Pharmacology and therapeutics

Antimicrobial efficacy of granulysin-derived synthetic

peptides in acne vulgaris
Hee-Sun Lim1, MD, Seung-Min Chun1, MD, Min-Gyu Soung2,3, MD, PhD,
Jenny Kim4, MD, PhD and Seong-Jin Kim1, MD, PhD

1
Department of Dermatology, Chonnam
National University Medical School,
Gwangju, South Korea, 2Department of
Applied Biology and Chemistry, College of
Agriculture and Life Science, Chungnam
National University, Daejeon, South Korea,
3
Cellicon Laboratory, Hannam University,
Daejeon, South Korea, and 4Division of
Dermatology, David Geffen School of
Medicine, UCLA

Correspondence
Seong-Jin Kim, MD, PHD
Department of Dermatology
Chonnam National University Medical
School
5 Hak-Dong, Dong-Gu
Gwangju 501-746
South Korea
E-mail: seongkim@chonnam.ac.kr
Conflicts of interest: None.

Abstract
Background Antimicrobial peptides are considered as a potential alternative to antibiotic
treatment in acne vulgaris because the development of a resistant strain of
Propionibacterium acnes is problematic. Granulysin can be regarded as an ideal substance
with which to treat acne because it has antimicrobial and anti-inflammatory effects.
Objectives This study was performed to explore the effectiveness of granulysin-derived
peptides (GDPs) in killing P. acnes in vitro under a standard microbiologic assay and to
evaluate their potential use in a topical agent for the treatment of acne vulgaris.
Methods Twenty different peptides based on the known sequence of a GDP were
synthesized and tested in vitro for antimicrobial activity. Thirty patients with facial acne
vulgaris were instructed to apply a topical formulation containing synthetic GDP to acne
lesions twice per day for 12 weeks.
Results A newly synthesized peptide in which aspartic acid was substituted with arginine,
and methionine was substituted with cysteine, showed the highest antimicrobial activity
against P. acnes. Moreover, it was effective against both Gram-positive and Gramnegative bacteria in vitro. After treatment with the topical formulation containing 50 ppm of
synthetic peptide for 12 weeks, a significant reduction in the number of pustules was
observed, regardless of the increase in the number of comedones. In addition, a significant
reduction in the clinical grade of acne based on the Korean Acne Grading System (KAGS)
was evident.
Conclusions Synthesized GDP shows strong antimicrobial activity against P. acnes in
vitro. The clinical improvement observed suggests a topical formulation containing the GDP
has therapeutic potential for the improvement of inflammatory-type acne vulgaris by its
antimicrobial activity.

Introduction
Antimicrobial peptides (AMPs) have long been considered
as a potential alternative to antibiotic treatment. Antimicrobial peptides are typically small, cationic peptides that
are part of the innate immune response of most plants,
insects, and animals.1 The analysis of clinical isolates of
Propionibacterium acnes, a major etiologic agent of
inflammatory acne vulgaris, has related to increasing
resistance to standard antibiotic therapies that makes the
treatment of acne more challenging.24 Thus, it is necessary to develop a new antimicrobial treatment which
effectively prevents the development of a resistant strain.
Granulysin is a human AMP of the saposin-like family,
a family of peptides that has been conserved from amoebas to humans, and a novel effector molecule released by
2015 The International Society of Dermatology

human cytotoxic T lymphocyte (CTL) and natural killer

(NK) cells.5,6 Antimicrobial peptides of human origin are
considered to be safer than those of plants, insects, and
animals. In addition, granulysin has been shown to be
important in the host defense of the skin.5 Furthermore,
the physicochemical nature of granulysin facilitates its
powerful bactericidal action, which can destroy the membrane of a microorganism instantly.6
Because it represents a natural form of host defense in
cutaneous infection, granulysin may be useful as a topical
agent in the treatment of acne. We sought to explore the
effectiveness of granulysin and granulysin-derived peptides (GDPs) in killing Propionibacterium acnes in vitro
under standard microbiologic assays. Because P. acnes is
located deep in the sebaceous gland below the mid-dermis,7 it is important that the peptide is delivered in an
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appropriate size in an effective formulation into the target

area. Therefore, in this study, we aimed to establish the
optimum antibacterial form of granulysin by modifying
its structure to make it smaller than in original sequences.
We also sought to explore the clinical effectiveness of
GDPs and to establish the one with the strongest antimicrobial activity that might be used in a topical agent for
the treatment of acne by inhibiting inflammation or killing bacteria.

