Thiosemicarbazones as inhibitors of tyrosinase enzyme

Article abstract of DOI:10.1016/j.bmcl.2017.05.057

In the search for compounds which may inhibit the development of melanomas, a series of thiosemicarbazones has been investigated as possible inhibitors of the tyrosinase enzyme. The results showed that all the thiosemicarbazones tested exhibited significant inhibitory effects on the enzyme. Thiosemicarbazones Thio-1, Thio-2, Thio-3 and Thio-4 substituted with oxygenate moieties, were better inhibitors (IC₅₀ 0.42, 0.35, 0.36 and 0.44?mM, respectively) than Thio-5, Thio-6, Thio-7 and Thio-8. For the better inhibitors, molecular docking results suggested that the oxygen present in the para position of the aromatic ring is essential for the tyrosinase inhibition, due its high ability for complexation with Cu²⁺ ions. Inside the active protein pocket, Thio-2 – the best studied inhibitor – is able to interact with the amino acid residues His-155, Gly-170 and Val-172 via hydrogen bonding and hydrophobic force. Thio-2, containing a substituent on the aromatic ring similar to the substrate L-DOPA, showed a competitive inhibition mechanism as viewed in a Lineweaver–Burk plot. The same results were observed in the UV–Vis curves.

Full text of DOI:10.1016/j.bmcl.2017.05.057

Accepted Manuscript
Thiosemicarbazones as Inhibitors of Tyrosinase Enzyme
Mariana A. Soares, Mariana A. Almeida, Carla Marins-Goulart, Otávio A.
Chaves, Aurea Echevarria, Márcia C.C. de Oliveira
PII:
S0960-894X(17)30544-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.05.057
BMCL 25002
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
31 March 2017
16 May 2017
17 May 2017
Please cite this article as: Soares, M.A., Almeida, M.A., Marins-Goulart, C., Chaves, O.A., Echevarria, A., de
Oliveira, M.C.C., Thiosemicarbazones as Inhibitors of Tyrosinase Enzyme, Bioorganic & Medicinal Chemistry
Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.05.057
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Bioorganic & Medicinal Chemistry Letters
Thiosemicarbazones as Inhibitors of Tyrosinase Enzyme
Mariana A. Soares, Mariana A. Almeida, Carla Marins-Goulart, Otávio A. Chaves, Aurea Echevarria,
*
Márcia C. C. de Oliveira
Department of Chemistry, Universidade Federal Rural do Rio de Janeiro, Seropédica, RJ, 23890-000, Brazil.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
In the search for compounds which may inhibit the development of melanomas, a series of
thiosemicarbazones has been investigated as possible inhibitors of the tyrosinase enzyme. The
results showed that all the thiosemicarbazones tested exhibited significant inhibitory effects on
the enzyme. Thiosemicarbazones Thio-1, Thio-2, Thio-3 and Thio-4 substituted with oxygenate
moieties, were better inhibitors (IC50 0.42, 0.35, 0.36 and 0.44 mM, respectively) than Thio-5,
Thio-6, Thio-7 and Thio-8. For the better inhibitors, molecular docking results suggested that
the oxygen present in the para position of the aromatic ring is essential for the tyrosinase
2
+
Keywords:
Melanin
Phenol oxidase
Melanome
Thiosemicarbazones
Molecular docking
inhibition, due its high ability for complexation with Cu ions. Inside the active protein pocket,
Thio-2 – the best studied inhibitor - is able to interact with the amino acid residues His-155,
Gly-170 and Val-172 via hydrogen bonding and hydrophobic force. Thio-2, containing a
substituent on the aromatic ring similar to the substrate L-DOPA, showed a competitive
inhibition mechanism as viewed in a Lineweaver–Burk plot. The same results were observed in
the UV-Vis curves.
2
017 Elsevier Ltd. All rights reserved.
Melanin pigments are found in mammalian skin and
extracutaneous tissue including the brain, eye and inner ear.
Cutaneous melanin is formed in neural crest-derived
melanocytes. Melanin produced in skin melanocytes
provides protection from the sun’s ultraviolet radiation.
