Conjugation with dihydrolipoic acid imparts caffeic acid ester potent inhibitory effect on dopa oxidase activity of human tyrosinase

Article abstract of DOI:10.3390/ijms19082156

Caffeic acid derivatives represent promising lead compounds in the search for tyrosinase inhibitors to be used in the treatment of skin local hyperpigmentation associated to an overproduction or accumulation of melanin. We recently reported the marked inhibitory activity of a conjugate of caffeic acid with dihydrolipoic acid, 2-S-lipoylcaffeic acid (LCA), on the tyrosine hydroxylase (TH) and dopa oxidase (DO) activities of mushroom tyrosinase. In the present study, we evaluated a more lipophilic derivative, 2-S-lipoyl caffeic acid methyl ester (LCAME), as an inhibitor of tyrosinase from human melanoma cells. Preliminary analysis of the effects of LCAME on mushroom tyrosinase indicated more potent inhibitory effects on either enzyme activities (IC₅₀ = 0.05 ± 0.01 μM for DO and 0.83 ± 0.09 μM for TH) compared with LCA and the reference compound kojic acid. The inhibition of DO of human tyrosinase was effective (Ki = 34.7 ± 1.1 μM) as well, while the action on TH was weaker. Lineweaver–Burk analyses indicated a competitive inhibitor mechanism. LCAME was not substrate of tyrosinase and proved nontoxic at concentrations up to 50 μM. No alteration of basal tyrosinase expression was observed after 24 h treatment of human melanoma cells with the inhibitor, but preliminary evidence suggested LCAME might impair the induction of tyrosinase expression in cells stimulated with α-melanocyte-stimulating hormone. All these data point to this compound as a valuable candidate for further trials toward its use as a skin depigmenting agent. They also highlight the differential effects of tyrosinase inhibitors on the human and mushroom enzymes.

Full text of DOI:10.3390/ijms19082156

International Journal of
Molecular Sciences
Article
Conjugation with Dihydrolipoic Acid Imparts Caffeic
Acid Ester Potent Inhibitory Effect on Dopa Oxidase
Activity of Human Tyrosinase
Raffaella Micillo 1 ID , Julia Sirés-Campos 2 ID , José Carlos García-Borrón 2, Lucia Panzella 1
,
ID
Alessandra Napolitano 1,
*
and Conchi Olivares 2,
*
ID
1
Department of Chemical Sciences, University of Naples Federico II, Via Cintia 4, I-80126 Naples, Italy;
raffaella.micillo@unina.it (R.M.); panzella@unina.it (L.P.)
Department of Biochemistry and Molecular Biology, School of Medicine, University of Murcia and Instituto
Murciano de Investigación Biosanitaria (IMIB), 30120 El Palmar, Spain; juliasc@um.es (J.S.-C.);
gborron@um.es (J.C.G.-B.)
2
*
Correspondence: alesnapo@unina.it (A.N.); mcolisan@um.es (C.O.);
Tel.: +39-081674133 (A.N.); +34-868-889439 (C.O.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 26 June 2018; Accepted: 20 July 2018; Published: 24 July 2018
Abstract: Caffeic acid derivatives represent promising lead compounds in the search for tyrosinase
inhibitors to be used in the treatment of skin local hyperpigmentation associated to an overproduction
or accumulation of melanin. We recently reported the marked inhibitory activity of a conjugate of
caffeic acid with dihydrolipoic acid, 2-S-lipoylcaffeic acid (LCA), on the tyrosine hydroxylase (TH)
and dopa oxidase (DO) activities of mushroom tyrosinase. In the present study, we evaluated a
more lipophilic derivative, 2-S-lipoyl caffeic acid methyl ester (LCAME), as an inhibitor of tyrosinase
from human melanoma cells. Preliminary analysis of the effects of LCAME on mushroom tyrosinase
indicated more potent inhibitory effects on either enzyme activities (IC50 = 0.05
0.83 0.09 M for TH) compared with LCA and the reference compound kojic acid. The inhibition
of DO of human tyrosinase was effective (Ki = 34.7 1.1 M) as well, while the action on TH was
weaker. Lineweaver–Burk analyses indicated a competitive inhibitor mechanism. LCAME was not
substrate of tyrosinase and proved nontoxic at concentrations up to 50 M. No alteration of basal
±
0.01 µM for DO and
±
µ
±
µ
µ
tyrosinase expression was observed after 24 h treatment of human melanoma cells with the inhibitor,
but preliminary evidence suggested LCAME might impair the induction of tyrosinase expression in
cells stimulated with α-melanocyte-stimulating hormone. All these data point to this compound as a
valuable candidate for further trials toward its use as a skin depigmenting agent. They also highlight
the differential effects of tyrosinase inhibitors on the human and mushroom enzymes.
Keywords: caffeic acid; dihydrolipoic acid; tyrosinase; inhibition mechanism; dopa oxidase;
skin lightening; depigmenting agent
1. Introduction
Melanin pigmentation is believed to be one of the main determinants of sensitivity to ultraviolet
light and susceptibility to sun damage [1]. Under physiological conditions, its synthesis is restricted
to melanosomes, membrane-bound organelles located in melanocytes, and is under complex
regulatory control by multiple agents interacting via pathways activated by receptor-dependent
and -independent mechanisms [
excess of pigmentation (or “hypermelanosis”) associated with disorders such as melasma, lentigo,
or postinflammatory hyperpigmentation [ ] whose medical and aesthetical impact has prompted a
2,3]. An overproduction or accumulation of melanin can lead to a local
2,4,5
constant search for new nontoxic depigmenting agents [6–10].
Int. J. Mol. Sci. 2018, 19, 2156; doi:10.3390/ijms19082156
www.mdpi.com/journal/ijms

