A derivative of 2-aminothiazole inhibits melanogenesis in B16 mouse melanoma cells via glycogen synthase kinase 3β phosphorylation

Article abstract of DOI:10.1111/j.2042-7158.2011.01305.x

Objectives: We have investigated whether KHG25855 (2-cyclohexylamino-1,3- thiazole hydrochloride) affected melanogenesis in B16 mouse melanoma cells, and the mechanisms involved. Methods: Melanin content and tyrosinase activity were measured using an ELISA reader after cells were treated with KHG25855. KHG25855-induced signalling pathways were examined using Western blot analysis. Key findings: KHG25855 decreased melanin production in a dose-dependent fashion, but KHG25855 did not directly inhibit tyrosinase, the rate-limiting melanogenic enzyme. The expression of microphthalmia-associated transcription factor, tyrosinase, and the related signal transduction pathways were also investigated. The effects of KHG25855 on the extracellular signal-regulated kinase and cAMP response element binding protein signalling pathways were determined, and KHG25855 was shown to have no effect on these signalling pathways. The Wnt signalling pathway is also deeply involved in melanogenesis, and so glycogen synthase kinase 3β (GSK3β) phosphorylation was assessed after KHG25855 treatment; KHG25855 caused GSK3β phosphorylation (inactivation), but the level of β-catenin was not changed by KHG25855. Furthermore, α-melanocyte stimulating hormone-induced tyrosinase expression was downregulated by KHG25855. Conclusions: We propose that KHG25855 showed hypopigmentary activity through tyrosinase downregulation via GSK3β phosphorylation. 2011 The Authors JPP

Full text of DOI:10.1111/j.2042-7158.2011.01305.x

Research Paper
JPP 2011, 63: 1031–1036
© 2011 The Authors
JPP © 2011 Royal
Pharmaceutical Society
Received November 3, 2010
Accepted April 18, 2011
DOI
A derivative of 2-aminothiazole inhibits melanogenesis in
B16 mouse melanoma cells via glycogen synthase kinase
3b phosphorylationjphp_1305 1031..1036
10.1111/j.2042-7158.2011.01305.x
ISSN 0022-3573
Su Yeon Kima, Hoh-Gyu Hahnb, Kee Dal Namb, Kyoung-Chan Parkc,
Hye-Young Yuna, Kwang Jin Baeka, Nyoun Soo Kwona and
Dong-Seok Kima
aDepartment of Biochemistry, Chung-Ang University College of Medicine, bOrganic Chemistry Laboratory,
Korea Institute of Science and Technology and cDepartment of Dermatology, Seoul National University
College of Medicine, Seoul, Korea
Abstract
Objectives We have investigated whether KHG25855 (2-cyclohexylamino-1,3-thiazole
hydrochloride) affected melanogenesis in B16 mouse melanoma cells, and the mechanisms
involved.
Methods Melanin content and tyrosinase activity were measured using an ELISA reader
after cells were treated with KHG25855. KHG25855-induced signalling pathways were
examined using Western blot analysis.
Key findings KHG25855 decreased melanin production in a dose-dependent fashion, but
KHG25855 did not directly inhibit tyrosinase, the rate-limiting melanogenic enzyme. The
expression of microphthalmia-associated transcription factor, tyrosinase, and the related
signal transduction pathways were also investigated. The effects of KHG25855 on the
extracellular signal-regulated kinase and cAMP response element binding protein signalling
pathways were determined, and KHG25855 was shown to have no effect on these signalling
pathways. The Wnt signalling pathway is also deeply involved in melanogenesis, and so
glycogen synthase kinase 3b (GSK3b) phosphorylation was assessed after KHG25855
treatment; KHG25855 caused GSK3b phosphorylation (inactivation), but the level of
b-catenin was not changed by KHG25855. Furthermore, a-melanocyte stimulating
hormone-induced tyrosinase expression was downregulated by KHG25855.
Conclusions We propose that KHG25855 showed hypopigmentary activity through tyro-
sinase downregulation via GSK3b phosphorylation.
