Anti-melanogenic effect of (Z)-5-(2,4-dihydroxybenzylidene) thiazolidine-2,4-dione, a novel tyrosinase inhibitor

Article abstract of DOI:10.1007/s12272-013-0184-5

We synthesized (Z)-5-(2,4-dihydroxybenzylidene)thiazolidine-2,4-dione (MHY498) as a potential tyrosinase inhibitor. MHY498 potently inhibited mushroom tyrosinase activity (mean IC₅₀ = 3.55 μM) in a dose-dependent manner. MHY498 was more potent than the well-known tyrosinase inhibitor, kojic acid (mean IC₅₀ = 22.79 μM). When tested in B16F10 melanoma cells treated with α-melanocyte stimulating hormone (α-MSH), MHY498 inhibited murine tyrosinase activity and decreased melanin production without inducing cytotoxicity. Docking models showed that the binding affinity of MHY498 to tyrosinase was higher than that of kojic acid, and docking simulation results indicated that the tyrosinase binding moieties of MHY498 and kojic acid were similar. Western blotting showed that tyrosinase inhibition by MHY498 partly resulted from the expressional modulations of tyrosinase and its transcription factor, microphthalmia-associated transcription factor, via the cAMP-PKA-CREB pathway. These findings suggest that MHY498 could be useful as an antimelanogenic agent for the prevention and treatment of diseases associated with skin pigmentation.

Full text of DOI:10.1007/s12272-013-0184-5

Arch. Pharm. Res.
DOI 10.1007/s12272-013-0184-5
RESEARCH ARTICLE
Anti-melanogenic effect of (Z)-5-(2,4-dihydroxybenzylidene)
thiazolidine-2,4-dione, a novel tyrosinase inhibitor
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So Hee Kim Young Mi Ha Kyoung Mi Moon Yeon Ja Choi
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Yun Jung Park Hyoung Oh Jeong Ki Wung Chung Hye Jin Lee
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Pusoon Chun Hyung Ryong Moon Hae Young Chung
Received: 24 March 2013 / Accepted: 6 June 2013
Ó The Pharmaceutical Society of Korea 2013
Abstract We synthesized (Z)-5-(2,4-dihydroxybenzylid-
ene)thiazolidine-2,4-dione (MHY498) as a potential tyrosi-
nase inhibitor. MHY498 potently inhibited mushroom
tyrosinase activity (mean IC50 = 3.55 lM) in a dose-
dependent manner. MHY498 was more potent than the well-
known tyrosinase inhibitor, kojic acid (mean IC50 = 22.79
lM). When tested in B16F10 melanoma cells treated with
a-melanocyte stimulating hormone (a-MSH), MHY498
inhibited murine tyrosinase activity and decreased melanin
production without inducing cytotoxicity. Docking models
showed that the binding affinity of MHY498 to tyrosinase
was higher than that of kojic acid, and docking simulation
results indicated that the tyrosinase binding moieties of
MHY498 and kojic acid were similar. Western blotting
showed that tyrosinase inhibition by MHY498 partly resul-
ted from the expressional modulations of tyrosinase and its
transcription factor, microphthalmia-associated transcrip-
tion factor, via the cAMP–PKA–CREB pathway. These
findings suggest that MHY498 could be useful as an anti-
melanogenic agent for the prevention and treatment of dis-
eases associated with skin pigmentation.
Keywords (Z)-5-(2,4-Dihydroxybenzylidene)
thiazolidine-2,4-diones ꢀ Melanogenesis ꢀ Tyrosinase
inhibitor ꢀ B16F10 melanoma cells ꢀ cAMP ꢀ PKA
Introduction
S. H. Kim ꢀ K. M. Moon ꢀ Y. J. Choi ꢀ K. W. Chung ꢀ
H. Y. Chung
College of Pharmacy, Pusan National University, Kumjeong-gu,
Busan 609-735, Republic of Korea
Skin pigmentation is commonly induced by ultraviolet
(UV) light, and is due to an accumulation of melanin in the
epidermis. Melanin synthesis takes place within specialized
intracellular organelles called melanosomes. In mammals,
two major types of melanin are produced, the eumelanins,
which are black/brown, and the pheomelanins, which are
yellow/red.
Y. M. Ha
Department of Chemistry, Dong-A University, Hadan2-dong,
Saha-gu, Busan 604-714, Republic of Korea
K. M. Moon ꢀ Y. J. Choi ꢀ H. O. Jeong ꢀ
K. W. Chung ꢀ H. Y. Chung (&)
Although melanogenesis is a necessary defense mecha-
nism against UV irradiation and excessive oxidative stress,
abnormal accumulations of melanin can cause hyperpig-
mentation, including melasma, freckles, and senile lentigi-
nes (Picardo et al. 1999; Brenner and Hearing 2008). In fact,
UV radiation, chronic inflammation, and the release of
abnormal a-melanocyte stimulating hormone (a-MSH) are
well-known triggering factors of hyperpigmentation and
inflammatory pigmentation (Choi et al. 2005; Im et al. 2002).