CFU assay was performed as previously reported.6

Gram-positive (P. acnes, Staphylococcus aureus,
Streptococcus mutans and Streptococcus pyogenes) and
Gram-negative (Escherichia coli) bacteria were grown under
anaerobic conditions in reinforced clostridial medium (Oxoid
Ltd., Basingstoke, UK) for two days and collected in mid-log
phase. The bacteria were washed three times with the assay
buffer, 10 mM sodium phosphate, pH 7.2, supplemented with
0.03% trypticase soy broth (TSB; Becton-Dickinson & Co.,
Cockeysville, MD, USA), and enumerated by applying a
conversion factor of 7.5 9 107 bacteria per ml = 1 OD unit at

Materials and methods

600 nm. Various concentrations of GDPs were incubated with

Recombinant granulysin

3.75 9 105 bacteria in a final volume of 30 ll at 37 C for

four hours. After incubation, 10-fold dilutions were prepared

Synthesis and purification of peptides

and plated on solid media comprised of Brucella broth (BD

Peptides were synthesized using standard 9H-

Biosciences, Inc., San Diego, CA, USA) with 5% sheep red

fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase

chemistry.8 Na-Fmoc deprotection was achieved with 20%

blood cells (Remel, Inc., Lenexa, KS, USA), 0.5 mg/L vitamin
K, and 5.0 mg/L hemin (Remel, Inc.). Plates were incubated

piperidine/N-methylpyrrolidone (NMP) (3 9 5 min). All standard

for four days at 37 C under anaerobic conditions, after which

Fmoc amino acid derivatives and resins were purchased from

ufelfingen, Switzerland). At the
Merck Biosciences AG (La

individual colonies were counted and the number of CFUs per

tube calculated.

completion of synthesis, resins were washed with

dichloromethane and dried (N2 flush, 10 min). Peptides were

Clinical assessment of a topical agent containing GDP 20

cleaved from the resin, and side chains were deprotected using
modified reagent K (trifluoroacetic acid [TFA], 95% [v/v];
triisopropylsilane [TIS], 2.5%; water, 2.5% [v/v]; for 3 h). Post-

Preparation of a topical agent

cleaving solutions were collected and peptides precipitated by

highest antimicrobial activity against P. acnes according to a

the addition of ice-cold diethyl ether. The precipitated peptides

CFU count with log scale. A topical formulation of GDP 20 was

were separated by centrifugation, washed using cold diethyl

synthesized by mixing GDP 20 at 50 ppm into a commonly

ether, spun down again, and dried in vacuo.

used water-soluble cream formulation (Picostec Co. Ltd.,

Peptides were purified by preparative reverse-phase high-

Of the synthesized GDP derivatives, GDP 20 showed the

Pyeongtak, South Korea). No other antimicrobial agents or

performance liquid chromatography (RP-HPLC) to >95%

antimicrobial preservatives were added.

homogeneity. The purity of peptides was evaluated by analytical

RP-HPLC using a Shimadzu LC Solution System (Shimadzu

Subjects and compatibility of test

Corp., Kyoto, Japan) connected to a photodiode array detector

A total of 30 acne patients, including men and women aged

(monitoring at 220 nm), using a 4.6 9 250 mm C18 TP column

19 years, were enrolled in the study. The volunteers with mild

(Grace-Vydac Separations Group, Inc., Hesperia, CA, USA)

(pore diameter: 300 
A; particle size: 5 lm) in a linear gradient

to moderate acne were recruited by public posting during March

from 5% to 90% (v/v) solvent B (0.1% trifluoroacetic acid [TFA]

therapy and any use of systemic antibiotics within one month of

in acetonitrile) in solvent A (0.1% TFA in water) over

entry to the study, use of systemic retinoids within the

30 minutes (flow rate: 1 ml/min). The molecular weight of each

peptide was confirmed using an HP 1100 Series LC/MSD

six months prior to study entry, pregnancy, and lactation.