Keywords a-melanocyte stimulating hormone; glycogen synthase kinase 3b; KHG25855;
melanogenesis; tyrosinase
Introduction
Human skin shows wide pigment gradations that range from white-to-black. This feature of
human skin is due to the amount of a chemically-stable pigment known as melanin, which
is produced in the skin.[1] Melanin synthesis takes place within specialized intracellular
organelles (melanosomes) that contain tyrosinase, the key enzyme in melanogenesis, and
tyrosinase catalyses the rate-limiting reaction of the melanogenic process.[2] Tyrosinase
catalyses the oxidation of tyrosine to dopaquinone and the oxidation of 3,4-
dihydroxyphenylalanine (DOPA) to dopaquinone.[3] Therefore, melanin production is regu-
lated primarily by the expression and activation of tyrosinase.[4,5]
The production of melanin pigment is stimulated by extracellular signals, such as ultra-
violet (UV) light, endothelin 1, a-melanocyte stimulating hormone (a-MSH), and a wide
variety of growth factors and cytokines.[1] a-MSH binds to melanocortin 1 receptor, which
stimulates melanogenesis through an increase in cAMP, followed by increased expression of
microphthalmia-associated transcription factor (MITF).[2] MITF plays an important role as a
melanocyte-specific transcription factor in melanocyte development and differentiation.[6] In
addition, MITF strongly stimulates tyrosinase expression, which suggests that MITF is an
important regulator of melanogenesis.[5,7]
Correspondence: Dong-Seok
Kim, Department of
Biochemistry, Chung-Ang
University College of Medicine,
221 Heukseok-dong
Dongjak-gu, Seoul 156-756,
Korea.
E-mail: ds_kim@cau.ac.kr
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Journal of Pharmacy and Pharmacology 2011; 63: 1031–1036
cAMP response element binding protein (CREB) phos- 2000) and anti-rabbit IgG (PI-1000) were purchased from
phorylation is known to induce the expression of MITF and to Vector Laboratories (Burlingame, CA, USA).
increase melanin synthesis.[8,9] Moreover, several reports indi-
Synthesis of KHG25855
cate that signalling pathways, such as the extracellular signal-
regulated kinase (ERK) and Akt pathways, are involved in KHG25855 was synthesized at the Organic Chemistry Labo-
melanogenesis.[10,11] Thus, inhibition of ERK and Akt results ratory (Korea Institute of Science and Technology, Seoul,
in an increase in melanin synthesis in melanocytes.[12,13]
Korea). A solution of cyclohexylisothiocyanate (1 g, 7 mmol)
MITF expression is also regulated by the Wnt signalling in ethanol (15 ml) was added to an ethanolic ammonia solu-
pathway. Wnt pathway activation results in glycogen synthase tion (28 mmol (7 ml 2.0 mol solution in ethanol)) at room
kinase 3b (GSK3b) inactivation and subsequent b-catenin temperature and the reaction mixture was refluxed for 4 h.
accumulation.[14] Furthermore, the accumulated b-catenin The reaction mixture was cooled and the precipitation was
moves into the nucleus and binds lymphoid-enhancing factor/ collected by filtration to afford cyclohexylthiourea (0.78 g
T-cell factor transcription factor, after which MITF expression (70%)). A solution of cyclohexylthiourea (0.2 g, 1.26 mmol)
is stimulated.[15] In contrast, it is known that activated GSK3b in acetone (3 ml) was added to 2-chloroacetaldehyde
leads to the ubiquitination and degradation of b-catenin.[16]
(0.24 ml, 1.26 mmol), and the reaction mixture was refluxed
Combinatorial chemistry serves as a powerful tool in lead for 24 h. The reaction mixture was cooled and the solvent was
discovery and optimization by allowing a rapid generation of removed by evaporation to give a dark blue oily liquid (0.26 g
potential candidates for screening. Lead optimization involves (94%)). This was separated by flash chromatography using
structural modifications of a hit compound to lead compound silica gel and a mixture of ethyl acetate and n-hexane (2 : 1,
that has demonstrated the desired biological or pharmacologi- v/v) as the eluent to obtain a yellow solid (0.23 g (83%)).