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Since control of pigmentation depends on several factors that can be regulated individually or
concomitantly, different control points are possible, e.g., tyrosinase activity, expression, and stability,
the use of chemicals to inhibit the reaction pathway leading to melanin, or interference with
melanosome transfer [11
tyrosinase (EC 1.14.18.1). This enzyme catalyzes the initial steps of melanogenesis [13
–
13]. One of the most common approaches involves the use of inhibitors of
15], namely
–
the hydroxylation of the monophenol L-tyrosine to the o-diphenol L-3,4-dihydroxyphenylalanine
(DOPA), and the oxidation of DOPA to the corresponding o-quinone, dopaquinone, via an electron
exchange with copper atoms present in the active site [16–18]. These two tyrosinase activities are
termed cresolase and catecholase activity or, more specifically in the case of melanogenesis, tyrosine
hydroxylase (TH) and DOPA oxidase (DO) activity. Oxidative polymerization of products deriving
from intramolecular cyclization of dopaquinone leads to eumelanin pigments [19,20].
A critical issue in the search for tyrosinase inhibitors is that most compounds, tested preliminarily
on mushroom tyrosinase, proved to be noneffective on human tyrosinase [13,21–23]. Currently only
a few tyrosinase inhibitors have been further developed for cosmetic and health care purposes as
several factors such as cytotoxicity, solubility, cutaneous absorption, and stability should be considered
as well.
Bioinspired structural manipulations of catechol systems from natural sources have been pursued
as a strategy to potentiate their properties for several applications in biomedicine and materials
science [24]. In a previous paper [25], we reported the inhibitory activity of a conjugate of caffeic acid
(CA) with dihydrolipoic acid (DHLA). Together with its oxidized form lipoic acid (LA), DHLA behaves
as a powerful antioxidant [26,27] that can exert its functions at membrane level and in aqueous
phases of cytoplasm, being soluble both in fats and water. The structure of these compounds
is shown in Figure 1. Both DHLA and LA per se have been reported to block the expression of
microphthalmia-associated transcription factor (Mitf) [28], a master regulator of the expression of
melanogenic proteins [29]. DHLA was shown to affect melanogenesis by trapping dopaquinone and
has therefore been included in topical formulations [30].
O
O
O
OH
HO
HO
S
SH
O
OH
OH
HO
HO
SH
SH
OH
Caffeic acid (CA)
Dihydrolipoic acid (DHLA)
2-S-lipoylcaffeic acid (LCA)
O
OH
S
SH
O
O
HO
HO
HO
HO
OMe
OMe
Caffeic acid methyl ester (CAME)
2-S-lipoylcaffeic acid methyl ester (LCAME)
Figure 1. Structures of the compounds used in this study.
The S-conjugate of DHLA and caffeic acid, the 2-S-lipoylcaffeic acid (LCA), showed a half maximal
inhibitory concentration (IC50) value of 3.22 and 2.0 M for the DO and TH activity of the mushroom
µ
enzyme, respectively, being a promising lead structure for the development of new catechol-based
bioinspired tyrosinase inhibitors [25]. Moreover, LCA was shown not to be a substrate of tyrosinase,
a critical issue for the toxicity of depigmenting agents in vivo as fully appreciated further to the case
of rhododendrol, a phenolic depigmenting agent whose oxidation products generated by the action