For melanin production, specific enzymes are required,
and the best characterized are tyrosinase, tyrosinase-related
protein 1 and dopachrome tautomerase. Tyrosinase is a
Molecular Inflammation Research Centre for Aging Intervention
(MRCA), College of Pharmacy, Pusan National University,
Kumjeong-gu, Busan 609-735, Republic of Korea
e-mail: hyjung@pusan.ac.kr
Y. J. Park ꢀ H. J. Lee ꢀ H. R. Moon (&)
Laboratory of Medicinal Chemistry, College of Pharmacy,
Pusan National University, Kumjeong-gu, Busan 609-735,
Republic of Korea
e-mail: mhr108@pusan.ac.kr
P. Chun
College of Pharmacy, Inje University, Gimhae,
Gyeongnam 621-749, Republic of Korea
123

S. H. Kim et al.
copper-containing enzyme and catalyzes the two initial
rate-limiting reactions of melanogenesis, that is, the
hydroxylation of tyrosine to give 3,4-dihydroxyphenylala-
nine (DOPA) and the oxidation of DOPA to DOPAquinone
(Busca and Ballotti 2000; Ebanks et al. 2009).
Materials and methods
Synthesis of (Z)-5-(2,4-dihydroxybenzylidene)
thiazolidine-2,4-dione (MHY498)
Because of its key role in melanogenesis, tyrosinase is
an attractive target for research on the biochemistry of
melanogenesis and management of hyperpigmentary dis-
orders. Over the last few decades, various tyrosinase
inhibitors, such as, hydroquinone (Garcia and Fulton
1996), ascorbic acid derivatives (Kameyama et al. 1996),
azeleic acid (Hermanns et al. 2002), retinoids (Yoshimura
et al. 2001), arbutin (Nakajima et al. 1998), kojic acid
(Cabanes et al. 1994), hydroxystilbene compounds (e.g.
resveratrol) (Ohguchi et al. 2003b; Shin et al. 1998; Kim
et al. 2002), and polyphenolic compounds (Ohguchi et al.
2003a; Roh et al. 2004; No et al. 2004) have been reported.
However, some well-known tyrosinase inhibitors, includ-
ing hydroquinone and kojic acid, have limited applications,
because of their undesirable side effects, which include
dermatitis and cytotoxicity. Therefore, safer and more
effective agents are needed.
To a stirred solution of substituted 2,4-dihydroxybenzalde-
hyde (200 mg, 1.45 mmol) and thiazolidine-2,4-dione
(204 mg, 1.74 mmol) in ethanol (4 mL) was added piperidine
(0.03 mL, 0.3 mmol).The reaction mixture was then refluxed
for 24 h (Fig. 2). After cooling, water was added, and the
precipitate so formed was filtered through a Buchner funnel
and washed with water/ethanol (5:1). For further purification,
the precipitate was subjected to flash silica gel column chro-
matography using chloroform/methanol (10:1) as eluent to
give (Z)-5-(2,4-dihydroxybenzylidene) thiazolidine-2,4-
dione (MHY498, 156.1 mg, 45.4 %) as a dark yellow solid.
Melting point, 169.4–171.6 °C; 1H NMR (400 MHz,
DMSO-d6) d 12.36 (br s, 1 H, NH), 10.40 (s, 1 H, OH), 10.13
(br s, 1 H, OH), 7.93 (s, 1 H, vinylic H), 7.13 (d, 1 H,
J = 8.8 Hz, 60-H), 6.38 (d, 1 H, J = 2.4 Hz, 30-H), 6.36 (dd,
1 H, J = 8.8, 2.4 Hz, 50-H); 13C NMR (100 MHz, DMSO-
d6) d 169.1 (C2), 168.5 (C4), 162.3 (C40), 160.0 (C20), 130.6
(benzylic C), 128.0 (C60), 117.6 (C5), 112.3 (C10), 109.0
(C50), 103.2 (C30); LRMS(ES) m/z 236 (M–H)-.
In this context, our laboratory has reported that com-
pounds with imidazole ring have significant inhibitory
activities against tyrosinase (Ha et al. 2011b). In addition,
we found that 2-hydroxy, 4-hydroxy or 2,4-dihydroxy
phenyl compounds had more potent inhibitory activities
against tyrosinase than other derivatives (Chung et al.