cal activity, often using an in-vitro assay system. Among the Crystallization from ethyl acetate and n-hexane gave a light-
1
broad range of templates, heterocyclic scaffolds represent yellow solid: mp 217–218°C; H NMR (300 MHz, CDCl3) d
the most promising molecules as leading structures for 1.27~2.31 (m, 10H, C6H11), 3.14 (methineCH, 1H, C6H11),
the discovery of novel synthetic drugs. In particular, a 6.46 (d, 3J = 4.0 Hz, 1H, CH), 7.06 (d, 3J = 4.0 Hz, 1H, CH),
2-aminothiazole core is found in numerous drugs, and clinical 9.90 (br s, 1H, NH), 13.57 (br s, 1H, HCl).
and preclinical candidates that address a broad spectrum of
targets.[17] For example, the 2-aminothiazole compounds bind
Cell culture
to enzymes, such as cyclooxygenases, kinases, and acetylcho- B16F10 murine melanoma cells were obtained from the
linesterase, and receptors, such as integrins and various Korean Cell Line Bank (Seoul, Korea). The cells were main-
members of the G-protein-coupled receptor family.[18–20]
tained in Dulbecco’s modified Eagle’s medium (DMEM),
As a part of an effort for exploring new hypopigmentary supplemented with 10% (v/v) fetal bovine serum (Hyclone,
compounds, we have examined our in-house indigenous Logan, UT, USA), 50 mg/ml streptomycin and 50 mg/ml peni-
chemical library. KHG25855 (2-cyclohexylamino-1,3- cillin (Hyclone) in 5% CO2 at 37°C.
thiazole hydrochloride), a 2-aminothiazole derivative, was
Cell viability assay
selected from the primary screening. Thus, we determined the
effects of KHG25855 on melanin synthesis and tyrosinase Cell viability was determined using a crystal violet assay.After
activity using B16 mouse melanoma cells. In addition, we incubating cells with KHG25855 for 24 h, the media were
determined the effects of KHG25855 on several signalling removed, and the cells were stained with 0.1% crystal violet in
pathways related to melanogenesis.
10% ethanol for 5 min at room temperature. The cells were
then rinsed four times with distilled water, and the crystal violet
retained by adherent cells was extracted with 95% ethanol.
Absorbance was determined at 590 nm using an ELISAreader
(VersaMax; Molecular Devices, Sunnyvale, CA, USA).
Materials and Methods
Materials
Measurement of melanin content
KHG25855 was dissolved in dimethyl sulfoxide (DMSO)
and stored at -20°C as a stock solution (50 mm). Synthetic Extracellular melanin release was measured as described
melanin, 3,4-dihydroxy-l-phenylalanine
(L-DOPA), previously, with a slight modification.[21] Briefly, B16F10 cells
(2′Z,3′E)-6-bromoindirubin-3′-oxime (BIO), and mushroom were incubated at a density of 1 ¥ 105 cells in 6-well plates
tyrosinase were purchased from Sigma-Aldrich Co. (St Louis, overnight. a-MSH (1 mm) was then added and cells were
MO, USA). Antibodies specific for phospho-ERK1/2 treated with increasing concentrations of KHG25855 in
(Thr202/Tyr204, #9101S), total (phosphorylated and non- phenol red-free DMEM for three days. Samples (200 ml) were
phosphorylated) ERK1/2 (#9102), phospho-CREB (ser133, then placed in 96-well plates and the optical density (OD)
#9198), total CREB (#9197), phospho-GSK3b (#9336), total of each culture well was measured using an ELISA reader
GSK3b (#9315), and b-catenin (#9581) were purchased from at 400 nm. The number of cells was then counted using a
Cell Signaling Technology (Beverly, MA, USA). Antibodies haemocytometer. Melanin production was expressed as the
specific for tyrosinase (C-19) and actin (I-19) were purchased percentage of a-MSH-treated controls.
