The hypopigmentary action of KI-063 (a new tyrosinase inhibitor) combined with terrein

Article abstract of DOI:10.1211/jpp.60.3.0009

Resorcinol derivatives are known to inhibit melanin synthesis. In this study, resorcinol derivatives were synthesized and screened for their activity on melanogenesis. KI-063 (a tyrosinase inhibitor) was examined for its effects on melanogenesis using a spontaneously immortalized mouse melanocyte cell line (Mel-Ab). In a cell-free system, KI-063 directly inhibited tyrosinase, the rate-limiting melanogenic enzyme. Moreover, in a cell system, it inhibited melanin synthesis in a concentration-dependent manner. In addition, KI-063 inhibited the activity of cellular tyrosinase. Thus, this study examined the effects of a combination of KI-063 with terrein, an agent that down-regulates microphthalmia-associated transcription factor. The data suggest that KI-063 has an additive effect in combination with terrein. Thus, the suppression of tyrosinase activity by KI-063 and the inhibition of tyrosinase production by terrein appear to be an optimal combination for skin whitening.

Full text of DOI:10.1211/jpp.60.3.0009

JPP 2008, 60: 343–348
© 2008 The Authors
Received June 3, 2007
Accepted November 13, 2007
DOI 10.1211/jpp.60.3.0009
ISSN 0022-3573
The hypopigmentary action of KI-063 (a new
tyrosinase inhibitor) combined with terrein
Dong-Seok Kim, Sangku Lee, Hyun-Kyung Lee, Seo-Hyoung Park,
In-Ja Ryoo, Ick-Dong Yoo, Sun-Bang Kwon, Kwang Jin Baek,
Jung-Im Na, Kyoung-Chan Park
Abstract
Department of Biochemistry,
College of Medicine, Chung-Ang
University, 221 Heukseok-Dong
Dongjak-Gu, Seoul 156-756,
Republic of Korea
Resorcinol derivatives are known to inhibit melanin synthesis. In this study, resorcinol deriva-
tives were synthesized and screened for their activity on melanogenesis. KI-063 (a tyrosinase
inhibitor) was examined for its effects on melanogenesis using a spontaneously immortalized
mouse melanocyte cell line (Mel-Ab). In a cell-free system, KI-063 directly inhibited tyrosinase,
the rate-limiting melanogenic enzyme. Moreover, in a cell system, it inhibited melanin synthesis
in a concentration-dependent manner. In addition, KI-063 inhibited the activity of cellular tyrosi-
nase. Thus, this study examined the effects of a combination of KI-063 with terrein, an agent that
down-regulates microphthalmia-associated transcription factor. The data suggest that KI-063 has
an additive effect in combination with terrein. Thus, the suppression of tyrosinase activity by KI-063
and the inhibition of tyrosinase production by terrein appear to be an optimal combination for skin
whitening.
Dong-Seok Kim, Kwang Jin Baek
National Cosmeceutical Research
Center, Korea Research Institute
of Bioscience and Biotechnology,
52 Oeun-Dong, Yuseong-Gu,
Daejeon 305-333, Korea
Sangku Lee, In-Ja Ryoo,
Ick-Dong Yoo
Department of Dermatology,
Seoul National University
College of Medicine,
Introduction
28 Yongon-Dong, Chongno-Gu,
Seoul 110-744, Republic of Korea
The production and distribution of melanin pigment is a major determinant of skin and
hair colour. Tyrosinase is a major melanosomal enzyme in melanogenesis, which catalyses
the rate-limiting reaction of the melanogenic process (Hearing & Jimenez 1989; Slominski
et al 2004). Therefore, melanin production depends mainly on the expression and activa-
tion of tyrosinase (Hearing & Tsukamoto 1991). Previous studies reported that resorcinol
(1,3-dihydroxybenzene) derivatives inhibit the activity of tyrosinase (Tasaka et al 1998).