2012; Ha et al. 2011a, c). On the basis of these results, we
synthesized the compound (Z)-5-(2,4-dihydroxybenzylid-
ene)thiazolidine-2,4-dione (MHY498) (Fig. 1), which was
substituted NH to S in imidazole ring based on their bio-
isostere and had 2,4-hydroxy phenyl moiety to expect the
potent inhibitory activity against tyrosinase. It has been
reported that the synthesis and inhibitory effects of (Z)-5-
(2,4-dihydroxybenzyli dene)thiazolidine-2,4-dione (MHY498)
on Mur ligase and oxidative stress (Cacic and Molnar 2011;
Jiang et al. 2011; Ha et al. 2012a; Shah and Singh 2012;
Materials
Antibodies against tyrosinase, microphthalmia-associated
transcription factor (MITF), phosphorylated Ser96 PKA
regulatory type IIa subunit (pPKA RIIa(Ser96)), b-actin,
and transcription factor IIB (TFIIB) were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Mushroom tyrosinase, L-tyrosine, 3,4-dihydroxyphenylal-
anine (L-DOPA), kojic acid, a-melanocyte stimulating
hormone (a-MSH), and other chemical reagents were
purchased from Sigma Chemical (St. Louis, MO, USA).
´
Tomasic et al. 2010), however, this is the first report on the anti-
Cell culture system
melanogenic effect of MHY498.
In this study, we investigated the inhibitory effects of
MHY498 on tyrosinase activity and melanin production,
and its regulatory effects on melanogenesis in B16F10
melanoma cells. Molecular studies of the inhibitory
mechanism of MHY498 on tyrosinase active site were
conducted using kinetic study and docking simulation.
B16F10 cells (from the Korean Cell Line Bank) were
cultured in Dulbecco’s modified Eagle’s medium (DMEM)
(Nissui Pharmaceutical Co., Tokyo) containing 10 % fetal
bovine serum (FBS, Sigma Chemical Co., USA) and pen-
icillin/streptomycin (100 IU/50 lg/mL) in a humidified
atmosphere containing 5 % CO2 (in air) at 37 °C. B16F10
Fig. 1 Chemical structure of
(Z)-5-(2,4-dihydroxybenzy-
lidene)thiazolidine-2,4-dione
(MHY498)
Fig. 2 Brief scheme of the synthesis of MHY498
123

Anti-melanogenic effect of MHY498
cells were cultured in 24-well plates for melanin quantifi-
cation and enzyme activity assays.
added to a 96-well plate into a total volume of 200 lL.
Initial rates of DOPAchrome formation were determined
by measuring increases in absorbance at 492 nm per min
(DOD492/min) using a microplate reader. The type of
inhibition of the compounds was determined by a modifi-
cation of the Michaelis–Menten equation and Lineweaver-
Burk plots at varying L-DOPA concentrations.
Cell viability
Cell survival was quantified by colorimetric 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) assay that measured mitochondrial activity in viable
cells. This method is based on the conversion of MTT
(Sigma Chemical Co., USA) to MTT-formazan crystals by
a mitochondrial enzyme, as previously described (Tada
et al. 1986). Briefly, cells were seeded at a density of
3 9 104 cells/well in a 48-well plate (Corning, NY, USA),
allowed to adhere overnight, and culture medium was
replaced with fresh serum-free DMEM. MTT was freshly
prepared at 0.5 mg/ml in phosphate-buffered saline (PBS).
After exposing cells to MHY498 at various concentrations
(0.5–32 lM) for 24 h, 500 lL aliquots of MTT stock
solution were added to each well, and the plate was incu-
bated at 37 °C for 2 h in a humidified 5 % CO2 incubator.
After 2 h, the medium was removed and 500 lL of etha-
nol–DMSO (1:1 mixture solution) was added to each well
to dissolve the formazan. After 10 min, the optical density
of each well was read at 560 nm using a spectrophotom-
eter. The results are the means of six experiments.
Tyrosinase activity assay in B16F10 melanoma cells
Tyrosinase activity was determined in B16F10 melanoma
cells by measuring the rate of L-DOPA oxidation. Cells
were seeded into the wells of a 24-well plate at a density of
5 9 104 cells/well, incubated in the presence or absence of
100 nM a-MSH, and then treated for 48 h with various
concentrations of MHY498 (0.5–16 lM) or 16 lM of kojic
acid. After washing, cells were lysed in 100 lL of 50 mM
sodium phosphate buffer (pH 6.5) containing 1 % Triton
X-100 (Sigma) and 0.1 mM phenylmethyl sulfonyl fluoride
(PMSF), and frozen at -80 °C for 30 min. After thawing,
cellular extracts were clarified by centrifugation at
12,000 g for 30 min at 4 °C, and 80 lL of the supernatants
obtained and 20 lL of L-DOPA (2 mg/mL) were placed
into the wells of a 96-well plate. Absorbances were read at
492 nm every 10 min for 1 h at 37 °C using an ELISA
plate reader. Final activities were expressed as DOD/min.
