Inhibition of tyrosinase by 4H-chromene analogs: Synthesis, kinetic studies, and computational analysis

Article abstract of DOI:10.1111/cbdd.13001

Inhibition of mushroom tyrosinase was observed with synthetic dihydropyrano[3,2-b]chromenediones. Among them, DHPC04 displayed the most potent tyrosinase inhibitory activity with a K_i value of 4?μm, comparable to the reference standard inhibitor kojic acid. A kinetic study suggested that these synthetic heterocyclic compounds behave as competitive inhibitors for the L-DOPA binding site of the enzyme. Furthermore, molecular modeling provided important insight into the mechanism of binding interactions with the tyrosinase copper active site.

Full text of DOI:10.1111/cbdd.13001

DR. CLÁUDIO NAHUM ALVES (Orcid ID : 0000-0001-6576-4229)
Article type : Research Article
Inhibition of Tyrosinase by 4H-Chromene Analogues: Synthesis, Kinetic
Studies and Computational Analysis
1,7
1
2
4
Edikarlos M. Brasil , Luciana M. Canavieira , Érica T. C. Cardoso , Edilene O. Silva ,
1
,3
2,3,*
5
Jerônimo Lameira , José L. M. Nascimento , Vera L. Eifler-Lima , Barbarella de Matos
2,3
6
7
1
Macchi , Dharmarajan Sriram , Paul V. Bernhardt , José Rogério Araújo Silva , Craig M.
7
1,3,*
Williams and Cláudio N. Alves
1
Laboratório de Planejamento e Desenvolvimento de Fármacos, Instituto de Ciências Exatas e
2
Naturais, Universidade Federal do Pará, 66075-110, Belém, PA, Brazil. Laboratório de
Neuroquímica Molecular e Celular, Instituto de Ciências Biológicas, Universidade Federal do Pará,
66075-110, Belém, PA, Brazil.
3
Programa de Pós-Graduação em Biotecnologia, Universidade Federal do Pará, 66075-110, Belém,
PA, Brazil.
4
Laboratório de Biologia Estrutural, Instituto de Ciências Biológicas, Universidade Federal do Pará,
66075-110, Belém, PA, Brazil.
5
Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Avenida Ipiranga 2752,
90610-000 Porto Alegre, Rio Grande do Sul, Brazil.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13001
This article is protected by copyright. All rights reserved.

