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Are Human Tyrosinase and Related Proteins Suitable Targets for Melanoma
Therapy?

Article  in  Current topics in medicinal chemistry · February 2016

DOI: 10.2174/1568026616666160216160112

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Are Human Tyrosinase and Related Proteins Suitable Targets for Mela- Impact
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Elina Buitragoa, Renaud Hardréb, Romain Haudecoeurc, Hélène Jameta, Catherine Bellea,
Ahcène Boumendjelc, Luigi Bubaccod and Marius Réglier*,b
Current Topics in Medicinal Chemistry

a
Universite Grenoble Alpes, CNRS, DCM UMR 5250, Grenoble 38000, France; bAix Marseille Universite, Centrale
Marseille, CNRS, ISM2 UMR 7313, Marseille 13397, France; cUniversite Grenoble Alpes, CNRS, DPM, UMR 5063,
Grenoble 38000, France; dDepartment of Biology, University of Padova, 35121 Padova, Italy

Abstract: Among the human copper-containing monooxygenases, Tyrosinase (Ty)

is an important enzyme involved in the determinant step of the biosynthetic pathway
of melanin pigment. In this pathway, Ty catalyzes the tyrosine monooxygenation
ARTICLE HISTORY into L-DOPA-quinone, which is the precursor of the skin pigment melanin. Ty in-
Received: April 26, 2016
hibitors/activators are a well-established approach for controlling in vivo melanin
Revised: June 06, 2016 production, so their development has a huge economical and industrial impact.
Accepted: July 10, 2016
Moreover, recent publications highlight that targeting tyrosinase with inhibi-
DOI: 10.2174/1568026616666160216 tors/activators to treat melanogenesis disorders is one of many possible approaches,
160112
 due to the complex biochemical reaction involved in the melanin synthesis. 

Keywords: Melanoma, Tyrosinase, TRP-1, TRP-2, Tyrosinase effectors.

1. INTRODUCTION
Representing at least 40% of cancer cases, skin cancer is
the most common form of cancer globally [1]. Three main
cancer types compose skin cancer: basal cell cancer,
squamous cell cancer and melanoma [2,3]. Melanoma ac-
counts for less than 2% of skin cancer cases but it is the most
aggressive form and causes the majority (75%) of skin can-
cer related deaths [4]. The recent epidemiological data used
to assess the incidence and consequences of melanoma [5,6],
indicate that, each year worldwide, about 130,000 melano-
mas are diagnosed and that approximately 37,000 people die
(28%) with an annual growth rate of new cases evaluated
around 8%.
The primary cause of melanoma is the excessive expo-
sure to ultraviolet light in conjunction with the level of skin
pigmentation in the analyzed population. The comparison
between European and global data highlights significant dif-
ferences both in the incidence of melanoma and in the risk of
death. In Europe, people of the Mediterranean region with
predominantly dark skin type have the lowest incidence of
melanoma (e. g. Greece: men 2.8 10-3 % vs. women 3.9 10-3
%), while the highest incidence is reported in the Scandina-
vian region (e. g. Norway: men 16.1 10-3 % vs. women 15.7
10-3 %). At the global level, it is in Australia and New Zea-
land that the risk factor is higher (men 39.8 10-3 % vs.
women 32.3 10-3 %), while the lowest incidence is found in
Asia and Africa where dark skin predominates. While the
*Address correspondence to this author at the ISM2 UMR CNRS 7313,
Centre Scientifique de Saint Jérôme, Aix Marseille Université, 13397, Mar-
seille cedex 20, France; Tel/Fax: int. +33-4-912-888-23/+33-4-912-888-
284-40; E-mail: marius.reglier@univ-amu.fr
incidence of many tumor types has been decreasing since the
beginning of 21st century, the incidence of melanoma, is still
increasing worldwide [6] and it remains a potentially fatal
malignancy which result in an important socio-economic
problem.
In an advanced stage, the melanoma treatment passes
mandatorily by the surgical excision associated with adju-
vant systemic therapies [7,8]. As for other cancers, radio-
therapy alone or in association with surgery is used in mela-
noma treatment. However, since melanoma is radio-resistant
when compared to other cancers, radiotherapy plays a lim-
ited role in the treatment of melanoma [9]. Immunostimu-
lants can be used in adjuvant systemic therapies. While inter-
feron-α exhibits limited efficacy [10,11], the recombinant
interleukin-2 has been found to induce remission in 6% of
cases of metastatic melanoma [12]. Ipilimumab, a mono-
clonal antibody against cytotoxic T-lymphocyte–associated
antigen 4 that triggers a T-cell–mediated response against the
tumor, was found to improve importantly patient survival in
advanced-stage melanoma [13]. Drugs conceived to directly
targeting cell-signaling pathways are promising in melanoma
treatment [14]. Bevacizumab, an endothelial growth factor
antibody, and sorafenib, a BRAF cellular pathway inhibitor,
have shown some efficacy in early clinical trials [15,16].
Involved in the biosynthetic pathways of the melanin
pigment, the immunogenic enzyme tyrosinase (Ty, mono-
phenol, 3,4-β-dihydroxy-L-phenylalanine oxygen oxi-
doreductase, EC 1.14.18.1) has been demonstrated to be a
sensitive marker for melanoma. On one hand, Ty is over-
expressed during tumorigenesis [17,18] and the other hand
melanoma is usually associated with changes in Ty-mediated
pigmentation [19]. Recently, the role of myeloid-derived