Assay of mushroom tyrosinase inhibition
Determination of melanogenesis level in B16F10
melanoma cells
Mushroom tyrosinase was used throughout the study. Tyrosi-
nase activity was determined as described previously with
minor modification (No et al. 1999). Briefly, 20 lL of an
aqueous solution of mushroom tyrosinase (1,000 units) was
added to each well of a 96-well microplate (Nunc, Denmark), in
a total assay volume of 200 lL containing a 1 mM L-tyrosine
solution, and 50 mM phosphate buffer (pH 6.5). Assay mix-
tures were then incubated at 25 °C for 30 min, and amounts of
dopachrome produced were determined spectrophotometri-
cally at 492 nm (OD492) using a microplate reader (Hewlett
Packard). Tyrosinase activity is expressed as IC50, that is, the
concentration that caused 50 % inhibition, which was read off
the X-axis of the dose–response curve. In the present study,
dose-dependent inhibition experiments were performed in
triplicate to determine the IC50 values of MHY498 and kojic
acid. Log–linear curves and their equations were determined by
averaging inhibitions at three doses, 1, 5, and 10 lM of
MHY498 and 20, 30, and 40 lM of Kojic acid.
Melanin contents were determined using a modification of
the method described by (Bilodeau et al. 2001). In the
current study, the amount of melanin was used as an index
of melanogenesis. B16F10 melanoma cells (5 9 104 cells/
well) were seeded in a 24-well plate and incubated in the
presence or absence of 100 nM a-MSH. Cells were then
incubated for 48 h with various concentrations of MHY498
(0.5–16 lM) or 16 lM of kojic acid. After washing twice
with PBS, cells were detached by incubation in trypsin/
EDTA, and dissolved in 100 lL of 1 N NaOH. The sam-
ples obtained were then incubated at 60 °C for 1 h and
mixed to solubilize the melanin. Melanin contents were
determined by measuring absorbances at 405 nm.
Cell lysis and Western blot analysis
Cells were washed with PBS, and then treated with ice-cold
PBS. Pelletswereharvestedat1,0009gat4 °C for 5 min. The
pellets were suspended in buffer A (10 mM Tris, pH 8.0 with
1.5 mM MgCl2, 1 mM DTT, 0.1 % NP-40, 1 lM pepstatin,
and 1 mM p-aminobenzamidine), incubated on ice for
15 min, and then centrifuged at 14,000 g at 4 °C for 15 min.
The supernatants were used as the cytosolic fractions and the
Kinetics of tyrosinase inhibition
Various concentrations of L-DOPA (0.5–4 mM) substrate,
20 lL of an aqueous mushroom tyrosinase solution (1,000
units), and 50 mM of potassium phosphate buffer (pH 6.5)
with or without a test sample (0, 5, 10 lM MHY498) were
123

S. H. Kim et al.
pellets were resuspended in buffer C (10 mM Tris, pH 8.0
with 50 mM KCl, 100 mM NaCl, 1 lM pepstatin, and 1 mM
p-aminobenzamidine) and incubated on ice for 30 min, then
centrifuged at 14,000 g at 4 °C for 30 min. The resultant
supernatants were used as the nuclear fraction.
mushroom tyrosinase activity using L-tyrosine as a sub-
strate. As shown in Table 1, MHY498 inhibited tyrosi-
nase activity in a dose-dependent manner. In addition,
MHY498 (mean IC50 = 3.55 lM) was significantly
more potent than kojic acid, a strong tyrosinase inhibitor
(mean IC50 = 22.79 lM).
Cell lysates (containing 20 lg of protein) were boiled for
5 min in gel-loading buffer (0.125 M Tris–HCl, pH 6.8, 4 %
SDS, 10 % 2-mercaptoethanol, and 0.2 % bromophenol blue)
at a 1:1 ratio. Total protein equivalents were separated by
sodium dodecyl sulfate–polyacrylamide gel electrophoresis on
10 % acrylamide gels, transferred to polyvinylidene fluoride
(PVDF) membranes, and membranes were immediately placed
into blocking buffer (5 % non-fat milk) in 10 mM Tris, pH 7.5,
100 mM NaCl, and 0.1 % Tween 20. Blots were blocked at
room temperature for 1 h. Antibody labeling was detected using
West-zol Plus and chemiluminescence FluorchemTM SP
(Alpha Innotech Co., San Leandro, CA, USA). Pre-stained
protein markers were used for molecular weight determinations.
The kinetics of MHY498 in the inhibition of mushroom
tyrosinase
To explore the mechanism responsible for tyrosinase
inhibition by MHY498, we conducted a kinetic study. The
mode of enzyme inhibition was determined by Linewe-
aver-Burk plot analysis as shown in Fig. 3. MHY498 at 5
or 10 lM had Km values of 2.48 and 5.29 mM, respec-
tively, and the same Vmax values of 1.74 9 10-2 mM/min
(Table 2). As the concentration of MHY498 was increased,
the Km for tyrosinase increased in a dose-dependent man-
ner without changing Vmax, indicating MHY498 is a
competitive inhibitor of tyrosinase.
In silico docking of tyrosinase and MHY498
Among many tools available for in silico protein–ligand
docking, DOCK6.3 and AutoDock4.2 are the most com-
monly used due to their automated docking capability. In
the present study, we used AutoDock4.2 for the docking
simulation of tyrosinase and MHY498. The program per-
forms ligand docking by using a set of predefined 3D grids
of the target protein and a systemic search technique
(Moustakas et al. 2006; Morris et al. 2009). To prepare for
the docking procedure, we performed the following: (1)
conversed 2D into 3D structures, (2) calculated charges,
and (3) added hydrogen atoms using the ChemOffice pro-
gram (http://www.cambridgesoft.com).