Subjects were advised not to take or apply any medications

System mass spectrometer (Hewlett Packard, Inc., Santa Clara,

that might affect acne. All subjects were evaluated for allergic

CA, USA).

contact dermatitis by patch test prior to their participation in the

The peptides used in this study comprised a panel of

to May 2010. Exclusion criteria were a history of topical acne

study. To ensure the compatibility of GDPs with topical use, we

modified peptides originally based on the primary sequence of

performed an occlusive 48-hour patch test in 25 volunteers in

a-helix 2 (aH2) and a-helix 3 (aH3) granulysin.9 The amino acid

order to check for any irritability and toxicity in skin. The patch

sequences and physicochemical properties of peptides are

test was applied to one side of the participants back, without

summarized in Table 1.

additional irritation, and the other side of the back was

stimulated by 10 rounds of tape stripping. Then patches

Determination of antimicrobial activity

including the GDP 20 formulation were applied to both sides for

The antimicrobial activity of the peptides against P. acnes was

48 hours. The presence of any irritant reaction on the tested

examined using the colony-forming unit (CFU) method.6 The

area was checked at 48 hours and at 96 hours after the

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Pharmacology and therapeutics

Table 1 Peptide sequences and physicochemical properties of granulysin-derived peptides (GDPs)

Molecular weight
Granulysin
(2152)a
GDP
GDP
GDP
GDP
GDP
GDP
GDP
GDP
GDP
GDP
GDP
GDP
GDP
GDP
GDP
GDP
GDP
GDP
GDP
GDP

01d
02e
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20

21
PTQRSVSNAA
aH2a

31
TRVCRTGRSR

TRVSRTGRSR
TRVSRTGRSR

41
WRDVCRNFMR
aH3a

51
RY

WRDWSRNFMR
W
R WRDWSRNFMR
R WRDWYRNFMR
R WRRWYRRFMR
R WRRWWRRFMR
R WRDWYRN
R WRDWYR
R WRDWY
R WRRWY
DWYRNFMR
WYRNFMR
YRNFMR
YRRFMR
R WRKWYRKFMRf
K WKRWYKRFMKf
K WKKWYKKFMKf
R WRHWYRHFMRf
R WRRWYRRFCRf
R WRRWYRKFCRf

Calc
value

Obs
valueb

Net
charge

Retention
time, minc

Purity
(%)

2610.9
1361.5
1609.8
1685.9
1767.9
1791.6
1250.6
1136.6
980.4
1021.5
1186.5
1071.5
885.4
927.5
1754.1
1698.1
1642.1
1772.0
1781.1
1753.1

2611.7
1362.5
1610.9
1687.0
1769.0
1792.2
1251.8
1137.9
981.6
1022.8
1187.6
1072.7
886.5
928.7
1755.6
1699.8
1642.3
1773.2
1782.7
1754.5

+6
+4
+3
+3
+6
+6
+2
+2
+1
+3
+1
+2
+2
+3
+6
+6
+6
+6
+6
+6

17.3
13.2
13.0
12.7
13.3
13.5
15.0
12.5
13.1
15.4
13.4
15.5
13.3
13.4
14.2
14.1
14.1
14.2
14.3
14.2

96.6
98.9
95.5
98.7
95.5
95.6
95.9
95.3
96.0
98.7
96.7
96.9
99.9
95.5
95.5
96.6
95.7
96.8
95.3
97.4

Calc, calculated; Ob, observed.