People with black skin are well protected from the
mutagenic effects of ultraviolet radiation while those with
fair skin are at high risk for developing skin cancer.
Melanoma is the most aggressive and potentially lethal form
of skin tumor. It originates in pigment-producing
melanocytes that are found in the basal layer of the
epidermis and in the eye. The number of melanoma cases
and deaths worldwide has increased faster than many other
dihydroxyphenylalanine (L-DOPA), then oxidation of the
latter to an ortho-quinone (dopaquinone). Dopaquinone is
further transformed through several reactions to yield brown
4
to black melanin.
Several tyrosinase inhibitors have also been used in the
cosmetic industry as skin-whitening agents. Most of the
inhibitors of tyrosinase are, therefore, copper chelators or
phenolic compounds structurally analogous to the substrates
5
–7
L-tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA).
It is well known that tyrosinase can be inhibited by aromatic
aldehydes and aromatic acids, tropolone, alkoxybenzoic
acids and kojic acid; recently, dialkylphosphorylhydrazones
8
–12
have been reported as potential tyrosinase inhibitors.
Thiosemicarbazones have received considerable
attention because of their potential therapeutic activity, such
1,2
cancers though, lately, the trend has stabilized.
Pigmentary disorders occur for various reasons such as
loss of melanocytes and increased or decreased melanocyte
activity. Hyperpigmentation in the epidermis is caused by
excessive melanin synthesis (increased melanocyte activity)
which is controlled by the rate-limiting tyrosinase enzyme
1
3–16
as antitumoral, antibacterial and antimalarial.
Several
times, the transition metal complexes of thiosemicarbazones
showed greater biological activity than uncomplexed
1
7,18
ligands.
The aim of this study was to investigate seven
benzaldehyde-substituted thiosemicarbazones and
(
EC 1.14.18.1, phenol oxidase), which is found in
melanocytes localized to the membrane of the unique
3
melanocyte organelle, the melanosome.
pyridinecarboxaldehyde-thiosemicarbazone for tyrosinase
inhibitory capacity. Evaluation of the inhibitory activity was
made through kinetic study of the diphenolase inhibition and
the inhibition constants that characterized the system. The
inhibition mechanism involved was also investigated by two
The active site of tyrosinase consists of two copper
atoms and it is involved in the transformation of L-tyrosine
to dopaquinone which occurs through two steps:
hydroxylation
of
L-tyrosine
to
L-3,4-

methods,
Lineweaver–Burk
plot
and
UV-Vis
(IC50 0.26 mM) was used as positive control. As shown in
Table 1 all compounds exhibited tyrosinase inhibitory
effects, with IC50 values ranging from 0.35 to 1.42 mM.
spectrophotometry. In order to offer a molecular level
description for the inhibitory ability of the studied
compounds, and suggest the main amino acid residues
responsible for the stabilization of the interaction between
the enzyme and its best inhibitor, molecular docking
exercises were carried out.
The thiosemicarbazones were prepared according to the
general Scheme 1, using the thiosemicarbazide and
respective
pyridinecarboxaldehyde. The substituted benzaldehydes
used to prepare the target compounds were obtained from
commercial sources. All the compounds above were purified
by recrystallization from methanol. Additionally, all the
synthetic compounds were characterized by spectroscopic
data, which were in full accordance with their proposed
Among the assayed compounds, Thio-1, Thio-2, Thio-3 and
Thio-4 showed the best inhibitory effect, with IC50 values in
the range of 0.35 mM to 0.44 mM, at use L-DOPA as
substrate for diphenolase activity of mushroom tyrosinase.
The p-hydroxybenzaldehyde-thiosemicarbazone
was previously evaluated;
(
Thio-1
however, the results were very
different, but one of them was consistent with those
)
2
3,24
substituted
benzaldehydes
and
1
9
2
4
observed in this work for the same compound.
Table 1
Concentrations of thiosemicarbazones and ascorbic acid leading to 50%
activity loss (IC50) of tyrosinase and substrate L-DOPA
2
0,21
structures.
The compounds tested are presented in Table
Compounds
Substituents
IC50
1
.