6
Medicinal Chemistry and Antimycobacterial Research Laboratory, Department of Pharmacy, Birla
Institute of Technology and Science, Pilani, Hyderabad Campus, Jawahar Nagar, Hyderabad 500
078, India.
7
School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, 4072,
Queensland, Australia.
Keywords: Tyrosinase; Kojic Acid; Multicomponent Reaction; Inhibitor; Kinetic Assays;
Molecular Modeling.
Corresponding author:
*
Phone: (+55) 91-3201-8235. Fax: (+55) 91-3201-8235. E-mail: nahum@ufpa.br
Phone: (+55) 91-3201-1601. Fax: (+55) 91-3201-7633. E-mail: jlmn@ufpa.br
*
Running Title: Developing new inhibitors with Tyrosinase activity via synthetic and
computational techniques.
Abstract
Inhibition of mushroom tyrosinase was observed with synthetic dihydropyrano[3,2-
b]chromenediones. Among them, DHPC04 displayed the most potent tyrosinase inhibitory
i
activity with a K value of 4µM, comparable to the reference standard inhibitor kojic acid. A
kinetic study suggested that these synthetic heterocyclic compounds behave as competitive
inhibitors for the L-DOPA binding site of the enzyme. Furthermore, molecular modeling
provided important insight into the mechanism of binding interactions with the tyrosinase
copper active site.
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Introduction
Melanin is the major cellular component observed in many species of animals, plants,
fungi and bacteria (1). In humans it is responsible for skin color and protection against
radiation (2). However, abnormal accumulation of melanin induces pigmentation disorders,
among which, the most deadly form of skin cancer can form (i.e. melanoma) (3). Melanin
develops in pigment cells called melanocytes due to the overproduction of melanin, the
protective tanning pigment in the skin (4).
Melanin biosynthesis is conducted in melanocytes, found in the basal layer of the
epidermis and controlled by a binuclear copper enzyme called tyrosinase (TYR) (EC
1
.14.14.1) (5). TYR is ubiquitous and catalyzes the oxidation of both monophenols (cresolase
or monophenolase activity) and o-diphenols (catecholase or diphenolase activity) into
reactive o-quinones. In addition, it is a rate-limiting enzyme for controlling the production of
melanin. The melanogenesis begins with the conversion of the amino acid L-tyrosine to L-
3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by tyrosinase (TYR) (6). Therefore, the
regulation of the TYR activity is directly connected with melanin biosynthesis.
TYR is classified structurally as a type-3 copper protein in which each active-site
copper ion is coordinated by three histidine residues (7) (Figure 1). In general, the TYR
inhibition mechanism is frequently based on both specific TYR inhibitors and specific TYR
inactivators (8). A large set of potent TYR inhibitors from natural and synthetic resources
have been reported during the last few years (8-12). Kojic acid (KA) is a well-known TYR
inhibitor and is widely used as a popular cosmetic skin-lightening ingredient. Its capacity to
chelate copper ion at the active site of the enzyme may consistently explain its inhibitory
effect (13). However, it only showed slight inhibitory activity against pigmentation within
intact melanocytes or in clinical assays (14). Therefore, it remains necessary to find new TYR
inhibitors with higher activity and without off target side effects.
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Figure 1. Catalytic site of TYR. The bivalent copper ions are chelated by three ind ii vidual
histidine residues.
Dihydropyrano[3,2-b]chromenediones (DHPCs), which are considered 4H-
chromenes, are an important class of fused oxygenated heterocycles that are synthesised via a
one-pot three-component reaction of kojic acid, an aldehyde and a 1,3-dione (15) (Figure 2).
These heterocyclic compounds dis pp lay a considerable structural diversity, including a
2
strategic alcoholic hydroxyl functio nn al group (-CH OH), which in turn can be modi ff ied by
attaching biologically functional groups to develop more potent drug molecules.
Interestingly, 4H-chromene derivativ ee s present a wide range of biological properties, ss uch as
fungicidal (16), antitumor (17), antib aa cterial (18), antidepressant (19), antioxidative (20), and
antihypertensive (21) activity.
In this study, we report the synthesis (including X-ray characterisation), inhibitory
effect and computational modeling of dihydropyrano[3,2-b]chromenediones (DHP CC s) as
TYR inhibitors.