a
Predicted a-helix regions are underlined.
b
Observed molecular weight [M+H]+ of the purified peptide.
c
Linear gradient elution from 5% to 90% (v/v) solvent B (0.1% TFA in acetonitrile) in solvent A (0.1% TFA in water) over
30 min (flow rate: 1 ml/min).
d
Positive control peptide in McInturff et al.6
e
GDP 02 to GDP 20 are designations for each granulysin-derived peptide.
f
Peptide N-terminal acetylation and C-terminal amidation.

removal of the patch. No irritant reaction was observed in any

of the volunteers, and therefore the GDP 20 formulation is
regarded as safe for use in a clinical trial. Subjects were
advised to apply the topical cream containing GDP 20 to acne
lesions twice per day (in the morning and evening) for
12 weeks. Subjects were followed up at 2, 4, 8, and 12 weeks
to evaluate the efficacy and safety of the treatment. All of the

Statistical analysis
Wilcoxons signed rank test was used to compare findings
before treatment with those at 2, 4, 8, and 12 weeks. All
analyses were performed using SPSS Statistics for Windows
Version 12.0 (SPSS, Inc., Chicago, IL, USA). Statistical
significance was indicated by a P-value of <0.05.

procedures involved in the clinical study were explained in

detail, and informed consent was obtained from all participants.

Clinical outcome assessment

Clinical photographs of the subjects were taken before treatment
and at 2, 4, 8, and 12 weeks (Fig. 1). To determine the severity
of acne, one investigator counted the number of papules,
pustules, nodules, and microcomedones. The severity of acne
was also estimated using the Korean Acne Grading System
(KAGS) (Table 2).10 Surveys to investigate the occurrence of any
adverse reactions such as pruritus, irritation, rash, xeroderma,
burning sensation, scaling, burn, pain, and changes in
pigmentation were conducted at each follow-up appointment.

2015 The International Society of Dermatology

Results
Characteristic background of synthesized peptides

The crystal structure of granulysin reveals that it contains

five a-helical domains. The ability of granulysin to kill
Salmonella typhimurium, E. coli, and P. acnes is localized
at regions of a-helices 2 or 3.11 The modification of a
helixloophelix peptide, 3150, by substitution of a tryptophan for valine at amino acid 44 (peptide 3150v44w,
GDP 01) has been reported to increase its interaction
with bacterial surfaces and its antimicrobial activity.6
Therefore, we modified the structure of the peptide based

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(a)

Lim et al.

Antimicrobial synthetic peptide for acne

(b)

(d)

(c)

(e)

Figure 1 Clinical photographs of a patient (a) before and at (b) 2 weeks, (c) 4 weeks, (d) 8 weeks and (e) 12 weeks after
topical application of the GDP 20 formulation. Clinical improvements were observed on skin to which GDP 20 was applied

on the sequence of GDP (GDP 01), 3150v44w, some of

which included these regions (Table 1).
Antimicrobial activity of synthesized peptides under
structure modification

The N-terminus peptide (GDP 02) containing the helix

loophelix domain had no effect against P. acnes. By contrast, the C-terminus peptide (GDP 04) had slightly higher
antimicrobial activity than GDP 01. Overall, these data
indicate that granulysin can kill P. acnes and that its antimicrobial activity can be localized to a smaller region of
the molecule with a defined structural motif, the a-helix 3
region (Fig. 2a). Next, we set out to determine whether
modifications to GDP 04 (peptide 4050) could enhance
its antimicrobial activity against P. acnes. The structural
International Journal of Dermatology 2015, 54, 853862

change according to the properties12 of a side chain of

amino acid for GDP derivatives was composed. Firstly,
cysteine (Cys, C), which is the 45th hydrophilic amino
acid located in the hydrophobic region of the a-helix, was
changed into serine (Ser, S: GDP 03), which is a polar
amino acid, and tyrosine (Tyr, Y: GDP 04), which is a
hydrophobic amino acid. The result indicates that the
antimicrobial activity of GDP 04 against P. acnes is about
10-fold higher than that of GDP 03 at a low micromolar
range. This result indicates that in terms of structure, tyrosine corresponds to the hydrophobic region of the a-helix
structure better than serine. The amino acid change
(GDP 05 and GDP 06) was attempted in the hydrophilic
region of the a-helix using the same method. However,
antimicrobial activity was not found to improve (Fig. 2a).
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Pharmacology and therapeutics