(
mM)
1
2
R
R
Thio-1
Thio-2
Thio-3
Thio-4
Thio-5
Thio-6
Thio-7
HO
HO
H
OCH
H
0.42
0.35
0.36
0.44
0.82
1.42
0.84
1.19
0.26
3
OC
2
OCH
H
H
5
3
H
Scheme 1 Synthetic route for thiosemicarbazone synthesis.
H
Br
H
In the present study, each thiosemicarbazone and then
thiosemicarbazide was examined for its ability to inhibit
tyrosinase activity with L-DOPA, in several concentrations,
according to the assay protocol described by Caxeiro et
Cl
H
Thio-8
Ascorbic
acid
Pyridine 2 carboxaldehyde thiosemicarbazone
-
-
1
2,22
al.
The percentage inhibition values were calculated
from the equation:
As tyrosinase is a copper-containing enzyme, it is
expected that potential tyrosinase inhibitors should show
%
inhib = {[(B30 – B ) – (Am – Am )] / (B – B )}  100
2+
25
0
30
0
30
0
high binding affinity for Cu ions. In order to offer a
molecular level description for the inhibitory ability of the
studied compounds, molecular docking exercises for
where B , B , Am and Am , are absorbance of the control
0
30
0
30
sample at time zero, absorbance of the control sample after
0 min, absorbance of the test sample at time zero and
2
mushroom Agaricus bisporus tyrosinase (PDB 2Y9X) were
3
2
6
performed with GOLD 5.2 program (CCDC).
absorbance of the test sample after 30 min, respectively. In
the equation, the interference of the possible absorbance of
organic compounds was subtracted. An increase of dose-
dependent inhibition percentage was observed through
exponential model curves for all thiosemicarbazones. Fig. 1
shows the graphic obtained for Thio-2 - the compound that
showed the highest inhibitory effect.
Fig. 2A and 2B show the best docking poses inside the
tyrosinase active site, for Thio-1/Thio-4 and Thio-5/Thio-8,
respectively. All studied compounds are buried inside the protein
binding site and both are close to the dicopper center (center
responsible for the tyrosinase activity) with an average distance
of 1.18 Å – 2.26 Å, however the hydroxyl group from phenol
moiety for Thio-1/Thio-4 and the sulfur/nitrogen groups from
thiosemicarbazone moiety for Thio-5/Thio-8, are the main
Thio-2 (mM)
% inhibition
2.67
2.22
1.78
1.33
93.78
2
+
chemical groups responsible for the complexation with Cu ions.
94.64
92.84
89.21
As Thio-1/Thio-4 showed better tyrosinase inhibition than Thio-
5
/Thio-8 (Table 1), molecular docking results suggest that it
occurs probably due the presence of hydroxyl groups, that are
better chemical functions than sulfur/nitrogen for the
complexation with Cu ions presented inside the tyrosinase
0
.89
.44
79.16
59.33
0
2+
0
.29
.15
45.66
24.01
structure.
0
Fig. 2C depicted the overlap between the best tyrosinase
inhibitor (Thio-2) with the standard sample (ascorbic acid).
Note that one of the hydroxyl groups of the standard sample is
overlap with the hydroxyl group of the Thio-2. It suggests that
the same chemical group (-OH) of both samples can interact
2
+
equally with the Cu ions, resulting in a quite similar
experimental IC50 values. Fig. 2C also shows the main amino
acid residues responsible for the stabilization of the complex
tyrosinase/Thio-2. The molecular docking result suggest that the
amino groups from the thiosemicarbazone moiety can interact via
hydrogen bonding with the peptidic C=O oxygen and N-H
hydrogen of Gly-170 and Val-171 residues, within distances of
Fig. 1. Inhibitory effect of Thio-2 on the diphenolase activity of mushroom
tyrosinase for the catalysis of L-DOPA at 25°C.