Figure 2. The 4H-chromene scaffold (left) and the pyrano[3,2-b] motif (right).
Materials and Methods
Experimental Section
Materials
TYR of mushroom (M.W. 128 kDa), L-DOPA, dimethylsulfoxide (DMSO) and KA
were purchased from Sigma-Aldrich.
TYR Activity
TYR-inhibition activity of the DHPC01-04 compounds (Table 1) were performed by
using L-DOPA as a substrate according to Kubo and co-workers (22) with slight
modification. Incubation was carried out at 160 µl of different concentrations of the substrate
L-DOPA, 20 µl (2.4 U) of enzyme mushroom tyrosinase, 20 µl of KA and different
concentrations of DHPCs (25, 50 and 100µM). All solutions were prepared in Phosphate
Buffered Saline (PBS) pH 7.2.
The reaction was initiated by addition of enzyme to all wells simultaneously, which
was measured over the first 5 minutes in the microplate reader (ELx800, BioTek) with a 490
nm filter. Furthermore, a control reaction was also performed by incubating the enzyme with
KA or DHPCs in the absence of the substrate to demonstrate that there was no variation in
absorbance over time.
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i
Determination of Inhibition Constant (K )
The mode of inhibition on the enzyme was assayed by incubation of the different
concentrations of L-DOPA (0; 0.25; 0.5; 0.75; 1.0; 2.0; 3.0 and 4.0 mM) and inhibitors (0;
0.001; 0.025; 0.05; 0.1; 0.2; 0.4; 0.8 and 1.6 mM). The kinetic parameters (Km, Vmax and Ki)
were determined with the GraphPad Prism®5.0 software, according to the following
equations:
I. Competitive Inhibition:
ꢅ
ꢀꢁꢂꢃꢃ
= ꢀ · ꢄ1 +
ꢇ
ꢁ
ꢀꢆ
ꢉ
· ꢌ
ꢊꢋ
ꢁ
ꢈ =
ꢀꢁ
+ ꢌꢍꢎꢎ
II. Mixed Inhibition:
ꢉ
ꢁ
ꢊꢋ
ꢉ
ꢁ
=
ꢊꢋꢂꢃꢃ
ꢅ
ꢏ1 +
ꢓ
ꢐꢑ · ꢀꢆꢒ
ꢅ
ꢀꢁ
· ꢕ1 +
ꢅ
ꢖ
ꢓ
ꢀꢆ
ꢀ
ꢔꢁꢊꢋꢂꢃꢃ =
ꢏ1 +
ꢐꢑ · ꢀꢆꢒ
ꢉ
· ꢌ
ꢁ
ꢊꢋꢂꢃꢃ
ꢈ =
ꢀꢁꢂꢃꢃ
+ ꢌ
where Y, X and I denotes average absorbance change per minute, concentration of L-DOPA
and concentration of DHPCs, respectively. The parameter α determines mechanism, its value
determines the degree to which the binding of inhibitor changes the affinity of the enzyme for
substrate.
Computational Section
Molecular Docking
The crystal structure of tyrosinase from A. bisporus (AbTYR) was used as the initial
point for computational procedures, it was obtained from the Protein Data Bank (PDB) under
code 2Y9X (23), which contains tropolone (TRO) as a crystal inhibitor. In order to validate
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the protocol and computational program used for molecular docking simulations for the
proposed inhibitors, the crystal inhibitor (TRO) was re-docked in the active site of TYR.
Then, the KA and DHPCs derivatives were submitted for docking calculations.
The molecular docking procedures were performed using the Molegro Virtual Docker
(MVD) program (24). It has two docking search algorithms; MolDock Optimizer and
MolDock SE (Simplex Evolution). The first is the default search algorithm in MVD (25),
which is based on an evolutionary algorithm. However, MolDock SE performs better on
some complexes where the standard MolDock algorithm fails (26). Accordingly, for previous
computational analysis performed for TYR systems, the MolDock SE can be applied with
success for this type of complex (27). The default parameters for the search algorithm were
used to carry out molecular docking analysis. The detailed theory behind the MVD program
and its characteristics are described elsewhere (24, 27).
Results and Discussion
4H-Chromene Analogues Synthesis
The synthesis of the dihydropyrano[3,2-b]chromenediones was successfully achieved
by employing principles of “green chemistry”, and these included inexpensive off-the-shelf
catalysts, short reaction times, solvent-free conditions, straightforward work-up, and good-to-
high yielding syntheses (28).
For this study DHPCs were prepared in racemic form (mixture of enantiomers) via a
three-component condensation reaction of an aromatic aldehyde, dimedone, and KA (28). To
synthesize DHPC03 and DHPC04, chlorokojic acid (29) was used instead (Scheme 1). All
1
13
compounds were fully characterized by H NMR, C NMR, IR, HRMS and in some cases X-
ray crystallography (see SI file).
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Scheme 1. Synthesis of DHPC01 (R == H, X=OH), DHPC02 (R=4-CN, X=OH), DHPC
00 3
(R=2-F, X=Cl), and DHPC04 (R=4- FF , X=Cl).
Experimental Kinetic Assays and I nn hibition
The kinetic analysis was det ee rmined from the equation of the Lineweaver- BB urk to
obtain the inhibition constant (K ). The kinetic parameters showed that DHPC01 had a lower
i
effect in TYR activity, and therefor ee , DHPC01 did not appear to inhibit mushroo mm TYR.
Whereas, TYR activity was prominently inactivated by DHPC02, DHPC03 and DHPC04.
The kinetic analysis showed that the value of Vmax remained the same and the value of K
m
increased with increasing inhibitor cc oncentrations, indicating that these molecules i nn duced
competitive inhibition (Figure 3). D HH PC substrates might directly bind to the copper at the
active site and thus compete with the LL -DOPA substrate during catalysis.
Figure 3. Plot of Lineweaver-Burk on the oxidation of L-DOPA by TYR and DHPC02,
DHPC03 and DHPC04, respectively.