Table 2 Korean Acne Grading System

Grade

Definition

1
2
3
4
5
6

10 papules
11~30 papules
31 papules, 10 nodulesa
11~20 nodules  mild ongoing scars
21~30 nodules  moderate ongoing scars
31 nodules  severe ongoing scars  sinus tracts

Nodule diameter: >5 mm.

Next, the modification focused on reducing the amino

acid of, respectively, the C- and N-terminals in the
GDP 04 peptide (GDP 07GDP 14). As a result, the antimicrobial activity of short peptides was drastically
reduced (>1000 times). This result indicates that the
amphoteric structures of the a-helix, including more than
11 amino acids, are necessary to improve antimicrobial
activity (Fig. 2b).
Lastly, amino acid changes were attempted randomly
in the amphoteric structure of the a-helix (GDP 15
GDP 20). Among the six GDP derivatives, GDP 20
(D43 ? R43, V44 ? W44, C45 ? Y45, N47 ? K47,
M49 ? C49) showed the highest antimicrobial activity
against P. acnes (Fig. 2c). The antimicrobial activity of
GDP 20 was dramatically improved at least 1000-fold
over that of GDP 04 at 32 lM.
Figure 3 schematizes the helical wheel representations of
natural granulysin (4050) and modified granulysin
(GDP 20). According to their characteristics,12 in GDP 20,
aspartic acid (Asp, D), which is the 44th acidic amino acid,
was changed into arginine (Arg, R), which is a basic polar
amino acid, and methionine (Met, M), which is an amino
acid of the methyl sulfide group, was changed into an
amino acid of an uncharged polar cysteine (Cys, C).
Antimicrobial activities of GDP 20 in vitro

Antimicrobial activities of GDP 20 were examined against

Gram-positive (S. aureus, S. mutans and S. pyogenes) and
Gram-negative (E. coli) bacteria. The results are displayed
in Figure 4. Antimicrobial activities of GDP 20 against
other bacteria excluding S. pyogenes occurred in the concentration range of 0.657 lM. These results indicate that
the modified granulysin, GDP 20, can be applied as a
therapeutic alternative for the treatment of bacterial
diseases in humans.

(a)

(b)

(c)
Figure 2 Antimicrobial activities of granulysin-derived
peptides (GDPs) against Propionibacterium acnes. (a)
Various concentrations (050 lM) of GDPs (GDP 0106)
were incubated with Propionibacterium acnes for 4 h and
tested for antimicrobial activity using the colony-forming
unit (CFU) assay. (b) Synthetic GDPs (GDP 0714) predicted
to conform to a helixloophelix motif (a-helix 3 region, 41
51) were incubated with P. acnes at various concentrations
(050 lM) for 4 h and antimicrobial activity was determined
by CFU assay. (c) The antimicrobial activities of GDPs
(GDP 1520) were compared with those of GDP 01 and
GDP 04 in a qualitative CFU assay

Clinical outcome

Subjects
Of the 30 participants in this clinical study, one dropped
out for personal reasons and 29 completed 12 weeks of
2015 The International Society of Dermatology

treatment (16 men, 55.2%; 13 women, 44.8%). Their

mean age was 22 years (range: 1927 years). According
to the KAGS, the severity of acne was recorded as grade
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Figure 3 Predicted helical wheel representations of natural granulysin (4050, left) and modified granulysin (GDP 20, right).
Amino acids are colored by residue type: basic (blue); acidic (red); uncharged polar (brown), and non-polar hydrophobic
(green)