The IC50 values obtained from equations generated
through percentage inhibition against concentration curves
for each thiosemicarbazones are summarized in Table 1. For
comparison between the studied compounds, ascorbic acid
1
.84 Å and 3.07 Å, respectively. Hydrophobic force, via π-

stacking, between the aromatic ring of the ligand with the amino
acid residue His-155, was also detected by molecular docking,
within a distance of 3.21 Å.
where K , K and K are the Michaelis–Menten constant,
m map i
the apparent Michaelis-Menten constant and the inhibition
constant, respectively. Vm is the maximum velocity in the
presence of each inhibitor. Table 2 shows the Kmap, K and
i
Vm values, as well as the mechanism of tyrosinase
inhibition. The presence of sulfur and nitrogen atoms in
thiosemicarbazone structures may explain the inhibitory
effect; however, the competitive mechanism of inhibition
due to increased affinity for copper ions present in the active
site was due to the presence of substituents that favored this
interaction.
Fig. 2 (A) Overlap among the thiosemicarbazones Thio-1, Thio-2, Thio-3
and Thio-4, inside the tyrosinase active site. (B) Overlap among the
thiosemicarbazones Thio-5, Thio-6, Thio-7 and Thio-8, inside the tyrosinase
active site. (C) Overlap between the best tyrosinase inhibitor (Thio-2) with
the standard compound (ascorbic acid). Tyrosinase structure is in cartoon
representation (PDB: 2Y9X); Carbon atoms for Thio-1, Thio-2, Thio-3,
Thio-4, Thio-5, Thio-6, Thio-7, Thio-8 and ascorbic acid are in moss green,
beige, violet, light blue, dark green, lilac, purple, orange and gray,
respectively. The selected amino acid residues are in cyan. Copper ions (Cu )
are represented as spheres in brown. Element colors: hydrogen: white;
oxygen: red; nitrogen: dark blue; sulfur: yellow, chloro: limon and bromo:
firebrick.
Fig. 3 Lineweaver–Burk plots of tyrosinase and L-DOPA in the presence and
absence of thiosemicarbazones at 1.6 mM.
2+
Table 2
i m m m
Type of inhibition mechanism, as well as Kmap, K and V values. K and V
in the tyrosinase-catalyzed oxidation of L-DOPA were 0.87 mM and 30.58
mM/min.
The inhibitory mechanism of thiosemicarbazones on
mushroom tyrosinase for oxidation of L-DOPA was
determined from Lineweaver–Burk double reciprocal plots.
Compound
at 1.6 mM
Inhibitory
mechanism
K
map
K
i
V
m
(mM)
0.7
(mM)
7.3
(mM/min)
9.5
Thio-1
Thio-2
Thio-3
Thio-4
Thio-5
Thio-6
Thio-7
Thio-8
Uncompetitive mixed
Competitive
Graphs were constructed and some are shown in Fig. 3
.
22.4
21.7
26.3
0.8
0.06
0.06
0.05
4.5
48.1
The equations used to determine the inhibition constant
Competitive
38.4
K for competitive, uncompetitive and uncompetitive mixed
i
2
8
Competitive
34.8
inhibition are described below.
Uncompetitive mixed
Uncompetitive
Competitive
18.9
Competitive inhibition: Kmap = [(K /K )[I] + K ]
m
i
m
0.5
0.6
16.6
1.9
1.3
39.7
Uncompetitive inhibition: 1/V = [(1/V K ) [I] + 1/V ]
m
m
i
m
Competitive
8.3
0.2
27.1
Uncompetitive mixed inhibition: −1/Kmap = −(1 + [I]/K )/K
i
m

It was interesting to note that Thio-2
showed higher competitiveness with the active site (when
compared to other compounds with the same mechanism of
,
Thio-3 and Thio-4
Kinetic studies of the steady state of the pathway showed
the low catalytic efficiency of tyrosinase on monophenols
2
8
than o-diphenols. The thiosemicarbazones were used in the
evaluation of enzyme activity to determine the enzymatic
kinetics. The evolution of the enzyme activity was
monitored by readings taken with an UV-Vis
spectrophotometer at 475 nm for 60 min at 10 min intervals.