1873-5294/16 $58.00+.00 © 2016 Bentham Science Publishers

3034 Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 27
suppressor cells (MDSCs) in the maintenance of the mela-
noma microenvironment have been investigated in immune
suppression of T cells in an antigen-specific B16 melanoma
murine system, using a novel synthetic Ty DNA vaccine
therapy in both prophylactic and therapeutic models [20,21].
This Ty vaccine induced a robust and broad immune re-
sponse and also reduced the number of MDSCs in the tumor
microenvironment. This novel synthetic DNA vaccine sig-
nificantly reduced the melanoma tumor burden and increased
survival in vivo, due likely, at least in part, to the facilitation
of a change in the tumor microenvironment through MDSC
suppression [21]. Because of its over-expression mainly con-
fined in melanocytes during tumorigenesis, Ty is a potential
molecular target in melanoma therapy [22,23].
2. MELANIN BIOSYNTHESIS
Tys are ubiquitous metalloenzymes found in mammals,
plants, fungi, and bacteria [24], where they play critical roles
in melanin pigment biosynthesis [25]. Ty catalyzes the two-
step oxidation of phenolic compounds into corresponding
catechols (phenolase activity) and ortho-quinones (catecho-
lase activity) (Scheme 1).
 rosinase [33]. The additional function of melanins is as anti-
Scheme 1. Reactions catalyzed by Ty and CaOx.
In bacteria, Ty was reported to be involved in several
processes, such as pathogenic virulence and redox processes
[26]. In fungi and plants, Ty is a key enzyme in the browning
process that occurs upon tissues damage and long-term stor-
age of fruit and vegetables. In mammals, Ty biological func-
tion is to convert L-tyrosine into L-DOPA quinone, which is
the key product for melanin pigment biosynthesis (Scheme
2). Melanin is the primary determinant of skin, eyes and hair
color and the absence or inactivation of Ty leads to some
forms of albinism. Human skin is our largest organ and it is
divided into three layers, the outer epidermis, which is the
external layer, the underlying dermis and finally the hypo-
dermis, which mostly consists of fatty acids that connect the
dermis to the underlying components. The skin is a barrier
against diverse insults that could affect the body, it contrib-
utes to regulation of body temperature and it generates a loss
of fluid and electrolytes. In relation to the subject discussed
here, the skin acts as a defense system against UV radiation
(UVR, 100-400 nm) [27]. Melanogenesis, the biosynthesis of
melanin pigments, takes place in melanosomes, which are
organelles within cells called melanocytes found in the epi-
dermis of the skin. The key proteins involved in pigmenta-
tion are located on the membrane of the melanosomes that
are transported to keratinocytes via dendrites. Melanogenesis
can consequently be separated into two discrete steps, the
Buitrago et al.
formation of the melanocytes and the subsequent biochemi-
cal transformation of L-tyrosine to melanin [28,29].
Melanocytes are developed from melanoblasts that are
generated from the neural crest during the second embryonic
month. Then they migrate to their specific target areas,
namely dermis and epidermis and hair follicles. The melano-
blasts differentiate into melanocytes, and those in the epi-
dermis proliferate and start producing melanins while those
in the dermis decrease in number. Then the differentiated
melanocytes start producing the melanosomes in which the
melanogenesis takes place [30]. The epidermis contains
keratinocytes and melanocyte cells, where the protective
melanin pigments are located, while the dermis is a layer of
connective tissue mostly consisting of extracellular matrix.
Melanins are heterogeneous polyphenol-like biopolymers
with complex structures and colors varying from yellow to
black, resulting from the autopolymerisation of very reactive
quinones formed by oxidization of phenol and catechol pre-
cursors [31]. In mammals, the biosynthetic pathway involves
the synthesis of eumelanins (black to brown insoluble sub-
groups of melanin pigments) and pheomelanins (yellow to
reddish brown, alkali-soluble and sulfur containing sub-
groups) (Scheme 2). Human skin color depends primarily on
the size, shape and activity of melanosomes and on the rela-
tive amounts and distribution of eumelanins and pheome-
lanins. Neuromelanins (NM), found in the brain are dark
pigments produced within neurons by the oxidation of do-
pamine and others catecholamine precursors.
Melanins play a photoprotecting role and exposure to
UVR stimulates melanin formation (the most common ex-
ample is tanning response after sun exposure) [32] by an
increase in melanocytes formed and up-regulation of ty-
oxidants for its capacity to neutralize reactive oxygen species
(ROS), such as hydroxyl radicals, superoxide or hydrogen
peroxide [34]. Eumelanins are more efficient UV blockers
than pheomelanins and function also as radical scavengers as
the quinol/quinone redox transformation of indole monomers
is responsible of the anti-oxidant properties [35]. Lighter
skin has a larger fraction of pheomelanins and is conse-
quently more susceptible to UVR induced damage. UV ex-
posure leads to increased production of melanin but it can
also damage the melanocytes, which can then grow out of
control into tumors. Melanin may be protective against can-
cer as well as carcinogenic when irradiated by UVA. It has
been shown that mice without melanin do not develop mela-
noma when irradiated by UVA [36]. Upon UVA exposure,
melanin is excited to a high energy triplet state that induces
cyclobutane pyrimidine dimers by energy transfer to DNA
[37]. There are indications that melanin intermediates as well
as pheomelanins can enhance UV-induced DNA damage due
to their mutagenic properties, and it has been suggested that
the higher fraction of less effective pheomelanins in light
skin is partly responsible for the higher incidence of UV-
induced melanoma [38]. Moreover melanins have a high
affinity for redox active metal ions such as iron and copper,
which can affect the ability of native melanins to protect
cells from ROS or photochemically generated radicals [39].
The first step in the biosynthesis of melanins (Scheme 2),
which is also the rate limiting step, is the oxidative o-
Are Human Tyrosinase and Related Proteins Suitable Targets Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 27 3035