Figure 4 Antimicrobial activities of granulysin-derived

peptide (GDP 20) against Gram-positive (Propionibacterium
acnes, Staphylococcus aureus, Streptococcus mutans and
Streptococcus pyogenes) and Gram-negative (Escherichia
coli) bacteria. Various concentrations (0, 0.6, 5.7 and 57 lM)
of GDP 20 were incubated with the Gram-positive and
Gram-negative bacteria for 4 h and tested for antimicrobial
activity using the colony-forming unit assay

1 in eight participants, grade 2 in 11, grade 3 in nine,

and grade 4 in one.

increased at week 8 and week 12. Interestingly, the

mean  SD number of pustules decreased during the
study period from 2.93  4.44 at baseline to 0.97  2.35
at two weeks, 0.90  2.02 at four weeks, 0.62  1.61 at
eight weeks, and 0.24  0.58 at 12 weeks. In comparison
with baseline, these decreases in pustule count were significant (P = 0.002 at 2 weeks, P = 0.000 at 4 weeks,
P = 0.002 at 8 weeks, P = 0.000 at 12 weeks). The average number of nodules also consistently decreased from
week 2, although the difference was not statistically significant in comparison with the baseline count (1.90 
2.73) (Fig. 5a).
Changes in acne grade
Based on scores on the KAGS, the average grade of acne
severity decreased from 2.00  0.76 at baseline to
1.69  0.76 at two weeks, 1.66  0.67 at four weeks,
1.79  0.73 at eight weeks, and 1.69  0.60 at
12 weeks. In comparison with baseline, these differences
were statistically significant at weeks 2 and 4 (P = 0.007
at week 2, P = 0.018 at week 4, P = 0.175 at week 8,
P = 0.058 at week 12) (Fig. 5b).
Safety assessment

Changes in numbers of acne lesions (microcomedones,

papules, pustules, and nodules)
The average number of microcomedones started to
increase at week 4 and reached a mean  standard deviation (SD) of 23.97  9.33 and 22.41  9.93 at weeks 8
and 12, respectively. The differences between these counts
and the baseline count (17.10  9.76) were statistically
significant (P = 0.001 at 8 weeks; P = 0.007 at 12 weeks).
However, the mean  SD number of papules decreased
from week 2, and the reduction was statistically significant in comparison with the baseline count (15.59 
10.23) (P = 0.020 at week 4). Numbers of papules then
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No side effects or adverse reactions were observed during

the study. Of the 29 participants, six experienced mild
transient symptoms such as a burning sensation, erythema, irritation, pruritus, or xeroderma. However, these
symptoms resolved gradually after four weeks without
complications (Table 3).
Discussion
Antimicrobial peptides are known to suppress bacteria
through various mechanisms. Some peptides destabilize
the cytoplasmic membrane or extracellular membrane of
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Pharmacology and therapeutics

(a)

(b)
Figure 5 (a) Changes in numbers of acne lesions after topical application of the GDP 20 formulation. Although counts of
comedones were significantly increased, counts of other lesions were reduced by application of the GDP 20 formulation.
Statistical analysis shows a significant reduction in pustules noted at all follow-up visits. (*P < 0.05 vs. baseline). (b) Changes
in scores on the Korean Acne Grading System after topical application of the GDP 20 formulation. Acne severity in patients
using GDP 20 was reduced. Statistical analysis shows significant reductions at 2 weeks and 4 weeks (*P < 0.05 vs. baseline)

Table 3 Incidences of adverse skin reactions during the

clinical study
Patients, n (%)

Burning sensation
Erythema
Irritating sensation
Pruritus
Dry skin

2 weeks (%)