Compounds with a 4-substituted resorcinol skeleton are potent inhibitors of tyrosinase
(Shimizu et al 2000; Kim & Uyama 2005; Song et al 2007). Some of these polyphenols
can act as both inhibitors and substrates through a complex reaction involving tyrosinase
(Espin et al 1997; Fenoll et al 2003). Indeed, several polyphenols act as suicide substrates,
causing a mechanism-based inactivation of tyrosinase (Garcia Canovas et al 1987; Tudela
et al 1988; Haghbeen et al 2004; Chang 2007). Moreover, we have recently reported that
4-n-butylresorcinol strongly inhibits melanogenesis (Kim et al 2005), and this agent has
been useful as an experimental treatment for melasma (Khemis et al 2007). Thus, resorcinol
derivatives (including KI-063) were synthesized and screened for their inhibitory effects
on tyrosinase.
Microphthalmia-associated transcription factor (MITF) is known to regulate tyrosi-
nase expression and melanin synthesis at the transcription level (Yasumoto et al 1997;
Bertolotto et al 1998; Lin & Fisher 2007). Hence, mutations in the MITF gene cause
abnormal pigmentation of the skin and hair (Hughes et al 1994; Tassabehji et al 1994;
Tachibana 1997). Activation of the extracellular signal-regulated kinase (ERK) pathway
has been shown to decrease levels of MITF and to inhibit melanin synthesis (Englaro et al
1998; Kim et al 2002, 2003). We have shown that terrein, a bioactive fungal metabolite,
down-regulates MITF and tyrosinase through activation of ERK and thus reduces melanin
synthesis (Park et al 2004). We therefore hypothesized that the inhibition of tyrosinase
Hyun-Kyung Lee, Seo-Hyoung
Park, Jung-Im Na,
Kyoung-Chan Park
Welskin Co. Ltd,
28 Yongon-Dong, Chongno-Gu,
Seoul 110-744, Korea
Sun-Bang Kwon
Correspondence: Kyoung-Chan
Park, Department of
Dermatology, Seoul National
University Bundang Hospital,
300 Gumi-Dong, Bundang-Gu,
Seongnam-Si, Kyoungki-Do
463-707, Korea. E-mail:
gcpark@snu.ac.kr
Acknowledgements: This study
was supported by a grant
(A050432) from the Korea
Health 21 R&D Project, Ministry
of Health and Welfare, Republic
of Korea. We also wish to
acknowledge the support of the
Welskin Corporation (Seoul,
Republic of Korea).
343

344
Dong-Seok Kim et al
production by terrein might strongly potentiate the inhibitory the cells were treated with KI-063 at various concentrations
effect of tyrosinase inhibitors on melanin synthesis.
for 4 days. The cell pellets were dissolved in 1 mL 1 m NaOH
This study examined the effects of KI-063 on melanin at 100ꢁC for 30 min and centrifuged for 20 min at 16,000 g.
synthesis and tyrosinase activity in a spontaneously immor- The optical density of the supernatants was measured at
talized mouse melanocyte cell line (Mel-Ab), alone and in
combination with terrein.
400 nm using an ELISA reader (TECAN). Before measuring
the melanin content, the cells were observed under a phase-
contrast microscope (Olympus Optical Co., Tokyo, Japan)
and photographed with a CoolSNAPcf digital video camera
system (Roper Scientific, Inc., Tucson, AZ, USA) supported
by RS Image software (Roper Scientific).
Materials and Methods
Materials
KI-063 and terrein were synthesized at the Korea Research
Institute of Bioscience and Biotechnology (Lee et al 2005).
Cholera toxin (CT), 12-O-tetradecanoylphorbol-13-acetate
(TPA), synthetic melanin, l-DOPA, and mushroom tyrosi-
nase were obtained from Sigma (St Louis, MO, USA).