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Scheme 2. Schematic biosynthetic pathways leading to eumelanins and pheomelanins production (the pathway from 2-S-CysteinylDOPA is
not represented) with representative units found in eumelanin, derived at least in part from L-DOPA via indolic intermediates and pheomela-
nin, derived from the oxidation of cysteinylDOPA precursors via benzothiazine and benzothiazole intermediates.

hydroxylation of L-tyrosine to form L-dihydroxy-
phenylalanine (L-DOPA) catalyzed by Ty using dioxygen as
the oxidant. L-DOPA is then subsequently oxidized to L-
DOPA quinone, which then proceeds to L-DOPAchrome by
auto-oxidation. Ty catalyzes both initial oxidation steps, and
it is also associated to the oxidation of 5,6-dihydroxyindole
(DHI) into indole-5,6-quinone. This multistep process leads
to the formation of eumelanin, which is the most protective
melanin. If cysteine or glutathione are present, DOPAqui-
none is converted to cysteinylDOPA, which in turn leads to
the formation of pheomelanin [40,41].
The involvement of the two tyrosinase-related proteins
(TRP-1 and TRP-2) in the biosynthesis of melanin is now
well established leading to a much more complex scheme of
melanogenic pathway [42]. In an alternative chemical path-
way existing only in mammals, the L-DOPAchrome is trans-
formed into 5,6-dihydroxyindole-2-carboxylic acid (DHICA)
by TRP-2, a zinc-containing enzyme also known as L-
DOPAchrome tautomerase (DCT). DHICA is further oxi-
dized by a redox reaction with the enzyme TRP-1, a copper-
containing enzyme functionally homologous with catechol
oxidase (CaOx) enzyme, which regulates the fraction of car-
3036 Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 27
boxylated subunits in the melanin synthesis, to form indole-
5,6-quinone carboxylic acid. The role of TRP-1 is not clear.
In mice the transformation of DHICA is performed by TRP-
1 [43,44] while in human the same transformation is cata-
lyzed by Ty [45].
NM-containing neurons have been reported to be distrib-
uted throughout the entire brainstem. However, the more
significant clusters were found in the substantia nigra pars
compacta, the ventral tegmental area and the locus coeruleus
[46]. The biosynthesis involves oxidative polymerization of
dopamine and noradrenaline. In the human brain, Ty is ex-
pressed at low level but in the absence of tyrosine hydroxy-
lase, the iron-containing enzyme that catalyzes the transfor-
mation of tyrosine into DOPA in the brain, Ty can do the
conversion and can be linked to catecholamine neurotoxicity
[47,48]. NM formation is thought to have a protective role,
incorporating toxic catechol compounds into the polymer
structure and binding potentially toxic metal ions [46,49]. In
patients with Parkinson’s disease it has been noted that neu-
rons that express NM are the ones more prone to degenerate.
The main reasons for the degeneration of the NM containing
neurons is still unknown, but it has been proposed that it may
involves mitochondria dysfunction, protein degradation dys-
function, aggregation of α-synuclein to neurotoxic forms,
oxidative stress and neuroinflammation [50,51].
3. TYROSINASE, TRP-1 AND TRP-2: STRUCTURE
AND REACTIVITY
Tys belong to the type-3 copper-containing enzyme fam-
ily [52] that include also hemocyanin (Hc) [53-55], catechol
oxidase (CaOx, EC 1.10.3.1) [25,56], ortho-aminophenol
oxidase (APOx, EC 1.10.3.4) [57] and the new hy-
droxyanilinase (NspF) [58,59]. Named also coupled binu-
clear copper-containing proteins or coupled binuclear poly-
phenol oxidases (CB-PPO), they are characterized by an
active site composed by two copper centers close to each
other (the dCu-Cu is ranging from 2.9 to 4.9 Å) bridged by
aquo(hydroxo) ligands in a met state that provides an antifer-
romagnetic coupling between the cupric ions leading to elec-
tron paramagnetic resonance (EPR) silent behavior. In the
reduced state, type-3 copper-containing enzymes reversibly
bind dioxygen to then perform monooxygenation (Ty,
NspF), oxidation (Ty, APOx and CaOx) and simply dioxy-
gen transport (Hc).
In terms of structure–function relationships, there is low
homology in sequence alignments between the members of
the type-3 family (Fig. 1). For Tys, the human form shares
only 10% homology with the mushroom forms and about
30% with the bacterial forms, suggesting important differ-
ences in 3D structure, and therefore in functional behavior
[60]. These structural differences were highlighted by recent
definition of the crystallographic structures of four different
Ty sources. These are, the X-ray structures of two bacterial
Ty sources (BmTy, Baccilus megaterium Ty [61] and ScTy,
Streptomyces castaneoglobisporus Ty [62]), and two mush-
room sources (AbTy, Agaricus bisporus Ty [63,64] and
AoTy, Aspergilus oryzae Ty [65]). The Mushroom AbTy was
crystallized as an H2L2 (two heavy, two light subunits)
tetramer, including the copper-containing subunit H (392
residues), whereas mushroom AoTy and the bacterial BmTy
Buitrago et al.
were crystallized as homodimers (620 and 568 residues, re-
spectively). The bacterial ScTy on the other hand crystallizes
as a monomer (273 residues) in association with a caddy
protein. The X-ray structures of three CaOxs have also been
solved. Two of them come from plants, Ipomeas batatas
(IbCaOx) [66] and Vinus viniferas (VvCaOx) [67] and one
from mushroom, Aspergilus oryzae (AoCaOx) [68,69]. The
prophenoloxidase from insect Menduca sexta (MsCaOx) [70]
have been also published.
Homology modeling of type-3 copper-containing pro-
teins based on the published crystal structures and pair-wise
comparison led to some clues about the structural features
that govern the function of the active site, which despite the
low sequence homology is highly conserved among the type-
3 copper-containing family. Indeed, this analysis reveals a
conserved hydrophobic core comprising a four-helix bundle
with two sets of three histidine residues that coordinate the
two copper-atoms, CuA and CuB (Fig. 2A and 2B). While the
structure of the CuB site is highly conserved, the CuA site
exhibits more pronounced differences (Fig. 1).
The X-ray structures reveal that in the two CaOxs from
plant (IbCaOx and VvCaOx) a conserved Cu A histidine
ligand (highlighted in blue in Fig. 1) is cross-linked to a cys-
teine residue (highlighted in green in Fig. 1). This post-
translational modification is absent in the AoCaOx (Fig. 1).
In Ty, only the mushroom forms AbTy and AoTy exhibit this
post-translational modification but with a vicinal cysteine
residue (YCXHG). In the two bacterial Tys (BmTy and
ScTy), the insect MsTy and probably in the human form
where this consensus sequence YCXHG is absent, this post-
translational modification does not exist. The function of this
post-translational modification is not well understood.
In the reduced state, type-3 copper-containing proteins
are ideally designed to react with dioxygen to give a μ-η2:η2-
peroxo-dicopper(II) intermediate, which is the common oxy
state for these enzymes [71]. In Hc, a dioxygen carrier in
mollusks and arthropods, this peroxo intermediate serves to
deliver dioxygen to tissues, thanks to a reversible reaction,
while in Ty and CaOx this intermediate oxidizes phenols and
catechols, respectively. The reactivity of the μ-η2:η2-peroxo-
dicopper(II) intermediate has been widely investigated on
enzymes and on model compounds [72,73]. Several reports
[74-76] suggest that the deprotonated phenol binds to the
empty sixth coordination of CuA site (Fig. 3A) [77]. The
high structural flexibility of the dicopper core [62] allows the
rotation of the peroxo moieties to react with the phenol in an
electrophilic substitution mechanism. This reaction produces
a catecholate, and releases a quinone while restoring the re-
duced Ty state. The reactivity of the μ-η2:η2-peroxo-
dicopper(II) core is controlled by the proteic environment. In
Hc, the packaging of several subunits in the hemolymph of
animals blocks the active site allowing only the reversible
binding of dioxygen [78]. The structural basis to explain the
difference in the enzymatic activity of CaOx and Ty, remains
under debate.
In IbCaOx, a phenylalanine (F261) located
nearby the Cu A site blocks the entrance of the phenolic sub-
strate and confers CaOx to only having catecholase activity
[65-68] (Fig. 3B). On the other hand, a recent report present
crystal structures of IbCaOx and BmTy with bound substrates
showing that both monophenol hydroxylation and diphenol
Are Human Tyrosinase and Related Proteins Suitable Targets Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 27 3037