Int. J. Mol. Sci. 2018, 19, 2156
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of tyrosinase proved to be toxic and able to induce leukoderma [31
–35]. The nature of tyrosinase
inhibition was explored using Lineweaver–Burk plots suggesting a mixed type mechanism. Under the
same conditions, caffeic acid did not show any effect, indicating that the inhibitory activity stems likely
from the insertion of DHLA in the molecular scaffold.
Caffeic acid is a substrate of tyrosinase allegedly for its similarity to L-DOPA [36],
while its derivatives containing amino acid residues such as the caffeoyl-prolyl-hydroxamic acid
(CA-Pro-NHOH) proved to be good tyrosinase inhibitors [37], possibly because of the copper-chelating
properties and the hydrophobicity that provides the proper structure for binding to the active site of
tyrosinase. Actually, amino acids containing hydrophobic aliphatic side chains like leucine are known
to play an important role in tyrosinase inhibition, and amino acids containing a hydrophobic aromatic
ring like phenylalanine can interact with tyrosinase because of its similarity to tyrosine, the natural
substrate of the enzyme [38]. Also amidic derivatives of caffeic acid with serine or lysine, in particular
N-caffeoyl-O-acetylserine methyl ester, showed strong tyrosinase inhibitory activity [39].
On this basis, in the present study we examined the ability of LCA to inhibit tyrosinase from HBL
human melanoma cells. In order to assess the contribution of the addition of hydrophobic groups
to the tyrosinase inhibitory potency, the methyl ester of LCA, the 2-S-lipoylcaffeic acid methyl ester
(LCAME), was prepared and the dose-activity profile was analyzed in comparison with the parent
compound and a reference tyrosinase inhibitor as kojic acid. Moreover, Lineweaver–Burk plots were
obtained to disclose the mechanism of inhibition. The effects on tyrosinase expression and cell viability
were also analyzed.
2. Results and Discussion
2.1. Preparation of LCAME
LCAME was prepared by a procedure previously developed for LCA [25], involving in situ
generation of the o-quinone of caffeic acid ester by the regioselective hydroxylation of p-coumaric acid
methyl ester with 2-iodoxybenzoic acid (IBX), followed by addition of DHLA. LCAME was obtained in
pure form in 52% yield after preparative High Pressure Liquid Chromatography (HPLC) purification
and subjected to complete spectral characterization.
2.2. Inhibition of Mushroom Tyrosinase Activities by LCAME
The inhibition properties of LCAME were preliminarily investigated on mushroom tyrosinase
by spectrophotometric monitoring of dopachrome formation, a method which is routinely used for
evaluation of the activity of potential tyrosinase inhibitors [13
,21,22]. L-DOPA was used as the substrate
to test DO activity while L-tyrosine was used to test TH activity.
LCAME was incubated in 50 mM phosphate buffer in the presence of mushroom tyrosinase
(20 U/mL) at room temperature. After 10 min, L-DOPA or L-tyrosine (1 mM final concentration) were
added and the absorbance at 475 nm was measured at different times over 10 min. For comparative
purposes the effects of CAME were evaluated as well.
Table 1 reports the IC50 values obtained for LCAME and CAME in comparison with those reported
for LCA [25]. For the DO activity of tyrosinase, the inhibitory potency of LCAME was much higher
than that of CAME or LCA. Concerning TH activity, LCAME was as well the most effective inhibitor,
with LCA showing a comparable potency, whereas CAME was far less active. Comparing the effects of
LCAME and LCA, it appears that the former is more active on DO activity than on TH activity whereas
the reverse holds for LCA. These findings are in keeping with the occurrence of subtle differences in
the structural requirements for efficient recognition of mono- and diphenols by tyrosinase, previously
described for the mammalian enzyme [40]. Moreover, it should be cautioned that promising results
with fungal enzyme are not expected to be straightforwardly confirmed using mammalian tyrosinase.