i
Analysis of these kinetics parameters show that the K values of three compounds
were 6.00 µM, 9.00 µM and 4.00 µM for DHPC02, DHPC03 and DHPC04, respectively (see
Table 1). Therefore, these results show high rates of inhibition of TYR and the order appears
to be DHPC04 > DHPC02 > DHPC03.
The increase in the slope of the graph 1/[S] indicated the binding strength of the
competitive inhibitors. That is, the slope of the graph is increased by the factor (1+ [I] / Ki) in
the presence of the inhibitor. The kinetic analysis showed that these inhibitors have a single
inhibition site, possibly with small differences in the docking site. Alternatively, these
inhibitors can bind in multiple orientations within the catalytic site of the enzyme, giving an
unexpected interactions, which may reflect small differences in inhibitor affinities.
The high slope value in the analysis of the variation in K
m
as a function of inhibitor
2
2
concentration: 22.12 ± 1.98 and r = 0.98 for DHPC02; 19.92 ± 1.37 and r = 0.99 for
2
DHPC03; and 42.19 ± 7.58 and r = 0.94 for DHPC04 (see Figure S4), suggests that these
compounds have a single inhibition site of inhibition.
Finally, these findings quite reasonably suggest that the DHPC compounds bind to the same
binding site as the substrate L-DOPA. The results match the values of affinity enzyme-
i
inhibitor (K ) and the values of free interaction energy. It is therefore likely that these
inhibitors present kinetic parameters that show similar features derived from inhibitors with
chelating and competitive properties (30).
Molecular Modelling Analysis
Molecular docking techniques have successfully been used to propose the bind mode
and interactions occurring between the TYR enzyme and some potential inhibitors, such as,
isophthalic acid (30), hesperetin (31), oxymatrine (32), hydroxy-based thymol analogues
(33), KA, and KA analogues (27). In this sense, we have performed similar analyses using
the DHPC compounds derived from chemical synthesis. In order to validate the molecular
docking procedure, and the MVD program, a re-docking calculation was computed using the
crystal inhibitor (TRO) as a reference. Thus, comparing theoretical and experimental
complexes (TYR-TRO) revealed excellent agreement between theoretical and experimental
models. In this way, this molecular docking procedure was used to compute the favored
conformation of the DHPC compounds in the binding site of the AbTYR.
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According to the re-docking results, the TRO conformation obtained by molecular
docking was in good agreement with its crystal structure conformation (see Figure S1).
Furthermore, the DHPC compounds were also submitted to the same molecular docking
steps. Our results showed that these compounds are complexed to the same binding pocket of
the TRO inhibitor (see Figure S2), and that the MVD program can reproduce suitable
conformations for molecular like KA and DHPC compounds in complex with the TYR
enzyme.
During the synthesis of the DHPC compounds two enantiomers were obtained for
each KA derivative (R and S). However, it is not clear at this stage which enantiomer is
responsible for the TYR activity during kinetic analysis. In order to elucidate this aspect,
molecular docking calculations using both enantiomers for all the DHPC compounds were
undertaken. This information was taken into account with respect to the binding affinity
values, and the interactions between the compounds and the amino acids residues of TYR.
The binding affinity values obtained through the MOLDOCK scoring function
showed better affinity for the R enantiomer in all cases (Table 1). The binding affinity values
for the S enantiomers are provided in the SI file. The orientation of the aryl group for this
conformation allowed suitable interactions with part of TYR enzyme (Figure 4A). Since the
aryl group for the S enantiomer was exposed to solvent the interaction with the residues of
TYR disappeared (Figure 4B). Comparing KA with the DHPC compounds, all molecules
interacted directly with one copper ion in the catalytic site of TYR as well as the TRO
inhibitor (Figure 5), which supported the competitive inhibition proposal. Considering that
DHPC04 had the strongest TYR inhibitory effect, our discussion at this point will be focused
around this facet, although, it is representative of all other DHPC compounds.
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Table 1. Experiment aa l and theoretical binding affinity values.
MOLDOCK sc oo ring
Compound
i
K value*
–
1
(
kcal·mol )
TRO
0.80 μM**
5.00 μM
2.40 mM
6.00 μM
9.00 μM
4.00 μM
*
–59.36
KA
–71.56
DHPC01
DHPC02
DHPC03
DHPC04
–101.26
–107.82
–93.26
–102.32
Standard deviation for analyzed molecules are smaller than 0.01 (< 0.01) **Experimental
value obtai nn ed by Espín and Wichers (34).

Figure 4. Binding mode of (A) R-DHPC04 and (B) S-DHPC04 con catalytic site o ff TYR.
The loop which comprises residues Asn242-Asn252 are highlighted on the stick model. The
binding mode of other DHPCs are present within the SI file (Figure S3).
Figure 5. Binding mode of (A) KA and (B) R-DHPC04 catalytic site of TYR ranked by the
MOLDOCK scoring function. The carbon atoms of inhibitors are in cyan color. Just the side
2
+
chain of the His residues (stick model) and Cu ions (ball model) are shown for clarity. The
binding mode of other DHPCs are present within the SI file (Figure S2).
According to a previous computational analysis, the residues Met280 and AA sn260
were found to be important in the first stage of inhibitor recognition, and their fixation during
molecular docking and MD simulations (30, 35), respectively. Whether van der Waals
interactions promoted by these residues are responsible is uncertain (30, 31, 36, 3 77 ). Our
molecular docking results suggest that the same van der Waals interaction can be found,
where the carbamoyl group of Asn260 interacts with nonpolar groups in the para position of
KA and its derivatives for the R enantiomer.
Aside from the interactions promoted by the Asn260 residue, an interesting
observation was the interaction between the DHPCs and the His243 residue, which could
provide support for maintaining π-π interactions with the aryl ring of the DHPC com pp ounds