CuA-site

HsTy NDINIYDLFVWMHYY-VSMDALLGGSE-IWRDIDFAHEAPAFLPWHRLFL 69
HsTRP-1 ENISIYNYFVWTHYY-SVKKTFLGVGQESFGEVDFSHEGPAFLTWHRYHL 72
HsTRP-2 ANCSVYDFFVWLHYY-SVRDTLLGPGR-PYRAIDFSHQGPAFVTWHRYHL 71

SaTy KRTGRYDAFVTTHN------AFILGDTDNG-ER-TGHRSPSFLPWHRRFL 65
ScTy KRSGRYDEFVRTHN------EFIMSDTDSG-ER-TGHRSPSFLPWHRRFL 65
BmTy KEKGIYDRYIAWHG------AAGKFHTPPGSDRNAAHMSSAFLPWHREYL 67

AoTy TGEDSFFYLAGLHG---EPFRGAGYNNSHWWGGYCHHGNILFPTWHRAYL 97
AbTy HDYSSFFQLAGIHGLPFTEWAKERPSMNLYKAGYCTHGQVLFPTWHRTYL 94

IbCaOx DDPRNFYQQALVHCAYCN---GGYVQTDYPDKEIQVHNSWLFFPFHRWYL 124

VvCaOx DDPRSFKQQANVHCTYCQ---GAYDQVGYTDLELQVHASWLFLPFHRYYL 224
AoCaOx LCLQKLPSRTPAHLAPGARTRYDDFVATHINQTQIIHYTGTFLAWHRYFI 123