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Table 1. IC50 values for caffeic acid methyl ester (CAME) and lipoyl caffeic acid methyl ester (LCAME)
for Dopa Oxidase (DO) and Tyrosine Hydroxylase (TH) activities of mushroom tyrosinase.
Compound
IC50 (DO Activity)
IC50 (TH Activity)
CAME
LCAME
LCA [25]
155 ± 7 µM
0.05 + 0.01 µM
3.22 ± 0.02 µM
79 ± 7 µM
0.83 ± 0.09 µM
2.0 ± 0.1 µM
In order to investigate the effect of pre-incubation on the inhibitory activity, we performed a similar
assay adding L-DOPA immediately after LCAME at a final concentration of 0.050 M. Under these
µ
conditions a decrease of the inhibition from 50 ± 2% to 42 ± 1% (p = 0.02) was observed.
Separate experiments aimed at investigating the possibility that the decreased absorbance at
475 nm observed with the caffeic acid derivatives under study might actually be due to a redox
exchange process or to addition to dopachrome. The inhibitor was therefore added to the reaction
mixture 10 min after the start of the reaction of L-DOPA, when dopachrome formation was at its
maximum, but no effect was observed on the intensity of the absorbance at 475 nm.
Some tyrosinase inhibitors are also substrates of the enzyme and this is a critical issue because
their oxidation can lead to the formation of highly reactive, cytotoxic o-quinones [22
,41,42]. To rule
out this possibility, either LCAME or CAME at 100 M were exposed to mushroom tyrosinase, in the
µ
absence of L-DOPA, and after 10 min, the reaction mixtures were analyzed by HPLC. In the case of
LCAME, no significant consumption of the inhibitor was observed, suggesting that it is not a substrate
of the enzyme. The same result was obtained in the presence of L-DOPA. In the case of CAME,
the consumption of the inhibitor was almost complete but it was extremely low in the presence of
L-DOPA, suggesting a competition between the two compounds as substrates.
2.3. Effect of Lipoylconjugates on Human Tyrosinase Activity
In further experiments, the effect of LCAME and LCA was investigated on extracts from HBL
human melanoma cells expressing tyrosinase, and the inhibition of DO activity was evaluated.
DO activity was determined according to Winder and Harris [43] with some minor modifications: cell
extracts were incubated with 3-methyl-2-benzothiazolinone hydrazone (MBTH) and absorbance at
490 nm was measured every 10 min for 1 h.
These experiments showed a significant inhibitory effect on DO activity of both LCA and LCAME
employed at a fixed concentration of 25 µM, whereas the corresponding compounds lacking the lipoic
acid moiety showed a statistically significant but lower inhibition activity (Figure 2a). Next, the dose
dependence profile for inhibition of DO activity was established by determining the residual DO
activity at a fixed L-DOPA concentration, in the presence of increasing concentrations of the inhibitors.
The results are shown in Figure 2b, expressed as percentage of the maximal activity (in the absence of
the inhibitors). In the presence of LCA and LCAME, residual activity was far lower than that of kojic
acid (KA), the reference DO inhibitor. IC50 values were evaluated as 76 µM for LCA, and as low as
30 µM for LCAME (Table 2).

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Table 2. IC50 values for selected compounds versus DO activity of human tyrosinase.
Compound
IC50 (Mean ± SEM, µM)
CA
LCA
CAME
LCAME
Kojic acid
n.d.
76.2 ± 6.0
n.d.
30.1 ± 1.5
282.2 ± 1.8
IC50 values were calculated by a nonlinear regression of the semi-logarithmic plot (log[inhibitor] vs. response
curves) of the data shown in Figure 2b using the GraphPad Prism software. Results are expressed as mean
±
SEM
of two independent assays. “n.d.” stands for “not determined”, as the inhibitory action of CAME was too low to
enable an accurate estimate of the IC50, and CA did not significantly inhibit the DO activity of human tyrosinase at
the concentrations tested.
Figure 2. Effect of the caffeic acid derivatives included in this study on the DO activity of tyrosinase
from HBL human melanoma cells. The oxidation of L-DOPA by cell-free HBL cell extracts was
monitored spectrophotometrically at 490 nm for 1 h, in the presence of MBTH, as described in Materials
and Methods. (
LCAME. Data are represented as residual activity relative to the control activity measured in identical
conditions but in the absence of the inhibitors. The results shown are the mean SEM of 4 independent
experiments, ** p < 0.01. ( ) Dose-response curves for DO activity inhibition. The DO activity of HBL
cell extracts was measured in the presence of several concentrations (0 to 1000 M) of the indicated
compounds. Residual activities were determined as above and plotted against the logarithm of the
a) Residual DO activity of HBL extracts incubated with 25 µM CA, LCA, CAME or
±
b
µ
inhibitor concentration (
KA = kojic acid.
µM). Data are shown as the mean
±
SEM of three independent experiments.
On the other hand, incubation of LCAME at different concentrations in the presence of HBL cell
lysates and MBTH did not induce any change in the absorbance at 490 nm for over 1 h, suggesting that
the compound is not a substrate for human tyrosinase. Overall, these results indicated LCAME as the
most promising inhibitor and further experiments mostly focused on this compound.
In order to determine the type of inhibition induced by LCAME, DO activity was measured at
varying concentrations of L-DOPA (from 0.1 to 2.0 mM), in the presence of two fixed concentrations of
LCAME (10 or 25 µM) (Figure 3a) and Lineweaver–Burk plots were obtained (Figure 3b). No significant
changes in Vmax between the control conditions and the two series tested in the presence of the
inhibitor were detected and a progressive increase in the apparent Km (Kmapp) was observed (Table 3),
strongly suggesting a competitive inhibition. Analysis of the data assuming Michaelis-Menten kinetics
app
and a competitive inhibition profile where Km
= Km (1 + [I]/Ki), yielded a Ki of 34.7 ± 1.1 µM.
On this basis it appears that introduction of a methyl group in LCA increased its inhibitory potency
towards human tyrosinase.