In silico docking of tyrosinase and binding residues
that interact with MHY498
The docking simulation using AutoDock4.2 produced sig-
nificant scores. The binding energies of MHY498 and kojic
acid were -5.83 and -3.91 kcal/mol, respectively. The
docking score between a ligand and a receptor is repre-
sented by various energy terms, such as, electrostatic
energy and van der Waals energy terms, and the solvation
energy.
The docking simulation using AutoDock4.2 showed that
the binding affinity of MHY498 was greater than that of the
kojic acid, a positive control. Using AutoDock4.2, we
searched for binding residues within tyrosinase that were
close to the inhibitor. As shown in Fig. 4, both MHY498
Statistical analysis
Inhibitions of tyrosinase activities are expressed as percent
inhibitions, defined as: 100 - [(A 9 100)/B], where
A = OD492 with a test sample and B = OD492 without a
test sample. Values are presented as means. The statistical
significance of differences between groups was determined
by one-factor analysis of variance (ANOVA) followed by
Dunnett’s post hoc test. Statistical significance was
accepted for p values \ 0.05.
Table 1 IC50 values for tyrosinase inhibition by MHY498 and kojic
acid
Compound
MHY498
Concentration
(lM)
Inhibition
(%)a
IC50(lM)b
(average)
1
5
45.41 2.21
53.82 0.5
3.55
10
20
30
40
58.41 1.32
46.45 8.58
58.76 15.77
66.75 6.62
Kojic acid
22.79
Results
The inhibitory effect of MHY498 on mushroom
tyrosinase activity
Each value is mean SE (n = 3). Repeated experiments showed the
similar results
a
Tyrosinase inhibition was measured using L-tyrosine as substrate at
1 mM
b
(Z)-5-(2,4-Dihydroxybenzylidene)thiazolidine-2,4-dione
50 % Inhibitory concentration (IC50
)
(MHY498) was tested for its inhibitory effects on
123

Anti-melanogenic effect of MHY498
in these cells. After 48 h of incubation with MHY498,
tyrosinase activity was 216.35 % at 0.5 lM, 172.53 % at
4 lM, and 142.33 % at 16 lM, whereas in the control
group treated with a-MSH alone it was 248.52 %. At
16 lM, the tyrosinase activities of MHY498 and kojic
acid were 142.33 and 199.08 %, respectively, indicating
that MHY498 is a more potent inhibitor of tyrosinase
activity.
Inhibition of melanin production in B16F10 melanoma
cells by MHY498
To assess the inhibitory effect of MHY498 on melano-
genesis, we quantified the melanin contents of B16F10
melanoma cells treated with MHY498. The inhibitory
effect of MHY498 on melanin content is shown in Fig. 7.
Melanin contents in B16F10 melanoma cells treated with
MHY498 decreased dose-dependently; percentage melanin
contents were 101.53 % at 0.5 lM, 90.47 % at 4 lM, and
78.92 % at 16 lM, as compared with 128.25 % in cells
treated with a-MSH alone. These results show that
MHY498 inhibits melanogenesis at concentrations that do
not significantly affect cell viability. At 16 lM, the per-
centage melanin content in cells treated with MHY498 or
kojic acid were 78.92 and 84.67 %, respectively, indicating
that MHY498 is the more potent inhibitor of melanin
production.
Fig. 3 Lineweaver-Burk plot of mushroom tyrosinase in the presence
of MHY498. Results are mean 1/V values of three independent tests
performed using different concentrations of L-DOPA substrate
Table 2 Kinetic analysis of MHY498
Concentration (lM)
Vmax (mM/min)
Km (mM)
No inhibitor
MIIV498 5.0
MI1Y498 10.0
1.73 9 10-2
1.74 9 10-2
1.74 9 10-2
1.11
2.48
5.29
Each data is mean value of 1/V, the inverse of rate of change of
absorbance per minute at a wavelength of 492 nm (DA492/min), of
three independent tests performed using different concentrations of L-
DOPA as a substrate. The Lineweaver-Burk plot’s equation is:
1/V = Km/Vmax 9 1/[S] ? 1/Vmax where V is the reaction velocity
(the reaction rate), Km is the Michaelis–Menten constant, Vmax is the
maximum reaction velocity, [S] is the substrate concentration
Effects of MHY498 on the expression of tyrosinase
and on the cAMP-dependent signaling pathway
It has been shown that the cyclic AMP (cAMP) pathway
plays a pivotal role in a-MSH-induced melanogenesis.
Furthermore, it has been shown that the molecular events
associated with this pathway involve the activations of
protein kinase A (PKA) and cAMP responsive element
binding protein (CREB) transcription factor, and that these
activations lead to the up-regulation of microphthalmia
associated transcription factor (MITF) expression (Busca
and Ballotti 2000).
and kojic acid were found to hydrogen bond with the MET
280 residue of tyrosinase.