CuB-site
HsTy --IADASQSSMHNALHIYMNGT----------------MSQVQGSANDPI 239
HsTRP-1 --KYDPAVRSLHNLAHLFLNGT----------------GGQTHLSPNDPI 243
HsTRP-2 --TLDSQVMSLHNLVHSFLNGT----------------NALPHSAANDPI 238

SaTy ----------LHNRVHVWVGG-----------------QMATGVSPNDPV 209

ScTy ----------LHNRVHVWVGG-----------------QMATGVSPNDPV 209
BmTy ----------LHNRVHRWVGGQ----------------MGVVPTAPNDPV 220

AoTy ATSLAVPLESPHNDMHLAIGGVQIPGFNVDQYAGANGDMGENDTASFDPI 328

AbTy -------LESVHDDIHVMVGYGKIEG-----------HMDHPFFAAFDPI 277

IbCaOx NKGGGSIENIPHTPVHRWVGDVK-PRTQNGE------DMGNFYSAGRDIL 296

VvCaOx DPGAGTLEHVPHNIVHKWTGLADKPSEDMGN----------FYTAGRDPI 370
AoCaOx QGVPGSGSIGVHGGGHYSMGGD--PG-------------RDVYVSP---- 303

HsTy FLLH---------------------------------HAFVDSIFEQWLR 256

HsTRP-1 FVLL---------------------------------HTFTDAVFDEWLR 260
HsTRP-2 FVVISNRLLYNATTNILEHVRKEKATKELPSLHVLVLHSFTDAIFDEWMK 288

mFWLH -------------------------------------HAYIDKLWAEWQR 226

ScTy FWLH---------------------------------HAYVDKLWAEWQR 226
BmTy FFLH---------------------------------HANVDRIWAVWQI 237
AoTy FYFH---------------------------------HCFIDYLFWTWQT 345
AbTy FWLH---------------------------------HTNVDRLLSLWKA 294
IbCaOx FYCH---------------------------------HSNVDRMWTIWQQ 333
VvCaOx FFGH---------------------------------HANVDRMWNIWKT 387
AoCaOx FWLH---------------------------------HGMIDRVWWIWQN 320
Fig. (1). Multiple sequence alignment of amino acid sequences. The alignment was generated with ClustalW2. The histidine residues in-
volved in the coordination of copper are highlihted in yellow and blue. HsTy (NP_000363.1); HsTRP-1 (AAC15468.1); HsTRP-2
(ABI73976.1); ScTy (AAP33665.1); BmTy (ADF40229.1); AoTy (CAX65671.1) and AbTy (XP_006459626.1).
3038 Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 27
A) B)
Cu A
Cu B
Fig. (2). (A) Superimposition of the Ty forms: AbTy (yellow, PDB 2Y9W), AoTy (blue, PDB 3W6Q), BmTy (green, PDB 3NMT) and ScTy
(red, PDB 2AHL) showing the conserved hydrophobic core comprising a (B) four-helix bundle with two sets of three histidine residues that
coordinate the two copper-atoms, CuA and CuB. (C) HsTy structure obtained by homology modeling techniques.
oxidation occur at the same site, namely CuA [79]. It is sug-
gested that concurrent presence of a phenylalanine above the
active site and a restricting thioether bond on the histidine
coordinating CuA prevent hydroxylation of monophenols by
CaOxs. However, in the structure of the AoCaOx both cop-
per sites are accessible [69].
The human Ty (HsTy) is a single strand membrane pro-
tein with a short transmembrane domain and 90% of the
structure located in the inner melanosomal region including
the active site [80]. To date, no structure of a mammalian Ty
has been experimentally solved. Due to the low sequence
similarity with the available Ty structures, homology model-
ing of human Ty may prove to be difficult [81]. However, on
the base of the structure of ScTy, structure models of the met
[82-84] and the oxy forms [85] forms of the HsTy built by
homology modeling techniques were recently published
(Fig. 2C). A model of mouse Ty has also been reported [86].
Recently, a robust homology model built using structural
information from both ScTy and IbCaOx was published [87].
In this model, the active site was refined through an optimi-
zation protocol with specific force-field parameters for cop-
per coordination and charges calculated by quantum me-
chanical methods. The superimposition of the human homol-
ogy model on crystallographic structures of the Ty from
other species revealed similar overall backbone topologies,
active site conformations, and conserved water molecules. In
addition, the docking with N-Phenylthiourea (PTU), an in-
hibitor for both Ty and CaOx, into the solvated human active
pocket revealed a binding mode consistent with the crystal-
lographic information obtained.
Probably formed by gene duplication [88], the TRP-1 and
TRP-2 are strongly associated to Ty in the melanin biosyn-
thetic pathway (Scheme 2). TRP-1 and TRP-2 are found in
the membrane of melanosomes, where they are important to
maintain the stability of Ty. Ty, TRP-1 and TRP-2 share up
to 40% amino acid identity and approximately 70% amino
acid homology [89]. Despite this high level of similarity,
they show different functions and catalytic behavior [90].
TRP-1 is a putative copper-containing enzyme [42,91] and
TRP-2 is a zinc-containing enzyme [92]. While the role of
TRP-2 in tautomerisation of DOPAchrome into DHICA is
well established, the TRP-1 function and role are not yet
Buitrago et al.
C)
Cu A
Cu B
clear. TRP-1 seems to have a structural function in the stabi-
lization of Ty [93,94]. The function of TRP-1 is closer to
CaOx than Ty, since it exhibits a low phenolase activity but
high catecholase activity [95,96].
4. TYROSINASE EFFECTORS
Being Ty inhibition a well-established approach to con-
trol in vivo melanin production, the development of Ty in-
hibitors has had a huge economical and industrial impact in
medicine, cosmetic and agriculture as highlighted by the
increasing number of Ty inhibitors reported in the recent
literature [97-101]. A large part of this literature is devoted
to naturally occurring compounds such as flavonoids and
polyphenolic compounds, triterpens, alkaloids [102] as well
as mixture extracts from plants [102] and peptides [103].
The best way to selectively inhibit the Ty activity is to
target its unique binuclear active site. Some of the described
Ty inhibitors such as Thujaplicin [104,105] and tropolone
[64], Kojic acid (KA) [106,107], L-mimosin [108], PTU
[109,110] and thiosemicarbazones (TSC) [111-121], 2-
hydroxypyridine N-oxide (HOPNOH) [122] and 2-
pyridinethiol N-oxide (HSPNOH) [123], match this strategy.
Apart from PTU and TSC that are simple chelators of the
dicopper site, the others are named transition-state (TS) ana-
logues, because they have structural analogies to catechol but
are not oxidizable (Scheme 3).
However, despite the abundant literature, there are few
data on the selectivity of inhibitors towards Tys, their inhibi-
tion mode and how they interact with Ty. KA [124], a fungal
metabolite used as a reference molecule in Ty inhibition,
exhibits features of competitive inhibition in the catalyzed
oxidation of L-DOPA for the mushroom AbTy [124] and for
the bacterial ScTy [125] (Fig. 4). These results argue for a
competition between KA and L-DOPA for the binding to the
dicopper Ty center. A recent structure of the BmTy in com-
plex with bound KA, shows that KA is oriented with the
hydroxymethyl towards the active site at a relatively long
distance of 7 Å [61]. Thus, the position and orientation of
KA cause an obstruction of the active site that could account
for the mechanism of inhibition. Conversely, a docking study
of KA in the active site of ScTy predicted an orientation of
Are Human Tyrosinase and Related Proteins Suitable Targets Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 27 3039