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA),
and microphthalmia Ab-1 (C5, MS-771-P0) was obtained
Tyrosinase activity
from NeoMarkers (Fremont, CA, USA). Secondary antibod- Tyrosinase activity was assayed as DOPA oxidase activity.
ies specific for anti-goat IgG (PI-9500), anti-mouse IgG (PI- B16F10 cells were incubated at a density of 1 ¥ 105 cells in

Inhibition of melanogenesis by KHG25855
Su Yeon Kim et al.
1033
6-well plates and incubated with KHG25855 in DMEM for
three days. Cells were washed with phosphate buffered saline
(PBS) and lysed with lysis buffer (0.1 m phosphate buffer, pH
6.8, containing 1% Triton X-100). They were then disrupted
by freeze-thawing, and lysates were clarified by centrifuga-
tion at 13 000 rev/min for 30 min. After quantifying protein
content using a protein assay kit (Bio-Rad, Hercules, CA,
USA), the cell lysates were adjusted to the same amount
of protein with lysis buffer, 90 ml of each lysate was pipetted
into the wells of a 96-well plate and 10 ml 10 mm L-DOPA
was added. Control wells contained 90 ml lysis buffer and
10 ml 10 mm L-DOPA. After incubation at 37°C for 20 min,
dopachrome formation was monitored by measuring absor-
bance at 475 nm using an ELISA reader.
A cell-free assay system was used to determine the
direct effect of KHG25855 on tyrosinase activity. Phosphate
buffer (70 ml) containing KHG25855 was mixed with 20 ml
53.7 U/ml mushroom tyrosinase, and 10 ml 10 mm L-DOPA
was then added. Following incubation at 37°C for 20 min, the
absorbance was measured at 475 nm.
S
H
N
HCl
N
Figure 1 The structure of KHG25855.
(a)
120
100
**
80
60
40
20
0
**
**
0
Control
1
10
25
50
KHG25855 (μM)
Western blot analysis
a-MSH (1μM)
Cells were lysed in cell lysis buffer (62.5 mm Tris-HCl, pH 6.8,
2% sodium dodecyl sulfate (SDS), 5% b-mercaptoethanol,
2 mm phenylmethylsulfonyl fluoride, and protease inhibitors
(Complete; Roche, Mannheim, Germany), 1 mm Na3VO4,
50 mm NaF, and 10 mm EDTA). A20 mg sample of protein per
lane was separated by SDS-polyacrylamide gel electrophoresis
and blotted onto polyvinylidene fluoride membranes, which
were then saturated with 5% dried milk in Tris-buffered saline
containing 0.5% Tween 20. Blots were then incubated with the
appropriate primary antibodies at a dilution of 1 : 1000, and
further incubated with horseradish peroxidase-conjugated sec-
ondary antibody. Bound antibodies were detected using an
enhanced chemiluminescence plus kit (Amersham Inter-
national, Little Chalfont, UK). The images of the blotted
membranes were obtained using a LAS-1000 lumino-image
analyser (Fuji Film, Tokyo, Japan).
(b)
120
100
80
60
40
20
**
**
**
Control
0
1
10
25
50
KHG25855 (μM)
a-MSH (1μM)
Figure 2 Effects of KHG25855 on melanogenesis in B16 mouse
cells. B16 cells were treated with KHG25855 (1–50 mm) in the presence
of a-melanocyte stimulating hormone (a-MSH; 1 mm) for three days.
Melanin content (a) and tyrosinase activity (b) were measured. The
results shown are averages of triplicate experiments Ϯ SD. **P < 0.01
compared with a-MSH-treated controls (a and b) and compared with the
untreated control.
Statistics
The differences between the results (n = 3) were assessed
using a Kruskal-Wallis test followed by a Dunnett’s test.
P-values Յ0.01 were considered significant.
dose-dependent manner. Furthermore, KHG25855 treatment
significantly reduced tyrosinase activity, which was stimu-
lated by a-MSH (Figure 2b).