Tyrosinase activity
The tyrosinase activity was assessed using a slight modifica-
tion of the method described by Busca et al (1996). Briefly,
Mel-Ab cells were cultured in six-well plates and incubated
with KI-063 at various concentrations for 4 days. The cells
were then washed with ice-cold phosphate-buffered saline
and lysed with 0.1 m phosphate buffer (pH 6.8) containing
1% Triton X-100. Cells were then disrupted by freezing and
thawing, and the lysates clarified by centrifugation at 10 000 g
for 5 min. After quantifying protein levels and adjusting
the concentrations with lysis buffer, 90ꢃL of each lysate
containing the same amount of protein was placed in each
well of a 96-well plate and 10ꢃL 10mm l-DOPA added to
each well. The control wells contained 90ꢃL 0.1 m phosphate
buffer (pH 6.8) and 10ꢃL 10mm l-DOPA. After incuba-
tion at 37ꢁC, the absorbance at 475 nm was measured every
10 min for at least 1 h using an ELISA plate reader.
Synthesis of KI-063
KI-063 was synthesized through the esterification of
dihydrocaffeic acid with 4-n-butylresorcinol using NꢀNꢀ-
dicyclohexylcarbodiimide and 4-di(methylamino)pyridine.
NꢀNꢀ-dicyclohexylcarbodiimide (720 mg, 3.49 mm) and
4-di(methylamino)pyridine (60 mg, 0.49 mm) were added to
a solution of dihydrocaffeic acid (500 mg, 2.75 mm) and 4-
n-butylresorcinol (450 mg, 2.71 mm) in ethyl acetate (30 mL)
at room temperature. The reaction mixture was stirred for
24 h, concentrated, and purified by silica gel column chro-
matography (2:1 hexane/ethyl acetate) to give KI-063. Its
chemical structure was analysed by NMR as follows: 1H
NMR (CDCl3, 300 MHz), ꢁ (ppm) 7.04 (d, J = 8ꢂ4Hz, 1H),
6.78 (d, J = 8ꢂ4Hz, 1H), 6.76 (d, J = 3ꢂ0Hz, 1H), 6.68 (dd,
J = 8ꢂ1, 1.8 Hz, 1H), 6.62 (dd, J = 8ꢂ1, 2.7 Hz, 1H), 6.41 (d,
J = 2ꢂ4Hz, 1H), 5.19 (br s, 3H), 2.96 (t, J = 6ꢂ6Hz, 2H),
2.84 (t, J = 6ꢂ6Hz, 2H), 2.33 (t, J = 7ꢂ8Hz, 2H), 1.45 (m,
2H), 1.26 (m, 2H), 0.88 (t, J = 7ꢂ5Hz, 3H).
A cell-free assay system was used to examine the direct
effects of KI-063 on tyrosinase activity. Phosphate buffer
ꢄ70ꢃLꢅ containing various concentrations of KI-063 was
combined with 20ꢃL 10ꢃg mL−1 mushroom tyrosinase, and
10ꢃL 10mm l-DOPA was added to each well. After incu-
bation at 37ꢁC, the absorbance was measured at 475 nm.
Statistical analysis
Cell cultures
Differences between groups were assessed using the Kruskal–
Wallis test followed by Dunnett’s test. P values below 0.01
were considered significant.
The Mel-Ab cell line is a mouse-derived spontaneously
immortalized melanocyte cell line that produces large
amounts of melanin (Dooley et al 1994). Mel-Ab cells were
cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum, 100 nm TPA,
1 nm CT, 50ꢃg mL−1 streptomycin, and 50U mL−1 peni-
cillin at 37ꢁC in 5% CO2.
Results
Cytotoxicity of KI-063 in Mel-Ab cells
Cell viability
KI-063 is a resorcinol derivative prepared from 4-n-
butylresorcinol and dihydrocaffeic acid (Figure 1). To deter-
mine whether KI-063 can be used safely as a skin-whitening
agent, Mel-Ab cells were treated with 0ꢂ01–50ꢃm KI-063,
and the cell viability measured using a crystal violet assay.
KI-063 had no cytotoxic effect in Mel-Ab cells at the concen-
trations used (data not shown).