A) B)
His Phe261
OH His
O N
N N
N N O
His N
N O His
CuA N N N O
CuB CuA N
His N O CuB N
His His N O
N N His
N N
N N
N N N
N
His His
His His

C) D)

CuB CuA CuB CuA

F261

Ty CaOx

Fig. (3). μ-η2:η2-peroxo-dicopper(II) intermediates involved in Ty and CaOx mediated oxidations. In Ty, the CuA is accessible to phenolate
that binds it (A), while in IbCaOx the accessibility of the CuA site is blocked by a phenyl alanine residue (B) imposing the catecholate to bind
on CuB. Pictures C) and D) show the accessibility of the active site in the ScTy and IbCaOx (PDB 1BUG), respectively.

Scheme 3. Selected Ty inhibitors that target the dicopper active site; (A) transition-state analogues and (B) chelators.
3040 Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 27
Fig. (4). Aurone compounds as Tys effectors.
the hydroxymethyl moiety towards the dicopper center
[126]. A study of binding of TS-analogs KA [127] and
HOPNOH [122,123,128] on biomimetic model compounds
reveals different binding modes where the catechol-like
moieties bind the dicopper center. KA adopts a η2:η1 coordi-
nation mode to the dicopper center (Fig. 5A). This η2:η 1
coordination mode exhibits strong similarities between Ty-
KA model obtained using XAS [129] or NMR spectroscopy
[130] (Fig. 5B). The location of KA in BmTy could be an
intermediate binding site.
Human Ty is difficult to obtain in an active form by ge-
netic expression [130], consequentially most Ty inhibitors
were evaluated using the commercially available mushroom
AbTy, as paradigm of all Tys among which also the human
one. The assumption of similarity between the mushroom
AbTy and other Tys is rather hazardous because of the low
sequence homology between Tys of different origin. In addi-
Buitrago et al.
tion, the emerging differences in the binding pocket topolo-
gies, as recently evidenced by the resolution of the crystal
structures mentioned above, pose further questions on these
data. A comparative study between mushroom AbTy and the
bacterial Streptomyces antibioticus Ty (SaTy) [131] analyses
these differences [132]. This study used a small chemical
library composed of aurone compounds (Fig. 4, compounds
A-F). The reported data point out that aurones have different
reactivity toward AbTy and SaTy. In a previous study, it was
demonstrated that aurone compounds interact with the Ty
active site by the C-ring (Fig. 4). While the aurones A and B
were described as inhibitors in melanogenesis [133], with
AbTy and SaTy they behave as substrates, producing quinone
compounds. The main differences were observed with
aurones D and C. While these compounds behave as hyper-
bolic activators for AbTy, they behaved as poor inhibitors of
SaTy. Several activators of AbTy have been reported in the
Are Human Tyrosinase and Related Proteins Suitable Targets
A) B)
(His)N
O N(His)
Cu
(His)N
(His)N
KA
Fig. (5). (A) X-ray crystal structure of the KA adduct with a biomimetic model; (B) possible binding mode for Ty-KA according to X-ray
absorption spectroscopy studies with bound kojic acid in a η2:η2 mode and (C) QM/MM Binding of the kojic acid inhibitor to the active site
of a bacterial Ty.
literature [134-137]. AbTy is naturally present in two forms:
one active and one latent. To activate the latent form, nu-
merous activators have been reported: anionic detergents,
alcohols, fatty acids, acid shock, protease and pathogens
attack [138]. Considering that this activation proceeds slowly
via a lag period and depends on the nature of the activating
molecules, it has been suggested that this activation is actu-
ally a conformational change of the protein to reach its active
form. However, the activator versus inhibitor behaviors ob-
served with aurones D and C towards AbTy and SaTy, re-
spectively, can be explained by the significant differences in
the structures of AbTy and SaTy. Unlike Ty from sac-
chacaromyces, which are monomeric in solution, AbTy is
obtained as a heterodimer of a heavy subunit (containing the
active center) and a light subunit. An allosteric effect could
be responsible for this behaviour. Some pentapeptides have
been published as activators for both mushroom and mouse
tyrosinase. An allosteric activation of Ty has been proposed
as well [139,140]. Finally, aurones C behave as inhibitors of
AbTy and SaTy, but with significant IC50 variation (Fig. 4).
The replacement of the phenol C-ring of aurone by
HOPNOH moieties (compound F in Fig. 4) allows decreas-
ing the inhibition constants to the μM range [1-10 µM]. This
comparative study conducted on AbTy and SaTy with a series
of aurones as effectors reveals large differences in Ty behav-