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Table 3. Effect of LCAME on the kinetic parameters for the DO activity of human tyrosinase.
[LCAME] (µM)
Vmax (∆A490/min mg)
Km (mM)
0
10
25
0.19 ± 0.01
0.18 ± 0.02
0.19 ± 0.03
1.12 ± 0.06
1.43 ± 0.30
1.95 ± 0.50
Results are expressed as mean
using the GraphPad Prism software for Michaelis–Menten kinetics.
±
SEM and were obtained by nonlinear regression of the data shown in Figure 3a
(a) (b)
Figure 3. (a) Michaelis-Menten and (b) Lineweaver-Burk plots for human tyrosinase DO activity in the
presence of LCAME. The DO activity of HBL cell-free extracts was assayed using concentrations of
L-DOPA ranging from 0.1 to 2.0 mM as described in Materials and Methods, in the absence or presence
of 10 or 25
µM of LCAME. The reaction rate, normalized for protein content, was represented vs. the
substrate concentration (
a
), and the corresponding double reciprocal plots were obtained ( ) using the
b
GraphPad Software for Michaelis–Menten kinetics.
2.4. Effects on HBL Cell Viability
The effect of the inhibitors on cell viability was evaluated by phase contrast microscopy and by
means of the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. HBL cells
were treated with the compounds (25 or 50 µM) for 24 h. At the end of the incubation period,
phase contrast images were taken and the MTT assay was performed to test the cytotoxicity of each
compound. Cells treated with CA, LCA, or LCAME exhibited a similar morphology than control,
untreated cells (Figure 4a), whereas treatment with 50 µM CAME induced a significant change in
the cells, which became rounded, indicative of detectable toxicity. Concerning their proliferation,
data showed that CA, LCA, or LCAME were not cytotoxic at the concentrations used. Conversely,
CAME reduced cell viability when employed at the highest concentration tested (50
keeping with the observed changes in phase contrast micrographs.
µM) (Figure 4b) in

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Figure 4. Effect of caffeic acid derivatives on the viability of HBL cells. Cells were incubated for
24 h with the inhibitors at 25 or 50 M. ( ) Phase contrast microscopy images. Representative images
are shown (n = 8) Bar size: 50 m. ( ) MTT viability assay. Cells were grown in 96-well plates to
µ
a
µ
b
~80% confluence in the presence or absence of the indicated compounds. After 24 h, MTT was added
(1 mg/mL final concentration) and maintained in the culture media for 4 h. Cells were PBS-washed
twice and solubilized in DMSO. Absorbance at 562 nm was measured and represented as a percentage
of the control value. The mean ± SEM of 8 independent experiments is shown. **** p < 0.0001.
2.5. Effect of Inhibitors on Human Tyrosinase Expression
Both DHLA and LA have been reported to block the expression of Mitf [28], a master melanocyte
transcription factor that upregulates the expression of genes encoding for tyrosinase and other
melanogenic proteins [29]. Mitf expression is normally activated via cAMP signaling following binding
of
α
melanocyte-stimulating hormone (
α
MSH) to the melanocortin 1 receptor (MC1R), thus accounting
MSH [44 45]. Accordingly, it was
for the induction of tyrosinase activity in melanocytes stimulated by
α
,
of interest to determine whether in addition to the competitive inhibition of tyrosinase activity observed
in the kinetic analysis described above, LCAME might also lead to reduced expression of tyrosinase in
cells cultured in the presence of the compound. To this end, we used HBL human melanoma cells as a
convenient cellular model. HBL cells are wild type for the MC1R, NRAS and BRAF genes, which allows
them to respond to αMSH by activating the cAMP and ERK pathways responsible for the regulation
of MITF expression and stability, much like normal human melanocytes [46] Accordingly, they have
been widely used to study the molecular mechanisms accounting for the physiological responses to