Effect of MHY498 on B16F10 melanoma cell viability
Some well-known tyrosinase inhibitors, such as, hydro-
quinone and kojic acid, are cytotoxic, and thus, we
examined the cytotoxicity of MHY498 using B16F10
melanoma cells using an MTT assay. The results obtained
indicated that MHY498 is relatively non-cytotoxic to
B16F10 melanoma cells (Fig. 5).
To investigate the molecular events underlying the anti-
melanogenic effect of MHY498, we examined the levels of
PKA, pCREB (phosphorylated CREB, active form), MITF,
and tyrosinase in B16F10 melanoma cells treated with
MHY498 (0.5, 4, 16 lM). To determine the activation of
PKA, we measured the phosphorylation of regulatory
subunit of PKA at Ser 96 [pPKA RIIa (Ser96)] which
decreases the affinity of regulatory subunit for catalytic
subunit of PKA (Han et al. 2012; Mauban et al. 2009; No
et al. 2004; Yang et al. 2011). As shown in Figs. 8 and 9,
MHY498 dose-dependently suppressed pPKA RIIa
(Ser96), tyrosinase, pCREB, and MITF protein levels, and
Inhibition of tyrosinase activity in B16F10 melanoma
cells by MHY498
To investigate the mechanism whereby MHY498 inhibits
melanin biosynthesis, we examined the inhibitory effect of
MHY498 on cellular tyrosinase activity in B16F10 mela-
noma cells treated with a-MSH. As shown in Fig. 6,
MHY498 inhibited a-MSH-stimulated tyrosinase activity
123

S. H. Kim et al.
Fig. 4 Computational structure
prediction for tyrosinase and
docking simulations with
MHY498 and kojic acid.
(a) Docking simulation results
between the predicted 3D
structure of mushroom
tyrosinase and the chemical
structures of MHY498 (cyan
color) and kojic acid (magenta
color). Docking simulations
were performed using
AutoDock4.2. The binding
energies of MHY498 and kojic
acid were -5.83 and
-3.91 kcal/mol, respectively.
(b) The green lines represent
possible hydrogen bonds. MET
280 of tyrosinase was predicted
to hydrogen bond to MHY498
and to kojic acid
Fig. 5 The effect of MHY498 on the viability of B16F10 melanoma
cells. Cells were treated with various concentrations (0.5–32 lM) of
MHY498 for 24 h and subjected to an MTT assay. The result is
expressed as a percentage of the control, and each data is expressed as
mean SE (n = 6)
Fig. 6 The inhibitory effect of MHY498 on tyrosinase activity in
B16F10 melanoma cells in the presence of 100 nM a-MSH. B16F10
melanoma cells were treated with various doses of MHY498
(0.5–16 lM) for 24 h. The result is expressed as a percentage of
control and each data is expressed as mean SE (n = 6). Statistical
results of one-factor ANOVA: ###p \ 0.001 versus the control group,
***p \ 0.001, *p \ 0.05 versus the group stimulated with only a-
MSH, respectively
significant differences have not found in the total PKA and
total CREB.
Discussion
These results implied that reduced tyrosinase levels in
B16F10 melanoma cells treated with MHY498 was due to
a blockade of the cAMP-dependent signaling pathway
involving PKA, CREB, and MITF.
Melanin is principally responsible for skin color and plays an
important role in the prevention of skin injury under normal
physiological conditions, but on the other hand, abnormal
123

Anti-melanogenic effect of MHY498
Fig. 9 Effects of MHY498 on the levels of pCREB and MITF.
Nuclear protein levels were measured by Western blotting. Samples
were resolved on 10 % SDS-PAGE gels and transferred to nitrocel-
lulose membranes. TFIIB was used as a loading control
Fig. 7 Effect of MHY498 on melanin contentsin B16F10 melanoma
cells in the presence of 100 nM a-MSH. Amounts of melanin were
measured as described in methods. The result is expressed as a
percentage of control and each data is expressed as mean SE
(n = 9). Statistical results of one-factor ANOVA: #p \ 0.05 versus
the control group, ***p \ 0.001, **p \ 0.01 versus the group
stimulated with only a-MSH, respectively
10-2 mM/min (Fig. 3; Table 2). Most inhibitors of mela-
nogenesis, especially inhibitors, such as, tropolone (Fitzpa-
trick 1971) and kojic acid (Cabanes et al. 1994), chelate
copper in the tyrosinase active site, and thus, act as com-
petitive inhibitors. 4,40-Dihydroxybiphenyl (No et al. 2004),
protocatechuic aldehyde (No et al. 2006), and 3,4-dihy-
droxyacetophenone (Kim et al. 2006) share a hydroxy-phe-
nyl group, which possible means that their competitive
tyrosinase inhibitory activities are due to their structural
similarities with tyrosinase substrates like L-tyrosine and
L-DOPA.
pigmentation manifesting as freckles, age spots, or melasma
sometimes causes serious problems. Therefore, the modula-
tion of melanogenesis is viewed as an important strategy for
the treatment of abnormal skin pigmentation (Jones et al.