Results
Effects of KHG25855 on melanin synthesis and
tyrosinase activity
In addition, we determined whether KHG25855 had a
direct effect on tyrosinase activity using mushroom tyrosi-
nase; however, KHG25855 showed no direct effect on tyrosi-
nase activity (data not shown). These results indicated that the
inhibitory activity of KHG25855 on melanogenesis may have
resulted from inhibition of the melanogenic pathway involv-
ing tyrosinase expression, not from the direct inhibition of
tyrosinase.
The structure of KHG25855 is shown in Figure 1. B16 cells
were treated with KHG25855 at various concentrations
(1–100 mm) for 24 h and cell viability was determined using
the crystal violet assay. Our results showed that KHG25855
was not cytotoxic to B16 cells at concentrations of 1–100 mm
(data not shown). To determine the effects of KHG25855
on melanogenesis, cells were cultured with KHG25855
(1–50 mm) for three days in the presence of a-MSH (1 mm),
which increases melanin synthesis, and extracellular melanin
Effects of KHG25855 on the signalling pathways
related to melanogenesis
release was measured. As shown in Figure 2a, KHG25855 It is known that ERK and CREB phosphorylation regulates
treatment reduced a-MSH-induced melanin synthesis in a melanogenesis. Therefore, we determined whether ERK and

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Journal of Pharmacy and Pharmacology 2011; 63: 1031–1036
(a)
a-MSH (1 μM)
KHG25855 (25 μM)
0
24
48
72
(h)
KHG25855 (25 μ
-
-
+
-
+
-
+
M)
0
2
10
30
60 180 360 (min)
Phospho-ERK
MITF
Tyrosinase
Actin
ERK
Phospho-CREB
Figure 4 Effects of KHG25855 on the microphthalmia-associated tran-
scription factor and tyrosinase protein expression. B16 mouse melanoma
cells were treated with a-melanocyte stimulating hormone (a-MSH;
1 mm) and KHG25855 (25 mm) for 24–72 h. Whole cell lysates were
then subjected to Western blot analysis using antibodies against
microphthalmia-associated transcription factor (MITF) and tyrosinase.
Equal protein loadings were confirmed using anti-actin antibody.
CREB
(b)
KHG25855 (25 μM)
10 30 60 180 360 (min)
0
2
Phospho-GSK3b
(a)
GSK3b
120
100
80
60
40
20
0
**
b-Catenin
Actin
Figure 3 Effects of KHG25855 on the signal transduction pathways in
B16 mouse melanoma cells. After 24 h of serum starvation, B16 cells
were treated with 25 mm KHG25855 for the times indicated. Whole cell
lysates were then subjected to Western blot analysis using antibodies
against phospho-specific extracellular signal-regulated kinase (ERK) and
phospho-specific cAMP response element binding protein (CREB) (a) or
using antibodies against phospho-specific glycogen synthase kinase 3b
(GSK3b), total GSK3b, and b–catenin (b). Equal protein loadings were
confirmed using ERK, CREB, or actin antibody.
Control
–
–
+
–
+
+
KHG25855 (25 μM)
BIO (1 μM)
a-MSH (1 μM)
(b)
BIO (1 μM)
Con
+
–
+
KHG25855 (25 mM)
Phospho-GSK3b
CREB could be affected by KHG25855 in a time-course
experiment. KHG25855 showed no effects on ERK and
CREB phosphorylation (Figure 3a). In contrast, we showed
that GSK3b phosphorylation was clearly induced by 25 mm
KHG25855 (Figure 3b). However, we showed that the
b-catenin level was not changed by 25 mm KHG25855
(Figure 3b).
b-Catenin
Next, we evaluated changes in MITF and tyrosinase
protein levels in a time-course experiment after 24–72 h of
KHG25855 (25 mm) treatment in the presence of a-MSH
(1 mm). Interestingly, MITF levels did not change between 24
and 72 h of KHG25855 treatment compared with a-MSH-
treated cells. A slight decrease in MITF was observed at 48 h.
