine in position 25 of the interface by leucine
in analogs 4 and 5 increased the cytotoxicity, as evidence by the IC50
values of 3.90 and 10.70 μg/mL, respectively. The incorporation of SA
into the ε-amino group of lysine in position 18 (analog 5) improved
the cytotoxicity compared with peptide 1. Substitution of lysine in
position 25 by arginine in analog 6 did not enhance the cytotoxicity,
while acetylation of analog 6 increased the cytotoxicity of analog
7 (IC50 12.46 μg/mL). Replacement of arginine in position 23 by lysine
in analog 8 did not enhance the cytotoxicity. Arginine in position
23 was also substituted by leucine in analogs
9 and 10.
This substitution did not enhance the cytotoxicity of analog 9,
but enhanced the cytotoxicity of the acetylated analog
10 (IC50 13.92 μg/mL).
TABLE 5
DLF normal cells
IC50 (μg/mL) of the peptides for A549 cancer cells and
Insertion of the IIKK motif (23-26) in peptides 13 and 14, and
the LLKKL motif (23-27) in peptides 15 and 16 improved the cyto-
toxicity, with IC50 values of 12.92 μg/mL (peptide 14), 12.83 μg/mL
(peptide 15), and 9.8 μg/mL (peptide 16). Substitution of valine in
position 21 by leucine in analogs 17 and 18 did not enhance
cytotoxicity.
Peptides
A549
20.1
33.7
29.8
3.9
DLF
29.3
1 F17KRIV21QR23 24K25D26F27LR-NH2
I
2 L17KRIVQRIKDFLR-NH2
56.9
-
3 A17KRIVQRIKDFLR-NH2
. 4 Ac-FκRIVQRIL25DFLR-NH2
5 Ac-FK (SA)RIVQRIL25DFLR-NH2
6 FKRIVQRIR25DFLR-NH2
20.1
24.3
-
10.7
56.0
12.5
145.1
18.9
13.9
75.0
45.5
20.0
12.9
12.8
9.8
All the peptides were also tested against normal DLF cells. Lower
cytotoxicity was evident compared with A549 cells, as indicated the
significantly higher IC50 values (Table 4).
7 Ac-FKRIVQRIR25DFLR-NH2
8 FKRIVQκ23IKDFLR-NH2
9 FKRIVQL23IKDFLR-NH2
54.1
-
The collective data of Table 5 demonstrated that the incorpora-
tion of the IIKK and LLKKL motifs, which are present in many
defense peptides, into the native sequence 17-29 of the cathelicidin
LL-37 significantly improved cytotoxicity against cancer cell line
A549 cells as compared with the native sequence 17-29 of LL-37
(peptide 1). Substitution of the positively charged arginine in position
23 and lysine in position 25, which are located in the interface, by
aliphatic residues significantly increased the cytotoxicity of the acety-
lated analogs compared with the native sequence 17-29 of LL-37
(peptide 1). Substitution of phenylalanine, an aromatic residue, in
positions 17 and 27 of the hydrophobic side of the amphipathic helix
by aliphatic residues did not enhance the cytotoxicity of peptide
1. Peptide 4, Ac-FKRIVQRIL25DFLR-NH2, displayed the best
23.5
22.5
-
10 Ac-FKRIVQL23IKDFLR-NH2
11 FKRIVQRIKDL27LR-NH2
12 Ac-FKRIVQRIKDL27LR-NH2
13 FKRIVQI23IKK26FLR-NH2
14 Ac-FKRIVQI23IKK26FLR-NH2
15 FKRIVQL23L24KK26L27LR-NH2
16 Ac-FKRIVQL23L24KK26L27LR-NH2
17 FKRIL21QRIKDFLR-NH2
18 Ac-FKRIL21QRIKDFLR-NH2
-
27.0
15.7
23.7
17.3
76.8
160.9
19.2
29.2
Note. (−) not active.

10 of 13
TZITZILIS ET AL.
FIGURE 6 Schematic representation
(SAR) of the % helical content of the
peptides 1( ), 4( ), 14( ), 15( ), and
16( ) in sodium dodecyl sulfate (SDS)
against antimicrobial and anticancer
activity. The experiments were performed
in triplicate
cytotoxicity (IC50 3.90 μg/mL), highlighting the significance of posi-
tion 25 in the interphase, as well as the importance of the N-
terminus neutralization.
1 was not toxic for human erythrocytes but was cytotoxic to A549
cells (IC50 20.13 μg/mL).
Selected substitutions were performed on residues R23, K25
,
V21, and F27 of the interphase and the aromatic residue F17. The
IIKK and LLKKL motifs were inserted into the 17-29 fragment,
and their antimicrobial/anticancer reactivity was studied. The
substituted peptide Ac-FKRIVQRIL25DFLR-NH2 (4) exhibited the
best cytotoxicity against A549 cancer cells, with an IC50 value of
3.90 μg/mL, probably due to its increased helical content. The
peptide was not toxic for human erythrocytes and displayed rela-
tively low antibacterial activity, with an MIC of 100 μg/mL for both
gram-negative (E. coli DH5a) and gram-positive (B. subtilis DELTA)
bacteria.
4
| CONCLUSIONS
In this study, we present the design, synthesis, and analysis of cationic
amphipathic peptide analogs of the cathelicidin 17-29 segment.
The aim of this study is to further contribute to the development of
new effective antimicrobial/anticancer compounds. The native
sequence 17-29 of LL-37 (peptide 1) showed weak antimicrobial
activity with an MIC of 200 μg/mL against the gram-negative bacte-
rium E. coli DH5a and fungus C. parapsilosis and an MIC of 50 μg/mL
against the gram-positive bacterium M. smegmatis mc2 155. Peptide
The peptides Ac-FKRIVQI23IKK26FLR-NH2 (14), FKRIVQL23L24
KK26L27LR-NH2
(15),
and
Ac-FKRIVQL23L24KK26L27LR-NH2

TZITZILIS ET AL.
11 of 13
FIGURE 7 Schematic representation
(SAR) of the % helical content of the
peptides 1( ), 4( ), 14( ), 15( ), and
16( ) in trifluoroethanol (TFE)/
phosphate-buffered saline (PBS)
50/50 v/v against antimicrobial and
anticancer activity. The experiments were
performed in triplicate
(16) displayed potent antimicrobial activity against the gram-
negative species P. aeruginosa PAO and Z. mobilis 10,988, with MIC
values ranging from 3 to 7.5 μg/mL, were not toxic for human
erythrocytes and were cytotoxic for A549 cancer cells (IC50 values
of 12.9, 12.8, and 9.8 μg/mL, respectively). All the peptides were
also tested against normal DLF cells. Toxicity was lower as com-
pared with A549 cancer cells, as indicated by their significantly
higher IC50 values. Schemes were added in understanding the SAR
studies and the conclusion achieved (Figures 6 and 7).
against the negatively charged cancer and microbial cells, whereas
changes of the side chain stereochemical volume (Ala, Ile, Val) did not
affect the overall reactivity of the peptides. N-terminal acetylation,
which increased the peptide stability, increased the antibacterial and
anticancer activity of most of the peptide analogs.
ORCID
Eugenia Panou-Pomonis
https://orcid.org/0000-0002-1249-1308
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