iors and that could be hazardous to use the commercially
available mushroom tyrosinase as a model for the human
form.
5. MANAGEMENT OF MELANOMA BY EFFECTORS
AND SUBSTRATES OF TYROSINASE
Regulation of Ty activity is a longstanding, effective ap-
proach for controlling melanin production. In this context,
several stages of Ty’s life can be targeted, such as the tran-
scription of its mRNA, its maturation, the catalytic activity
of the mature protein and its degradation [141]. The catalytic
activity was extensively studied and addressed biological,
cosmetic, food and medical fields, leading to the discovery
of a large helter-skelter pool of Ty effectors. In humans, Ty
modulators can be used against accumulation of high levels
of melanin associated to a variety of disorders. The literature
analysis clearly demonstrates that, if numerous compounds
were identified through a hazardous extrapolation of mush-
room Ty inhibition activity, others were selected for their
Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 27 3041
C)
N(His)
Cu
O N(His)
O
ability to inhibit melanogenesis directly in human melano-
cytes, paving the way for a well-established strategy relying
on mammals Ty activity as a potent lever for controlling
melanogenesis in vivo [142]. Therefore, the role of Ty modu-
lators in melanoma therapy mainly depends on established
relationships between melanin biosynthesis and melanoma
progression, resistance and general behavior.
The high lethality of cutaneous melanoma is mainly due
to strong resistances, in late stages III and IV of the disease,
to all current forms of therapy, such as chemotherapy, radio-
therapy, phototherapy and immunotherapy. Indeed, unlike
normal melanocytes, melanoma cells retain the synthesized
melanin, instead of excreting it to proximate keratinocytes.
Hence the presence of concentrated melanin pigments in
melanomas: i) protects them endogenously from radiother-
apy by heavy absorption of radiations; ii) produces interme-
diates of melanogenesis such as L-DOPA and reactive qui-
nones which lead to a strong immunosuppressive effect,
jeopardizing any kind of immunotherapy directed against
melanoma [143]; and iii) acts as an antioxidant, detoxifying
agent, and reactive oxygen species (ROS) scavenger, neutral-
izing photodynamic therapy or chemotherapy effects [144].
All these elements combine to associate increased melano-
genesis to shortened overall and disease-free survival of pa-
tients [145]. Thus, targeting Ty with inhibitors able to stop
melanin production in melanoma could restore its sensitivity
to external insults and constitute a promising adjuvant ap-
proach to therapies (Fig. 6).
To date, few studies focused on the impact of using Ty
inhibitors as therapy sensitizers against melanoma. A recent
report highlighted that a decrease of melanogenesis induced
by PTU, a known Ty inhibitor, was associated with an in-
crease of melanoma sensitivity in vitro to ionizing radiations
[146]. The authors further demonstrated that the inhibition of
Ty by PTU could also lead to a sensitization of melanoma
cells to the chemotherapeutic action of cyclophosphamide, a
well-known alkylating agent, leading to a killing of treated,
depigmented cells at 10-6 M, versus 10-3 M for pigmented
counterparts [147]. Similarly, while pigmented melanoma
cells are resistant to peripheral blood lymphocyte-mediated
cytotoxicity despite the presence of melanoma-associated
antigens (<0.1% of cells killed), the same cells depigmented
by treatment with PTU showed a dramatic increase of sensi-
3042 Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 27 Buitrago et al.

tivity (50% of cells killed under the same conditions) [147]. [159], amide [160] or hydrazine [157] linker to an oxidizable
Two more recent studies reported a significant decline in moiety (i.e. a phenol group), which acts as the substrate for
melanoma cell viability upon hypericin-mediated photody- Ty. The reaction mediated by Tys leads to the formation of a
namic therapy when a prior depigmentation was undertaken corresponding ortho-quinone, and a subsequent, often cycli-
using a known Ty inhibitor (either PTU or KA). A strong zation-based mechanism finally allows release of the cyto-
increase of ROS production was also recorded concomitantly toxic agent.
[148,149]. Collectively, these preliminary results are encour-
aging for a potential use of Ty inhibitors in radio-, chemo-, 2+
immuno- and phototherapy. 2+
2
+2 2
2(W
Various cell
damages 2
Restored +2
sensitivity 2+ 20H
2
Release of to therapies (WK\OK\GUR[\EHQ]RDWH
cytotoxic 20H
agents 70(&*
20H
Reduced
Melanocyte
melanin
production Keratinocyte
Ty 7\ 2