The good inhibitors maintained inhibition for the majority of
inhibition). These compounds with –OH, CH O– and
3
CH CH O– moieties in the aromatic ring do not hydrogen
3
2
bond with histidine residues present in the active site of the
enzyme, favoring the interaction of Thio-2 Thio-3 and
Thio-4 in the active site. Thio-6, with bromo as substituent,
showed uncompetitive inhibition; this is probably because
the bulky halide that causes distortion in the active site,
making the enzyme catalytically inactive.
,
the time (Fig. 5)
.
Interestingly, a strong decrease of Thio-1 inhibition with
time was observed in the kinetic graphs, and inhibitors with
similar UV-Vis behavior also showed similar kinetic curves.
Further, the low K value, indicate a high inhibition at a
i
Despite of the IC50 values for Thio-2, Thio-3 and Thio-4
given concentration of substrate and inhibitor; therefore, the
being higher than for the others inhibitors, these compounds
showed good enzymatic kinetics, since the inhibition power
remained beyond 30 min, similarly to the positive control
results for this constant for Thio-2, Thio-3 and Thio-4
corroborate to the above discussion.
The results obtained from double reciprocal Lineweaver–
Burk plots were compared with UV-Vis curves obtained
from the thiosemicarbazone without and with the presence
of enzyme + L-DOPA.
Generally, the methods used to determine the mechanism
of inhibition of enzymes are Lineweaver–Burk and Dixon.
In both methods, inhibitors and different substrate
concentrations are required, as well as the addition of the
increase in the enzyme consumption. Therefore, UV-Vis
curves can indicate the type of inhibition mechanism in
short time and with economy of reagents when compared to
the traditional techniques. In this case, the substrate was L-
2
8
ascorbic acid
.
(A)
0
0
0
0
0
.40
L-DOPA
.30
.20
.10
.00
Thio 8
Thio 2
Thio 7
Thio 3
Thio 4
DOPA and the thiosemicarbazones Thio-1
Thio-6 showed similar mechanisms of inhibition, according
to Lineweaver–Burk plots (uncompetitive and
, Thio-5 and
uncompetitive mixed). The UV-Vis curves were
superimposed onto Thio-5 and Thio-6; however, these two
thiosemicarbazones did not present similarity with the
substrate L-DOPA. Furthermore, the bromine moiety in
Thio-6 could cause distortion of the enzyme active site.
Thio-5 showed a tendency to a competitive type of
inhibition mechanism, when compared to pure
uncompetitive, as well as Thio-1, which has a hydroxyl
moiety on the aromatic ring.
0
10
20
30
40
50
60
70
Time (min)
0.40
(B)
L-DOPA
Thio 1
0.30
Thiosemicarbazones substituted with oxygenate moieties
(
Thio-2, Thio-3 and Thio-4), which can form complexes
with copper ions, had the same UV-Vis profile and the same
mechanism of inhibition (competitive). Furthermore, the
curves for the chloro-substituted thiosemicarbazone (Thio-
0
.20
Thio 6
7
) and the pyridine 2 carboxaldehyde thiosemicarbazone
Thio-8) (compounds with different molecular structure
compared with substrate) were similar, due to interaction of
the side chain with the active site of the enzyme (Fig. 4)
Thio 5
0.10
(
.
0.00
0
10
20
30
40
50
60
70
Time (min)
Fig. 5 Course for oxidation of L-DOPA (0.17 mM) by mushroom tyrosinase
in the absence and presence of inhibitors (1.6 mM). (A) competitive
inhibitors; (B) uncompetitive and uncompetitive mixed inhibitors.
In this study, a series of thiosemicarbazones was
synthesized and their inhibitory activity against tyrosinase
enzyme was evaluated, by experimental and theoretical
methods. The thiosemicarbazones with oxygenate
substituents in the aromatic ring exhibited strong inhibition
on the activity of mushroom tyrosinase and IC50 values were
similar to the standard ascorbic acid. Molecular docking
results suggest that it occurs probably due the higher affinity
Fig. 4 UV curves of thiosemicarbazone with competitive inhibition type for
Lineweaver–Burk.

2
+
Chem. 2008, 43, 1983.