Int. J. Mol. Sci. 2018, 19, 2156
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α
MSH [47–49]. Moreover, their high basal pigmentation and tyrosinase activity make them well-suited
for the analysis of potential depigmenting agents.
Control HBL cells or cells grown in the presence of CAME (25
µ
M) or LCAME (25 or 50
µM) were
stimulated for 48 h with a potent analogue of
of the 50 M concentration of CAME was avoided since this concentration was somewhat cytotoxic
as shown above. Expression of tyrosinase was then evaluated by Western blot using a specific
antibody directed against tyrosinase ( PEP7). Tyrosinase displayed the expected electrophoretic
α α
MSH ([Nle4,D-Phe7]- -MSH (NDP-MSH), 10−7 M). Use
µ
α
pattern corresponding to the presence of several N-glycosylation forms [50]. No significant changes in
basal tyrosinase expression were observed for cells treated with either CAME or LCAME (Figure 5).
Moreover, in control cells and in cells grown in the presence of 25 µM CAME, a compound lacking a
lipoic acid-derived moiety, tyrosinase expression was similarly induced by NDP-MSH. Conversely,
LCAME apparently blocked NDP-MSH-mediated stimulation of tyrosinase, suggesting that its lipoic
acid moiety retained the ability to interfere with Mitf expression. However, other mechanisms such as
interference with activation and/or functional coupling of the melanocortin 1 receptor responsible for
α
MSH actions cannot be ruled out and a complete study of these possibilities is beyond the scope of
this work. Accordingly, interference of LCAME with MITF expression or activity remains speculative
at this stage. In any case, this preliminary observation deserves confirmation using cultured normal
human melanocytes and further mechanistic analysis, as it may further support the potential use of
LCAME as a skin depigmenting agent. Indeed, LCAME may act at two different levels, on one hand
by inhibiting preexisting tyrosinase by a competitive mechanism, and on the other hand by interfering
with αMSH-dependent induction of tyrosinase expression.
Figure 5. Effect on tyrosinase expression of CAME (25 µM) and LCAME (25 and 50 µM). HBL melanoma
cells were incubated with or without the indicated compounds and stimulated with NDP-MSH
−7
(10 M). After 48 h of treatment, tyrosinase levels were compared by Western blotting, performed as
described in Materials and Methods. Comparable loading was assessed by staining the membranes
with an anti-ERK2 antibody. For each experimental condition, the fold-increase of tyrosinase abundance
upon NDP-MSH treatment relative to the corresponding control was determined by densitometric
analysis (ImageJ software) and is shown below the figure.
3. Materials and Methods
3.1. Materials
Iodobenzoic acid, oxone®, (
L-3,4-dihydroxyphenylalanine (L-DOPA)
±
)-lipoic acid (LA), sodium borohydride, p-coumaric acid, L-tyrosine,
bovine serum albumin, phenylmethylsulfonylfluoride
,
(PMSF), Igepal CA-630 NP 40, and 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate
(MBTH) were purchased from Sigma-Aldrich (Milan, Italy). Trichloroacetic acid, glycine, Tris, EDTA,
and monosodium and disodium phosphate were from Merck (Darmstadt, Germany). Acrylamide,
bisacrylamide, TEMED, Tween 20, and ammonium persulfate were obtained from Bio-Rad (Hercules,
CA, USA).

Int. J. Mol. Sci. 2018, 19, 2156
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High-performance liquid chromatography (HPLC) grade solvents (VWR, Milan, Italy) and
Milli-Q® water were used.
2-Iodoxybenzoic acid (IBX) [51], DHLA [52], 2-S-lipoylcaffeic acid (LCA) [25] were synthesized
as reported.
3.2. Methods
Nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz in deuterated solvents on
a Bruker instrument (Milan, Italy).
Assays of mushroom tyrosinase activity were performed recording spectra on a UV Jasco V-730
Spectrophotometer (Lecco, Italy).
HPLC purification was performed on an Agilent 1100 binary pump instrument (Agilent
Technologies, Milan, Italy) equipped with a UV detector set at 254 nm using an Econosil C18 column
(22 mm × 250 mm, 10 µm) and 0.1% formic acid: methanol 35:65 v/v as the eluant, at 15 mL/min.
HPLC analyses for determination of the consumption of CAME and LCAME in the presence of
mushroom tyrosinase were performed on a Agilent 1100 binary pump instrument equipped with a
UV-visible detector using an octadecylsilane-coated column, 250 mm
×
4.6 mm, 5 µm particle size
(Phenomenex Sphereclone ODS, Bologna, Italy) at 0.7 mL/min, using the following gradient: 0.1%
formic acid (eluant a)/ methanol (eluant b): 40% b, 0–10 min; from 40 to 80% b, 47.5–52.5 min; from 80
to 40% b, 52.5–57.5 min. Detection wavelength was set at 280 nm.
Liquid chromatography-mass spectrometry (LC-MS) analysis of LCAME was performed on an
Agilent HPLC 1100 VL instrument (Agilent Technologies, Milan, Italy), equipped with an electrospray
ionization source (positive ion mode, ESI+). An Eclipse XBD-C18 column (150 mm
×
4.60 mm, 5 µm)
was used, adopting the same eluant used for the HPLC analysis (flow rate: 0.4 mL/min). Conditions
were set up as following: nebulizer pressure 50 psi; drying gas (nitrogen) flow 10 L/min, 350 ◦C, and
capillary voltage 4000 V.
3.3. Synthesis of p-Coumaric Acid Methyl Ester
p-coumaric acid methyl ester was prepared by reacting p-coumaric acid (600 mg) in methanol
(6 mL) with 96% sulfuric acid (600 µL) under reflux. After 2 h the mixture was diluted with water,
extracted with ethyl acetate, and washed twice with a 5% sodium bicarbonate solution. The organic
layer was dried over sodium sulfate and taken to dryness to afford the product (410 mg, 58% yield) as
a pale-yellow powder.
3.4. Synthesis of 2-S-Lipoylcaffeic Acid Methyl Ester (LCAME)
A solution of p-coumaric acid methyl ester (180 mg, 0.90 mmol) in methanol (12 mL) was treated
with IBX (384 mg, 1.38 mmol) under vigorous stirring at room temperature. After 7 min a solution of
DHLA (774 mg, 3.66 mmol) in methanol (12 mL) was added dropwise, and after additional 15 min the
reaction mixture was concentrated and purified by preparative HPLC (209 mg, 52% yield).
1
ESI+/MS: m/z 401 ([M + H]+); UV:
λ
max
(CH3OH) 251, 318 nm; H-NMR (CD3OD):
δ
(ppm)
1.38 (m, 1H), 1.54 (m, 1H), 1.54 (m, 2H), 1.42 (m, 1H), 1.62 (m, 1H), 1.78 (m, 1H), 1.81 (m, 1H), 2.26 (m,
2H), 2.88 (m, 1H), 2.90 (m, 1H), 2.96 (m, 1H), 3.78 (s, 3H), 6.33 (d, J = 16 Hz, 1H), 6.84 (d, J = 8.4 Hz, 1H),
7.22 (d, J = 8.4 Hz, 1H), 8.40 (d, J = 16 Hz, 1H). 13C NMR (CD3OD):
δ (ppm) 25.7 (CH2), 27.6 (CH2),
34.1 (CH2), 34.7 (CH2), 39.3 (CH2), 39.6 (CH2), 41.3 (CH), 57.5 (CH3), 117.0 (CH), 117.1 (CH), 120.0 (CH),
122.4 (C), 130.7 (C), 145.3 (CH), 148.3 (C), 148.6 (C), 169.6 (C), 177.4 (C).
3.5. Mushroom Tyrosinase Activity Inhibition Assay
Stock methanolic solutions of LCAME or CAME, ranging in concentration from 0.02 to 20 mM
concentration were prepared dissolving a proper amount of the compound of interest in methanol.
100
µL of these solutions were added to 2 mL of 50 mM phosphate buffer at pH 6.8 (final concentrations
range: 0.001–1 mM), in the presence or in the absence of 20 U/mL mushroom tyrosinase. After 10 min