4 weeks

8 weeks

12 weeks

3
2
1
1
2

2 (6.90%)
1 (3.45%)
0
0
0

2 (6.90%)
1 (3.45%)
0
0
0

0
1 (3.45%)
0
0
0

(10.34)
(6.90)
(3.45)
(3.45)
(6.90)

susceptible bacteria and form pores and channels or conduct clearing activity.13,14 These activities change the permeability and stability of the membrane, and thus
antimicrobial cytotoxicity is achieved. This leads to the
seepage of cell contents and cell death.15 In addition,
some AMPs inhibit synthesis of DNA or protein and
intracellular function binding with molecules, which
2015 The International Society of Dermatology

represents a key factor in the survival of bacteria.16,17

Recently, a multi-hit mechanism was reported to be
responsible for the activity of AMPs at the various targets.18
In addition to antimicrobial activity, AMPs have antiinflammatory properties either through direct action on
the cell or through the suppression of bacteria-induced
inflammation.19 They bind to bacterial factors such as
lipoteichoic acid by interaction with various pathogenassociated molecular patterns (PAMPs) and suppress production of proinflammatory cytokines.20 They also inhibit
the cellular release of proinflammatory cytokines including tumor necrosis factor-a (TNF-a) and interleukin-1
(IL-1).19 Because of these characteristics, AMPs are considered to represent a viable treatment option in conditions in which both bacteria and inflammation are
causative factors. Previously a cationic peptide MX594AN was developed for clinical use and significantly reduced inflammatory lesions in patients with acne
vulgaris.19
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Granulysin is a distinctive AMP because it is released

by T cells into the skin rather than being produced in epithelial cells.15 In this sense, it is more appropriate to classify granulysin as an effective molecule of the adaptive
immune system rather than of the innate immune system.
It is a granule protein and a member of the saposin-like
protein family and contains lysine, which is found in the
NK cell. Granulysin is synthesized as a 15-kD precursor
protein and processed to a 9-kD active form for release.21
It has a single amino acid sequence with cationic charge
on the surface and an amphiphilic structure.22 Granulysin
is known to have broad-spectrum antimicrobial activity
against Gram-positive and Gram-negative bacteria, acidfast bacteria, and fungi. It can mediate the lysis of various
tumor cells and induce apoptosis.23,24
McInturff et al.6 conducted a thorough investigation of
the efficacy of granulysin in P. acnes using a CFU assay
with synthesized granulysin. The peptides containing
helixloophelix domains, which represent the crystal
structure of granulysin, and amino acid residues 135
and 3150 were found to significantly reduce the number
of viable P. acnes.6 The substitution of a tryptophan for
valine at amino acid 44, which is located between helix 2
(amino acids 2336) and helix 3 (amino acids 4251),
further increased its antimicrobial activity against
P. acnes. This was probably attributable to increased
hydrophobicity by the modification (peptide 3150v44w).
When the activity of the D-type amino acid of peptide
3150v44w (D-3150v44w) was compared with that of
widely used antibiotics, tetracycline and clindamycin,
findings showed that the antibiotics exerted bacteriostatic
activity, whereas D-3150v44w used bacteriocidal activity
to reduce P. acnes.6
Human skin is an excellent physicochemical barrier,
and most substances cannot penetrate deeply into it.
Because P. acnes resides considerably deeply inside the
skin at the sebaceous gland in the mid-dermis, peptides to
be delivered into this target area must be of an appropriate size.4 Bos et al.25 stated that the molecular weight of
topical agents should be <500 Da to penetrate skin easily.
As the size of the molecule increases, it becomes more difficult for it to penetrate the stratum corneum. Therefore,
modifying the peptide to a small molecular size is a very
important part of developing an effective topical agent.
This study tried to modify the existing sequence of the
peptide 3150v44w (GDP 01) discovered previously into
a smaller structure and also attempted to identify the
ideal structure-related activity. When each terminus of
GDP 01 was reduced, the N-terminus peptide (GDP 02)
lost antimicrobial activity, whereas the C-terminus peptide (GDP 03) maintained antimicrobial activity. This
indicates that some important structure is located around
a-helix 3. When further structural reduction was
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performed, all of GDP 0714 lost antimicrobial activity,