0. Yi, W.; Cao, R. H.; Chen, Z. Y.; Yu, L.; Ma, L.; Song, H. C.
Chem. Pharm. Bull. 2009, 57, 1273.
of hydroxyl groups than sulfur/nitrogen groups for Cu
2
ions. For the best tyrosinase inhibitor (Thio-2), it was able
to interact with His-155, Gly-170 and Val-172 residues. The
21. Coxon, G. D.; Craig, D.; Corrales, R. M.; Vialla, E.; Gannoun-
Zaka, L.; Kremer, L. PLoS ONE 2013, 8, e53162.
values of Kmap and K were in accordance with the inhibitory
i
2
2. The organic compounds were solubilized in dimethylsulfoxide
VETEC) (10 mM) and different aliquots of the stock solution
power and the type of inhibition mechanism investigated for
each compound. Thio-3 and Thio-4 showed the best
inhibitory effects at maintained the inhibition for a
significant time. Curves obtained by UV-Vis spectroscopy
indicated inhibition of the competitive type of mechanism
for compounds which structurally resembled the substrate
used, but in other cases the double reciprocal technique was
more appropriate. Further investigations are warranted to
explore their potential application in the management of
hyperpigmentation and melanoma cases.
(
were added to the reaction medium containing the tyrosinase
mushroom enzyme (50–100 units), EDTA (0.022 mmol/L) and
L-DOPA (0.17 mmol/L), all acquired for Sigma-Aldrich, in
PBS (50 mmol/L, pH 6.8) at room temperature. The time for
diphenolase to oxidize L-DOPA was 30 min and the readings
were performed in a UV-Vis spectrophotometer (Shimadzu,
model Mini 1240, Japan) at 475 nm.
2
3. Liang-Hu, C.; Yong-Hu, H.; Wei, S.; Kang-Kang, S.; Xuan, L.;
Yu-Long, J.; Jiang-Xing Z.; Qing-Xi, C. Agric. Food Chem.
2012, 60, 1542.
4. Wei, Y.; Rihui, C.; Wenlie, P.; Huan, W.; Qin, Y.; Binhua, Lin,
Z. M.; Huacan, S. Eur. J. Med. Chem. 2010, 45, 639.
5. Chaves, O. A.; de Barros, L. S.; de Oliveira, M. C. C.;
Sant’Anna, C. M. R.; Ferreira, A. B. B.; da Silva, F. A.;
Cesarin-Sobrinho, D.; Netto-Ferreira, J. C. J. Fluor. Chem.
2
2
Acknowledgments
2
017, 199, 30.
6. Thiosemicarbazones structures (Thio-1
Thio-5 Thio-6 Thio-7 and Thio-8) were built and energy-
The authors sincerely acknowledge the Brazilian
agencies: Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES), Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) and
Fundação de Amparo à Pesquisa do Estado do Rio de
Janeiro (FAPERJ) for the financial support. The authors also
thank Prof. Carlos M. R. Sant’Anna (Department of
Chemistry-UFRRJ) for the molecular docking facilities.
2
, Thio-2, Thio-3, Thio-
4
,
,
,
minimized with the Density Functional Theory (DFT), method
Becke-3-Lee Yang Parr (B3LYP) with the standard 6-31G*
basis set, available in Spartan'14 program (Wavefunction, Inc.).
The molecular docking exercises were performed with GOLD
5.2 program. Hydrogen atoms were added to the protein (PDB
2
Y9X) according to the data inferred by the GOLD 5.2
program on the ionization and tautomeric states. A 10 Å radius
spherical cavity around the dicopper center which is
-
responsible for the tyrosinase activity - was defined as the
binding site for the molecular docking calculations. The
scoring function used was ‘GoldScore’, due the best result
obtained by the redocking studies - ligand tropolone. The
figures of the docking poses for the largest docking score value
were generated with the PyMOL Delano Scientific LLC
program.
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Thiosemicarbazones as Inhibitors of Tyrosinase Enzyme
Mariana A. Soares, Mariana A. Almeida, Carla Marins-Goulart, Otávio A. Chaves, Aurea Echevarria, Márcia C.
*
C. de Oliveira
S
O
Tyrosinase H3C
N
NH
NH2
Thio-2 (IC = 0.35 mM)
50
HO
Inhibition