Int. J. Mol. Sci. 2018, 19, 2156
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incubation at room temperature, 20
µL of a 100 mM solution of L-DOPA or L-tyrosine in 0.6 M HCl,
were added (final concentration: 1 mM). Spectrophotometric analysis was performed by measuring
the absorbance at 475 nm for 10 min at 2 min intervals. In control experiments, the reaction was run in
the absence of LCAME. When required, the experiment was performed as above but adding L-DOP A
soon after the addition of LCAME (0.050
µM). In separate experiments, the assay was run as above
with LCAME or CAME at 100
was analyzed by HPLC.
µM, in the presence or absence of L-DOPA, and after 10 min the mixture
3.6. Cell Culture
HBL cells were kindly provided by Prof. G. Ghanem (Laboratory of Oncology and Experimental
Surgery, Université Libre de Bruxelles, Brussels, Belgium). Cells were grown in a water-saturated
5% CO2 atmosphere in plates of the required size to ~80% confluence. The culture medium was
DMEM-GlutaMAXTM (Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum, penicillin
(100 U/mL) and streptomycin (100 µg/mL).
3.7. Enzyme Activity Determination
Cells were solubilized in 1% Igepal, 1% phenylmethylsulfonyl fluoride, 50 mM phosphate buffer
(pH 6.8) and centrifuged at 2000 rpm for 5 min.
DO activity determinations were performed according to Winder and Harris [43] with minor
modifications. Briefly, cell extracts (usually 50–100 µg protein/assay) were incubated with 5 mM MBTH
and 2.5 mM L-DOPA in 50 mM phosphate buffer pH 6.8 in the presence of the indicated concentrations
of the compound of choice in a 96-wells plate. DO activity was measured spectrophotometrically in a
microplate reader every ten minutes for one hour and was expressed as the slopes calculated with the
GraphPad Prism 7 software (Linear Regression, San Diego, CA, USA).
For the analysis of kinetic parameters, the assays were run as above, using different
concentrations of L-DOPA (from 0.1 to 2.0 mM) and of the inhibitor (0, 10 and 25
the Lineweaver–Burk plot.
µM) to build
3.8. Immunochemical Techniques
After protein separation by SDS-PAGE, they were transferred from the gel to the membrane
and immunochemical detection with anti-PEP7 was performed as previously described [53]. Briefly,
the filters were blocked for 1 h in 2% bovine serum albumin in 1% Tween 20 PBS and incubated
◦
overnight at 4 C with primary antibodies (1:4000 dilution). The filters were washed and further
incubated with peroxidase-labeled mouse anti-rabbit IgG (1:5000 dilution). Staining and detection were
done with ECL Plus Western Blotting Detection System chemiluminescent substrate (Thermo Fisher
Scientific, Waltham, MA, USA). Images were acquired in a Fusion Solo.6S system (Vilber-Lourmat,
Marne La Vallée, France). Signal intensities were estimated with ImageJ (National Institutes of Health,
Bethesda, MA, USA) in independent blots, and normalized with the corresponding loading controls
(obtained by staining the membranes with an anti-ERK2 antibody).
3.9. MTT Assay
Cell viability was measured by the MTT assay [54]. HBL cells were seeded into the wells of a
96-wells plate at a density of 10,000 cells in 200
µ
L cell culture media. The seeded cells were incubated
for 18 h at 37 ◦C, then treated with the compounds (25 or 50
µM) for 24 h or left untreated. At the end
of the incubation period, images were taken and the MTT assay was performed to test the cytotoxicity
of the different caffeic acid derivatives.