(Figure 6). Interestingly, the DHPC compounds shown play a role in preventing the entry of
the substrate into the active site. Once the aryl ring on the enantiomer covers most of the
R
TYR catalytic site, they maintain interactions with a loop of TYR which comprises the
residues Asn242, His243, Gly244, Ala245, Val246, Val247, Gly248, Ala249, His250,
Ala251 and Asn252 (Figure 4A). At this point, it is propose that the DHPC compounds
influence the molecular behavior of this part of the TYR enzyme, by preventing the entrance
and recognition of the natural substrate.
Figure 6. Interactions between the DHPC04 compound and residues His243 and Asn260 on
the catalytic site of TYR. Carbon atoms of DHPC04 are in cyan color. The same interactions
have been found for the other DHPC
R enantiomers.
All DHPC compounds showed interactions with the copper ion in the catalytic site of
TYR (Figure 5B). Moreover, van der Waals interactions were found between the aromatic
groups of the DHPC compounds and residues Val247, Met256, Asn259, Phe263 and Val282
(Figure 7).

Figure 7. Van der Waals interactions between the DHPC04 compound and TYR residues.
Carbon atoms of DHPC04 are in cyan color. The same interactions have been found for the
others DHPC
R enantiomers. In 2D scheme, colored dash circles represent van der Waals
interactions; dashed line indicates copper ion chelated by carbonyl-oxygen atom.
Finally, Table 1 summarizes the affinity energy values computed by the MVD
program for the TYR-inhibitor complexes. Despite, the MOLDOCK scoring function (26)
not describing the relationship between the in silico and experimental data sufficiently well, it
was found that the computed values are suitable for qualitative analysis in the case when
multiple orientations are observed for the same compound. It is important to clarify that in the
present work, the interest was only focused on predicting the position of the analogues within
the active site of tyrosinase. Furthermore, for this application it was shown herein that the
docking approach using the MVD program (24) provides suitable predictions for docked
structures into the active site of tyrosinase. The computed energy values suggest that
compounds DHPC02 and DHPC04 exhibit the highest affinity for the TYR catalytic site,
with the same tendency as KA and the TRO inhibitors, which emphasise an inhibitory
activity comprising a competitive mode [as well as their interaction with one of the catalytic
2
+
copper ions (Cu A)]. These results suggest that the binding site occupied by the DHPC
compounds are very close to that found in previous studies (31, 35), therefore, the most
probable inhibition action occurs via competition with the substrate L-DOPA in the TYR
active site, which is in agreement with Ki values for competitive inhibition
.

Conclusion
The inhibitory effect of various DHPC compounds were investigated against the TYR
enzyme, which were found to induce changes in k . This is consistent with competitive
inhibition interactions with the substrate (L-DOPA) at the active site. Correspondingly,
changes in V indicated binding of the test compounds provided very effective to the
m
m
inhibition of TYR in a competitive inhibitory manner. The inhibitory mechanism of such
compounds is in accordance with the copper ion chelator concept, in that, analogues to kojic
acid compete with L-DOPA in the active site,, which induces changes in the hydrophobic
surfaces.
Therefore, a combination of inhibition kinetics and computational modeling may
facilitate the testing of new potential TYR inhibitors and the prediction of their inhibitory
mechanisms, as shown in the present study. Further biological evaluation of DHPCs and
detailed toxicity studies are ongoing and will be reported in due course.
Supplementary Materials
Supplementary
materials
can
be
accessed
at:
http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1747-0285
Acknowledgments
The authors gratefully acknowledge financial support from Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES) for financial support. As well as the Center for High
Performance Computing (CHPC) in Cape Town—South Africa—for computational support.
Financial support by the University of Queensland, and the Australian Research Council
(ARC) is gratefully acknowledged [C.M.W. (FT110100851)].
Conflicts of Interest
The authors declare no conflict of interest.
This article is protected by copyright. All rights reserved.

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