Cell viability was determined using a crystal violet assay
(Dooley et al 1994). After incubating with KI-063 for 24 h,
the culture medium was removed and the cells were stained
with 0.1% crystal violet in 10% ethanol for 5 min at room
temperature, then rinsed four times with distilled water. The
crystal violet retained by the adherent cells was then extracted
with 95% ethanol. The absorbance was determined at 590 nm
using an ELISA reader (TECAN, Salzburg, Austria).
KI-063 decreased melanogenesis in Mel-Ab cells
To investigate the hypopigmentation effect, KI-063
ꢄ0ꢂ1–50ꢃmꢅ was added to cultured Mel-Ab cells for 4 days.
Measurement of melanin content and microscopy
The melanin content was measured using a slight modifica- The Mel-Ab cells were then photographed under a phase-
tion of the method described by Tsuboi et al (1998). Briefly, contrast microscope. Cells treated with KI-063 showed much

Inhibition of melanogenesis by KI-063
345
Combination of KI-063 with terrain
O
OH
OH
We have previously reported that terrein down-regulates
MITF and tyrosinase and thus reduces melanin synthesis
(Park et al 2004). KI-063 (10 or 20ꢃm) was used in combi-
nation with terrein (1, 10 or 20ꢃm) to examine the combined
effects of the tyrosinase inhibitor and the tyrosinase produc-
tion inhibitor. Terrein had no cytotoxic effect on Mel-Ab cells
at the concentrations used (Park et al 2004). After 4 days,
the Mel-Ab cells were photographed using phase-contrast
microscopy. Cells co-treated with KI-063 and terrein showed
much less pigmentation than cells treated with KI-063 or
terrein alone (Figure 3). Melanin content and tyrosinase
activity were measured after 4 days’ treatment with KI-063
and terrein. Consistent with the microscopic observations,
the combination of KI-063 and terrein had an additive effect
and significantly inhibited melanin synthesis and the tyrosi-
nase activity, respectively, at concentrations above 10ꢃm
(Figures 4 and 5). The addition of 1ꢃm terrein to KI-063
did not have any synergistic effects. This suggests that the
potent inhibition of tyrosinase activity by KI-063 works in
combination with the reduced level of tyrosinase induced by
terrein.
OH
O
O
OH
HO
H3C
KI-063
Figure 1 The structures of KI-063 and terrein.
Terrein
less pigmentation than untreated cells (Figure 2). Melanin
content was measured after 4 days’ treatment with KI-
063. Consistent with the microscopic observations, KI-063
ꢄ10–50ꢃmꢅ significantly inhibited melanogenesis (Table 1).
Tyrosinase is a critical enzyme in melanogenesis and is
involved in the rate-limiting step. We therefore measured
tyrosinase activity after treatment with KI-063 (same concen-
trations), which was found to decrease as a function of the
KI-063 dose (Table 1). This suggests that KI-063 inhibits
tyrosinase activity and subsequently suppresses melanin
synthesis in Mel-Ab cells.
Discussion
Tyrosinase is a key enzyme that regulates the rate-limiting
step of melanin synthesis (Ando et al 2007). In addition,
resorcinol derivatives have been reported to inhibit tyrosi-
nase activity (Tasaka et al 1998). KI-063 is a synthetic resor-
cinol derivative. In this study, KI-063 inhibited tyrosinase
activity and, as a result, significantly suppressed melanin
synthesis. Thus, it appears that a decrease in tyrosinase
activity may contribute to the lower pigment content in the
KI-063-treated cells.
KI-063 directly inhibited tyrosinase in a cell-free system
To determine whether KI-063 inhibits tyrosinase activity
directly, its effect on mushroom tyrosinase was measured
in a cell-free system. These results showed that KI-063
had a strong concentration-dependent inhibitory effect on
tyrosinase activity (Table 1). This indicates that KI-063 can
inhibit melanogenesis, possibly through direct inhibition of
tyrosinase.
MITF is known to regulate the expression of tyrosinase
(Busca & Ballotti 2000; Tachibana 2000; Lin & Fisher 2007).