and molecular weight could not be reduced further. These
results illustrate that more than 11 amino acids of positive structures of the a-helix are required to enhance antimicrobial activity.
GDP 04 is produced by modifying the amino acid side
chain of GDP 03. The antimicrobial activity of GDP 04
was increased 10-fold. This may be because tyrosine (Tyr,
Y), a hydrophobic amino acid, reacted more actively with
the hydrophobic site of the a-helix structure than serine
(Ser, S) from the structural perspective. GDP 20 has the
highest antimicrobial activity against P. acne and was
found by randomly substituting the amino acid structure
of GDP 04. Antimicrobial activity analysis of GDP 20
revealed broad-spectrum activity against bacteria. Therefore, GDP 20 represents a candidate peptide for the treatment of inflammatory lesions caused by acne.
With regard to the inflammatory lesions of acne, the
topical agent containing GDP 20 reduced the number of
pustules significantly in comparison with numbers of
comedones. Propionibacterium acnes plays a key role in
inducing the inflammatory reaction in acne by releasing
diverse cytokines, including IL-12, p40, MCP-1, IP10,
and MDC, and stimulating human monocytes.6 GDP 01,
the basic structure of the AMPs discussed in this study, is
known to suppress those cytokines.6 As GDP 20 is a
derivative of GDP 01, it is speculated to inhibit the cytokines that induce the inflammatory response in acne.
In a previous in vitro experiment with isolated microcomedones from human donors, GDP 01 reduced the
number of P. acnes.6 However, with respect to the noninflammatory lesions of acne, microcomedones increased
in number after treatment in the present study. Firstly, we
should consider several possible sources of this comedogenic effect. The basic formulation used in the preparation of the topical agent in this research consisted of a
commonly used cream that is not comedogenic. In addition, a comedogenicity test of GDP 20 was carried out
and showed a negative result in comparison with a positive control. Therefore, we believe that neither the vehicle
nor GDP 20 are comedogenic. Thus, we can relate the
major pathogenetic mechanism of acne to an increase in
microcomedones, as confirmed by this study. The main
pathogenetic mechanism of acne is initiated from the microcomedone, and comedones result from hyperkeratinization, sebum production, and activation of the androgen
receptor.26 However, GDP 20, which is effective against
the proliferation of P. acnes, the other component
involved in the pathogenesis of acne, is unable to prevent
the clinical development of comedones. An anti-keratotic
agent is more useful than an anti-inflammatory agent to
deter the development of comedones. Further studies are
required to investigate the effectiveness of GDP 20 in
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Lim et al.

acne of various environmental causative factors and to

compare its efficacy with that of other known topical
agents.
In the present study, the topical agent containing
GDP 20 reduced the number of nodules (the most
advanced acne lesions) but without statistical significance.
According to KAGS classifications, GDP 20 reduced the
severity of acne in 2 to 4 weeks with statistical significance. However, KAGS scores subsequently increased.
Although this increase may reflect resistance to GDP 20,
we consider this unlikely because the count of pustules,
which represent the initial lesions of inflammatory acne,
maintained its improvement. Therefore, we think this
increase reflects the fact that the percutaneous absorption
of local agents depends not only on permeation but also
on the metabolism of the skin, affinity to the carrier, the
degree of release, and binding to unwanted mediators.
Further research into these factors is required. Although
GDP 20 has shown some restricted clinical efficacy
against the occurrence of acne lesions, investigations into
the use of synthetic peptides may represent a promising
avenue for further development.
Conclusions
Granulysin is considered an ideal substance with which to
treat acne because it has antimicrobial and anti-inflammatory effects. A new AMP, GDP 20, was synthesized by
reducing the molecular weight of GDP (peptide
3150v44w), which was reported to be effective in the
treatment of P. acnes. The present study has demonstrated an excellent antimicrobial effect of GDP 20
against P. acnes and revealed the potential of using a topical agent containing GDP 20 for inflammatory acne
treatment. In the near future, an AMP, granulysin, is
expected to play an important role as a novel therapeutic
agent in the treatment of acne, one of the most common
dermatologic conditions that affects over 85% of the population.
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