2002). Tyrosinase inhibition has been the subject of numerous
studies, and several inhibitors are currently used as cosmetic
additives and medicinal products for the treatment of hyper-
pigmentation. In addition, demands for safer and more
effective anti-pigmentation products continue to increase.
In the present study, we synthesized (Z)-5-(2,4-dihydr-
oxybenzylidene)thiazolidine-2,4-dione (MHY498) (Figs. 1,
2) by Knoevenagel condensation and investigated its tyros-
inase inhibitory effects. MHY498 was found to reduce
mushroom tyrosinase activity dose-dependently, and our
kinetic study showed that MHY498 is a competitive inhibitor
of mushroom tyrosinase with the Vmax value of 1.74 9
Docking simulation using AutoDock4.2 showed that
direct tyrosinase inhibition of MHY498 occurs at the active
site. Furthermore, the binding energy (-5.83 kcal/mol)
between tyrosinase and MHY498 calculated by the docking
program was higher than that (-3.91 kcal/mol) of kojic
acid. Although the inhibitory mechanism of MHY498
needs to be confirmed by a future X-ray crystallographic
study, this result suggests that MHY498 inhibits tyrosinase
more so than kojic acid. According to the docking program,
the binding residue interacting with MHY498 or kojic acid
was MET 280 of tyrosinase (Fig. 4), which indicates that
MHY498 might inhibit tyrosinase activity by binding at the
active site of mushroom tyrosinase.
Interestingly, MHY498 was not cytotoxic to B16F10
melanoma cells at the concentrations tested (Fig. 5), and
MHY498 (mean IC50 = 3.55 lM) was a 6.4-fold more
potent tyrosinase inhibitor than kojic acid (mean
IC50 = 22.79 lM). Furthermore, it was found in B16F10
melanoma cells, MHY498 dose-dependently inhibited both
tyrosinase activity and melanin production (Figs. 6, 7),
which suggests that the anti-melanogenic effects of
MHY498 are not associated with undesirable cytotoxicity.
In our previous studies, in order to search for potent skin-
whitening agents, we designed and synthesized several thia-
zolidine derivatives, such as, 5-(substituted benzylidene)
Fig. 8 Effects of MHY498 on the levels of tyrosinase and pPKA
RIIa (Ser96). Cytosolic protein levels were measured by Western
blotting. Samples were resolved on 10 % SDS-PAGE gels and
transferred to nitrocellulose membranes. b-Actin was used as a
loading control
thiazolidine-2,4-dione
derivatives
and
2-(substituted
123

S. H. Kim et al.
a signal transducer in cAMP-induced differentiation of melano-
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Bilodeau, M.L., J.D. Greulich, R.L. Hullinger, C. Bertolotto, R.
Ballotti, and O.M. Andrisani. 2001. BMP-2 stimulates tyrosinase
gene expression and melanogenesis in differentiated melano-
cytes. Pigment Cell Research 14: 328–336.
Brenner, M., and V.J. Hearing. 2008. The protective role of melanin
against UV damage inhuman skin. Photochemistry and Photo-
biology 84: 539–549.
phenyl)thiazolidine-4-carboxylic acid derivatives, and exam-
ined their tyrosinase inhibitory effects, kinetics, and effects on
melanin content in B16F10 cells. Subsequently, we found that
some of these derivatives inhibited mushroom tyrosinase at
levels similar to that of kojic acid (Ha et al. 2012a, b). However,
as compared with these derivatives, MHY498 has a very low
IC50 value (3.55 lM).
According to our previous studies and other related
studies, tyrosinase gene expression, and cAMP production
are enhanced during a-MSH-induced melanogenesis
(Busca and Ballotti 2000; Ebanks et al. 2009; Han et al.
2012; Gasperini et al. 2002; Bertolotto et al. 1998). cAMP
activates PKA, which increases the expressions of tyrosi-
nase and MITF. The second of these, MITF is a melano-
cyte-specific transcription factor and plays a pivotal role in
melanocyte survival, development, and differentiation,
which all occur via the activation of PKA. Activated PKA
phosphorylates CREB and this activates the expressions of
specific genes containing consensus CRE (cAMP respon-
sive element) sequences (50-TGACCTCA-30) in their pro-
moters (Busca and Ballotti 2000).
Busca, R., and R. Ballotti. 2000. Cyclic AMP a key messenger in the
regulation of skin pigmentation. Pigment Cell Research 13:
60–69.
Cabanes, J., S. Chazarra, and F. Garcia-Carmona. 1994. Kojic acid, a
cosmetic skin whitening agent, is a slow-binding inhibitor of
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tion of some novel 3-coumarinyl-5-aryliden-1,3-thiazolidine-
2,4-diones and their antioxidant activity. Zeitschrift fu¨r Naturf-
orschung 66: 177–183.