Int. J. Mol. Sci. 2018, 19, 2156
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3.10. Statistical Analysis
Results are given as mean
±
SEM for experiments performed at least twice, with independent
duplicates or triplicates (n
≥
4), unless specified otherwise. Statistical significance was assessed with
an unpaired two-tailed Student’s t-test, using the GraphPad Prism package (GraphPad Software,
San Diego, CA, USA). p values of <0.05 were considered statistically significant.
4. Conclusions
2-S-lipoylcaffeic acid (LCA), the S-conjugation product of caffeic acid and dihydrolipoic acid, and
its methyl ester derivative (LCAME) inhibited the TH and DO activities of mushroom tyrosinase, with
IC50 values much lower than those obtained for the reference inhibitor kojic acid. Under the same
conditions, caffeic acid (CA) and caffeic acid methyl ester (CAME) did not show any effect, pointing to
a critical role of the lipoyl moiety in determining the inhibitory activity.
The higher inhibitory potency of LCAME compared with LCA further points to improvement of
the inhibitory action as a result of enhancement of lipophilicity.
The inhibitory activity of lipoyl adducts was maintained on the DO activity of human tyrosinase,
with LCAME being more potent than LCA, whereas the effects of both compounds on the TH activity
were much smaller.
LCAME is not substrate of human or mushroom tyrosinase. In addition, the lack of toxicity
of LCAME on HBL human melanocytic cells further supports the perspective of its exploitation for
control of hyperpigmentation.
Analysis of the mechanism of action of LCAME on human tyrosinase indicated a competitive
mechanism. However, it is also possible that the lipoyl moiety might impair Mitf induction in
α
MSH-stimulated cells, thus contributing to tyrosinase inhibition by an additional mechanism based
on decreased tyrosinase gene expression.
The results of this study point to lipoyl conjugates, in particular LCAME, as good candidates for
further trials toward their use as skin depigmenting agent.
Author Contributions: R.M. performed the synthesis of inhibitors and mushroom tyrosinase inhibition
experiments; J.S.-C. performed the experiments with human tyrosinase; C.O. designed the experiments; C.O. and
L.P. supervised the experiments and analyzed data; A.N. and J.C.G.-B. prepared the original draft manuscript.
Funding: This research was funded by grants SAF2015-67092-R from the MINECO (Spain) and FEDER (European
Community) and 19875/GERM/15 from the Fundación Séneca, Comunidad Autónoma de la Región de Murcia
(Spain). J.S. holds a predoctoral fellowship from the MINECO (Spain).
Acknowledgments: R.M. gratefully acknowledges the scientific support to the research activity performed at the
Department of Biochemistry and Molecular Biology of University of Murcia generously provided by J.C.G.-B.
and his collaborators. HBL cells (LOCE-MM1) were established at the Laboratory of Oncology and Experimental
Surgery, LOCE, Université Libre de Bruxelles, Brussels, Belgium, and were a gift from G. Ghanem.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
α-MSH
CA
Melanocyte Stimulating Hormone
Caffeic acid
CAME
LCA
TH
DHLA
DO
Caffeic acid methylester
2-S-lipoylcaffeic acid
Tyrosine hydroxylase
Dihydrolipoic acid
Dopa oxidase

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DOPA
IBX
L-3,4-Dihydroxyohenylalanine
2-iodoxybenzoic acid
IC50
KA
Half maximum inhibitory concentration
Kojic acid
LA
Lipoic acid
LCAME
MBTH
Mitf
MTT
PMSF
2-S-lipoylcaffeic acid methylester
3-methyl-2-benzothiazolinone hydrazone
Microphthalmia-associated transcription
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
Phenylmethylsulfonylfluorid
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