Substrates of
Tyrosinase +2 +2 2 2
+
5KRGRGHQGURO
Inhibitors of
Tyrosinase

Fig. (6). Two general Ty-relying strategies for treating melanoma

cells. 2 7\ 2

Besides this promising melanogenesis suppressing strat-

egy, another Ty-relying approach emerged which takes ad-
vantage of the overexpression of Ty observed in melanoma
cells. Indeed, the latter can be used to selectively address
cytotoxic agents against melanoma, using a well-established
prodrug strategy in general cancer research [150]. Based on
the large amount of Ty in melanoma cells and the high turn-
over rate of its catalytic activity, the oxidation of alternative
substrates of the enzyme could release chemotherapeutics in
two distinct perspectives, called “Achilles heel approach”
and “Trojan horse approach” [23,151].
In the “Achilles heel approach”, the oxidation of Ty sub-
strates generates reactive quinones capable of leaking into
the cytosol and reacting with vital melanoma components,
thereby causing irreversible structural and functional dam-
age, followed by cell death. These phenolic substrates espe-
cially prevent the spontaneous, self-extinguishing cyclization
that occurs from dopaquinone in the normal melanogenesis
process, and allow an increased local diffusion of toxic com-
pounds (Fig. 6). Among a large number of such studied phe-
nols, ethyl 4-hydroxybenzoate [152], catechins [153] or rho-
dodendrol [154] were recently reported as promising agents
(Fig. 7). The Ty-mediated activation of TMECG, an epicate-
chin derivative, especially showed a strong antifolate activ-
ity, leading to thymidine pool depletion and melanoma cell
death in vivo [155,156]. In contrast, the “Trojan horse ap-
proach”, or MDEPT (Melanocyte-Directed Enzyme Prodrug
Therapy) consists in the Ty-mediated selective release of a
cytotoxic agent, such as phenol or nitrogen mustards
[157,158] or triazenes [159,160] (Fig. 8). Classically, the
anticancer drug is connected through a urea, carbamate
2 2 +2 2
&\WRWR[LFTXLQRQH
Fig. (7). Examples of “Achilles heel” phenol prodrugs; Example of
tyrosinase-mediated mechanism of the cytotoxic quinone formation.
In addition, recent findings tend to establish an implica-
tion of Ty-related proteins 1 and 2 (TRP-1 and 2) in mela-
noma progression and resistance. The levels of TRP-2 in
melanoma cells were especially positively correlated with
resistance phenomena to several chemotherapeutics [161],
while TRP-1 role is still poorly known, but the enzyme could
be involved in malignancy emergence processes and mela-
noma progression [162]. Despite the current lack of reported
effectors of TRP-1 and TRP-2, all these preliminary results
could lay the foundations for targeting Ty-related proteins
within the context of future therapies.
CONCLUSION
In summary, a large number of both inhibitors and sub-
strates of Ty were already designed to date, which could find
promising applications for fighting melanoma. The suppres-
sion of melanogenesis, which could restore cancer cell sensi-
tivity to various therapies, as well as the selective delivery of
toxic compounds or active drugs in melanoma cells, both
represent ongoing strategies based on the exploitation and/or
modulation of tyrosinase activity. In the same way as target-
ing the specific metabolism of melanoma cells, such as glu-
cose uptake and metabolism, folate metabolism or lipid sig-
Are Human Tyrosinase and Related Proteins Suitable Targets Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 27 3043

naling pathways, exploiting tyrosinase or related proteins AbTy = Agaricus bisporus tyrosinase
could ensure strong effectiveness and selectivity of treat-
AoTy = Aspergillus oryzae tyrosinase
ments for melanoma [163].
BmTy = Bacillus megaterium tyrosinase
2

+ 1+
HsTy = Homo sapiens tyrosinase
2 &+
1 1 1 MsTy = Manduca sexta tyrosinase
1 1
+ &O 2
1 ; SaTy = Streptomyces antibioticus tyrosinase
&O

2+ 2+
ScTy = Streptomyces castaneoglobisporus tyrosinase

+ &+
VvTy = Vitis viniferas tyrosinase
1 1 1 +2
1 2 TRP-1 = Tyrosinase-related proteins 1
2 &O
; 1 1 TRP-2 = Tyrosinase-related proteins 2
+ &O
+2
7\
TSC = Thiosemicarbazide
UVR = UV radiation
+ &+
1 1 1 CONFLICT OF INTEREST
1
;
2 2 +2
; The author(s) confirm that this article content has no con-
2 +2 1 +
1 1 flict of interest.
1
2
&+
ACKNOWLEDGEMENTS
The authors thank CNRS and the French Agence Nation-
ale pour la Recherche for financial support (ANR-09-BLAN-
+2
0028-01/02/03). R. Hardré and M. Réglier thank Aix-
+2 1 ; Marseille University and E. Buitrago, R. Haudecoeur, H.
+
Jamet, C. Belle and A. Boumendjel, are grateful to Labex
+ 1
1 1 Arcane for financial support (ANR-11-LABX-0003-01).
&2
&+
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