0.1 µM
1 µM
control
10 µM
20 µM
50 µM
Figure 2 Phase-contrast photomicrographs showing the effects of KI-063 on melanin synthesis in Mel-Ab cells (cultured with 0ꢂ1–50ꢃm KI-063
for 4 days).

346
Dong-Seok Kim et al
Table 1 The effects of KI-063 on melanin synthesis and tyrosinase activity in Mel-Ab cells
KI-063 concn ꢄꢃmꢅ
0
0.01
–
0.1
1
10
20
50
Melanin content
Tyrosinase activity
Mel-Ab cells
100ꢂ0 3ꢂ6
108ꢂ5 3ꢂ7
98ꢂ8 6ꢂ7
85ꢂ2 2ꢂ2∗
76ꢂ6 3ꢂ9∗
47ꢂ7 2ꢂ9∗
100ꢂ0 6ꢂ0
100ꢂ0 5ꢂ7
–
94ꢂ7 9ꢂ7
70ꢂ2 5ꢂ3∗
79ꢂ4 12ꢂ3
65ꢂ6 3ꢂ3∗
60ꢂ8 11ꢂ1∗
50ꢂ2 10ꢂ5∗
34ꢂ4 10ꢂ7∗
40ꢂ5 15ꢂ6∗
14ꢂ5 17ꢂ2∗
40ꢂ9 2ꢂ3∗
Cell-free system
79ꢂ4 1ꢂ6∗
Values are % of control activity, mean s.d. of three replicates. ∗P < 0ꢂ01vs untreated control.
KI-063
10
µ
M
20 µM
control
1
10
20
µ
µ
µ
M
M
M
Terrein
Figure 3 Phase-contrast photomicrographs showing the effects of KI-063 and terrein on melanin synthesis in Mel-Ab cells (cultured with 10 or
20ꢃm KI-063 without or with 1, 10, or 20ꢃm terrein for 4 days).
Previous studies suggest that ERK is an important kinase the structure of KI-063 suggests that it may act as a compet-
involved in melanin synthesis, as ERK activation induces itive inhibitor.
the phosphorylation and subsequent degradation of MITF
Many studies have focused on tyrosinase inhibitors for the
(Hemesath et al 1998; Wu et al 2000; Xu et al 2000). In addi- development of skin-whitening agents. Many such inhibitors
tion, ERK activation by terrein down-regulates MITF, which have been developed from studies using model enzymes
results in reduced tyrosinase levels and reduced melanogen- such as mushroom and plant tyrosinases (Kim & Uyama
esis (Park et al 2004). However, terrein does not inhibit the 2005; Chang 2007). Although these inhibitors have hypopig-
activity of tyrosinase directly. In this study, it was found that menting effects in-vitro, only a few substances were found
KI-063 directly inhibits tyrosinase (Figure 5). Although the to have effective hypopigmenting activity in clinical trials.
mechanism by which KI-063 inhibits tyrosinase is unclear, Moreover, they often induce skin irritation or permanent

Inhibition of melanogenesis by KI-063
347
P<0.01
P<0.01
P<0.01
P<0.01
Conclusions
These results showed that a combination of KI-063 and
terrein acted synergistically, indicating that the strong tyrosi-
nase inhibitory activity of KI-063 works in combination
with the reduced level of tyrosinase due to terrein. Because
melanogenesis in the skin is complex and involves different
stages of regulation, this combination suggests a new concept
for skin-whitening agents. Future studies should consider a
combination of KI-063 and MITF regulators to maximize the
hypopigmentary effect.
120
100
80
60
40
20
0
∗∗
∗∗
∗∗
∗∗
KI-063 ( M)
µ
0
10 20
0
10 20
0
10 20
0
10 20
Terrein 1
µ
M
Terrein 10
µ
M
Terrein 20 M
µ
Figure 4 Effects of KI-063 and terrein on melanin content of Mel-Ab
cells cultured with 10 or 20ꢃm KI-063 without or with 1, 10, or 20ꢃm
terrein for 4 days. Data are mean s.d. of three replicate experiments.
∗P < 0ꢂ01vs untreated control.
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