In contrast, a clear decrease in tyrosinase protein was demon-
strated after 48–72 h of treatment with KHG25855 (Figure 4).
Actin
Figure 5 Effects of KHG25855 on the glycogen synthase kinase 3b
phosphorylation and melanogenesis. B16 mouse melanoma cells were
treated with KHG25855 (25 mm) in the presence or absence of (2′Z,3′E)-
6-bromoindirubin-3′-oxime (BIO; 1 mm) after adding a-melanocyte
stimulating hormone (a-MSH; 1 mm) for three days. (a) Melanin content
was measured. Each determination was made in triplicate; the data shown
represent the mean Ϯ SD. **P < 0.01 compared with the KHG25855-
treated group. (b) Cells were cultured with KHG25855 (25 mm) for 6 h in
the presence or absence of BIO (1 mm). Con, control. Whole cell lysates
were then subjected to Western blot analysis with antibodies against
phospho-specific glycogen synthase kinase 3b (GSK3b) or b–catenin.
Equal protein loading was confirmed using actin antibody.
Effects of KHG25855 on GSK3b phosphorylation
and melanogenesis
We determined the involvement of GSK3b phosphorylation
by KHG25855 in melanogenesis of B16 cells using BIO, a
specific GSK3b phosphorylation inhibitor. Cells were treated
with KHG25855 for three days in the presence or absence
of BIO. BIO was shown to restore KHG25855-induced
melanin reduction (Figure 5a). Moreover, BIO blocked

Inhibition of melanogenesis by KHG25855
Su Yeon Kim et al.
1035
GSK3b phosphorylation in KHG25855-treated cells
(Figure 5b). These data clearly indicated that GSK3b phos-
phorylation was involved in the inhibition of melanogenesis
by KHG25855.
Conclusions
We have demonstrated that KHG25855 did not regulate the
ERK or CREB pathway, although KHG25855 decreased mel-
anogenesis. Rather, we showed that KHG25855 led to GSK3b
phosphorylation, which may have reduced the MITF binding
affinity and subsequently decreased the tyrosinase level.
We propose that KHG25855 may be useful as a new skin-
whitening agent which decreases the level of tyrosinase.
Discussion
Exploration of agents that act on the inhibition of melanin
synthesis would be of benefit in understanding and studying
the precise role of the related signal transduction pathways
and their functions in melanogenesis. Thus, the effects of
KHG25855, a 2-aminothiazole derivative, on melanin synthe-
sis and the related signalling pathways were studied using
B16 mouse melanoma cells.
Declarations
Conflict of interest
The Author(s) declare(s) that they have no conflicts of interest
to disclose.
In this study, we have shown that KHG25855 inhibited
melanin synthesis induced by a-MSH in B16 cells. Further-
more, we determined how KHG25855 regulated the molecu-
lar mechanism involved in melanogenesis. MITF is a major
regulator of the expression of tyrosinase.[22,23] The cAMP
pathway is involved in the regulation of melanogenesis, and
so we determined whether KHG25855 influenced CREB
phosphorylation.[24] Notably, we showed that KHG25855 did
not have an effect on CREB phosphorylation (Figure 3a). In
addition, several reports have suggested that cAMP upregula-
tion activated ERK in B16 melanoma cells and ERK is an
important regulator of melanin synthesis.[25,26] Furthermore,
Kim et al.[11] reported that ERK activation induced MITF
phosphorylation and subsequent degradation; however,
KHG25855 did not activate the ERK pathway in this study
(Figure 3a). These results indicated that KHG25855 did not
decrease melanin synthesis by regulating CREB or ERK acti-
vation. We investigated the possibility for direct inhibition of
tyrosinase by KHG25855. However, KHG25855 did not
inhibit tyrosinase directly. These results indicated that other
signalling pathways were involved in KHG25855-induced
hypopigmentation.
Funding
This study was supported by a grant (A100179) from the
Korea Healthcare Technology R&D Project, Ministry of
Health and Welfare, Republic of Korea.
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