Choi, H., S. Ahn, B.G. Lee, I. Chang, and J.S. Hwang. 2005.
Inhibition of skin pigmentation by an extract of Lepidium
apetalum and its possible implication in IL-6 mediated signaling.
Pigment Cell Research 18: 439–446.
Chung, K.W., Y.J. Park, Y.J. Choi, M.H. Park, Y.M. Ha, Y. Uehara,
J.H. Yoon, P.S. Chun, H.R. Moon, and H.Y. Chung. 2012.
Evaluation of in vitro and in vivo anti-melanogenic activity of a
newly synthesized strong tyrosinase inhibitor (E)-3-(2,4-dihydr-
oxybenzylidene)pyrrolidine-2,5-dione (3-DBP). Biochimica et
Biophysica Acta 1820: 962–969.
Ebanks, J.P., R.R. Wickett, and R.E. Boissy. 2009. Mechanisms
regulating skin pigmentation: the rise and fall of complexion
coloration. International Journal of Molecular Sciences 10:
4066–4087.
To determine the intracellular mechanism responsible for
theeffectsofMHY498,weattemptedtoidentifythosecellular
factors involved in melanogenesis that are expressionally
affected by MHY498. a-MSH increased the protein levels of
pPKA RIIa(Ser96), pCREB and MITF (as determined by
Western blotting), and MHY498 effectively and dose-
dependently reduced these levels (Figs. 8, 9), and led to the
suppression of tyrosinase expression (Fig. 8). These findings
suggest that the suppression of a-MSH-stimulated melano-
genesis by MHY498 is mediated by blockade of the cAMP/
PKA/CREB pathway and reduced MITF expression.
Fitzpatrick, T.B. 1971. The biology of pigmentation. Birth Defects
Orig Artic Ser 7: 5–12.
Garcia, A., and J.E. Fulton Jr. 1996. The combination of glycolic acid
and hydroquinone or kojic acid for the treatment of melasma and
related conditions. Dermatologic Surgery 22: 443–447.
Gasperini, S., L. Crepaldi, F. Calzetti, L. Gatto, C. Berlato, F.
Bazzoni, A. Yoshimura, and M.A. Cassatella. 2002. Interleukin-
10 and cAMP-elevating agents cooperate to induce suppressor of
cytokine signaling-3 via a protein kinase A-independent signal.
European Cytokine Network 13: 47–53.
Ha, Y.M., J.A. Kim, Y.J. Park, D.E. Park, Y.J. Choi, J.M. Kim, K.W.
Chung, Y.K. Han, J.Y. Park, J.Y. Lee, H.R. Moon, and H.Y.
Chung. 2011a. Synthesis and biological activity of hydroxyben-
zylidenyl pyrrolidine-2,5-dione derivatives as new potent inhib-
itors of tyrosinase. MedChemComm 2: 542–549.
Ha, Y.M., J.A. Kim, Y.J. Park, D.E. Park, J.M. Kim, K.W. Chung,
E.K. Lee, J.Y. Park, J.Y. Lee, H.J. Lee, J.H. Yoon, H.R. Moon,
and H.Y. Chung. 2011b. Analogs of 5-(substituted benzyli-
dene)hydantoin as inhibitors of tyrosinase and melanin forma-
tion. Biochimica et Biophysica Acta 1810: 612–619.
Ha, Y.M., J.Y. Park, Y.J. Park, D.E. Park, Y.J. Choi, J.M. Kim, E.K.
Lee, Y.K. Han, J.A. Kim, J.Y. Lee, H.R. Moon, and H.Y. Chung.
2011c. Synthesis and biological activity of hydroxy substituted
phenyl-benzo[d]thiazole analogues for antityrosinase activity in
B16 cells. Bioorganic and Medicinal Chemistry Letters 21:
2445–2449.
In conclusion, MHY498 was found to be a potent
tyrosinase inhibitor that suppresses tyrosinase activity and
total melanin contents in B16F10 melanoma cells without
adversely affecting cell viability. These findings suggest
that MHY498 could be a useful antimelanogenic agent for
the prevention and treatment of disorders related to skin
pigmentation. Furthermore, our molecular studies indicate
that the anti-melanogenic effect of MHY498 is probably
due to the direct inhibition of tyrosinase activity and the
inhibition of gene expressions of tyrosinase and MITF via
the modulation of the cAMP/PKA/CREB pathway.
Acknowledgments This work was supported by the Korean
National Research Foundation (NRF) funded by the Korean govern-
ment (MEST) (Grant no. 2009-0083538). We thank the Aging Tissue
Bank for providing us with research materials.
Ha, Y.M., Y.J. Park, J.A. Kim, D.E. Park, J.Y. Park, H.J. Lee, J.Y.
Lee, H.R. Moon, and H.Y. Chung. 2012a. Design and synthesis
of 5-(substituted benzylidene)thiazolidine-2,4-dione derivatives
as novel tyrosinase inhibitors. European Journal of Medicinal
Chemistry 49: 245–252.
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