Antimicrobial Efficacy of Granulysin-Derived Synthetic Peptides in Acne Vulgaris
Antimicrobial Efficacy of Granulysin-Derived Synthetic Peptides in Acne Vulgaris
Antimicrobial Efficacy of Granulysin-Derived Synthetic Peptides in Acne Vulgaris
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
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Lim et al.
Recombinant granulysin
blood cells (Remel, Inc., Lenexa, KS, USA), 0.5 mg/L vitamin
K, and 5.0 mg/L hemin (Remel, Inc.). Plates were incubated
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-
that might affect acne. All subjects were evaluated for allergic
CA, USA).
order to check for any irritability and toxicity in skin. The patch
summarized in Table 1.
Lim et al.
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
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.
Results
Characteristic background of synthesized peptides
855
856
(a)
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(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
Lim et al.
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
(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
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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)
Lim et al.
(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)
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
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23 Pe~
na SV, Krensky AM. Granulysin, a new human
cytolytic granule-associated protein with possible
involvement in cell-mediated cytotoxicity. Semin
Immunol 1997; 9: 117125.
24 Kaspar AA, Okada S, Kumar J, et al. A distinct pathway
of cell-mediated apoptosis initiated by granulysin.
J Immunol 2001; 167: 350356.
Lim et al.
25 Bos JD, Meinardi MM. The 500 Dalton rule for the skin
penetration of chemical compounds and drugs. Exp
Dermatol 2000; 9: 165169.
26 Harder J, Tsuruta D, Murakami M, et al. What is the
role of antimicrobial peptides (AMP) in acne vulgaris?
Exp Dermatol 2013; 22: 386391.
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