2-hydroxypyridine-N-oxide-embedded aurones as potent human tyrosinase inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.6b00369

With the aim to develop effective and selective human tyrosinase inhibitors, we investigated aurone derivatives whose B-ring was replaced by a non-oxidizable 2-hydroxypyridine-N-oxide (HOPNO) moiety. These aurones were synthesized and evaluated as inhibitors of purified human tyrosinase. Excellent inhibition activity was revealed and rationalized by theoretical calculations. The aurone backbone was especially found to play a crucial role, as the HOPNO moiety alone provided very modest activity on human tyrosinase. Furthermore, the in vitro activity was confirmed by measuring the melanogenesis suppression ability of the compounds in melanoma cell lysates and whole cells. Our study reveals that HOPNO-embedded 6-hydroxyaurone is to date the most effective inhibitor of isolated human tyrosinase. Owing to its low toxicity and its high inhibition activity, it could represent a milestone on the path toward new valuable agents in dermocosmetics, as well as in medical fields where it was recently suggested that tyrosinase could play key roles.

Full text of DOI:10.1021/acsmedchemlett.6b00369

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Letter
2-Hydroxypyridine-N-oxide-Embedded Aurones
as Potent Human Tyrosinase Inhibitors
Romain Haudecoeur, Marcello Carotti, Aurélie Gouron, Marc Maresca, Elina Buitrago, Renaud Hardre,
Elisabetta Bergantino, Helene Jamet, Catherine Belle, Marius Réglier, Luigi Bubacco, and Ahcene Boumendjel
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00369 • Publication Date (Web): 17 Nov 2016
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2-Hydroxypyridine-N-oxide-Embedded Aurones as Potent
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Romain Haudecoeur,*,†,§ Marcello Carotti,‡,§ Aurélie Gouron,∥,§ Marc Maresca,⊥ Elina Buitrago,†,∥
Renaud Hardré,⊥ Elisabetta Bergantino,‡ Hélène Jamet,∥ Catherine Belle,∥ Marius Réglier,⊥ Luigi
Bubacco,‡ and Ahcène Boumendjel†
†Univ. Grenoble-Alpes/CNRS, DPM UMR 5063, CS 40700, 38058 Grenoble, France.
‡Department of Biology, University of Padova, Via Ugo Bassi 58b, Padova, 35121, Italy.
∥Univ. Grenoble-Alpes/CNRS, DCM UMR 5250, CS 40700, 38058 Grenoble, France.
⊥Aix Marseille Univ, CNRS, Centrale Marseille, iSm2, Marseille, France.
KEYWORDS: Human tyrosinase, aurones, human melanoma, transition state analogues, QM/MM calculations.
ABSTRACT: With the aim to develop effective and selective human tyrosinase inhibitors, we investigated aurone deriva-
tives whose B-ring was replaced by a non-oxidizable 2-hydroxypyridin-N-oxide (HOPNO) moiety. These aurones were
synthesized and evaluated as inhibitors of purified human tyrosinase. Excellent inhibition activity was revealed and ra-
tionalized by theoretical calculations. The aurone backbone was especially found to play a crucial role, as the HOPNO
moiety alone provided very modest activity on human tyrosinase. Furthermore, the in vitro activity was confirmed by
measuring the melanogenesis suppression ability of the compounds in melanoma cell lysates and whole cells. Our study
reveals that HOPNO-embedded 6-hydroxyaurone is to date the most effective inhibitor of isolated human tyrosinase.
Owing to its low toxicity and its high inhibition activity, it could represent a milestone on the path towards new valuable
agents in dermocosmetics, as well as in medical fields where it was recently suggested that tyrosinase could play key roles.
Tyrosinases (Ty, EC 1.14.18.1) are type-3 copper-containing
metalloenzymes spread across a wide range of organisms,
especially mammals, fungi, bacteria and plants.1 These
include a binuclear active site, composed by two close
copper centers (dCu–Cu ≈ 2.9 to 4.9 Å) bridged by an
aquo(hydroxo) ligand in a met state, coordinated by six
histidine residues. Ty catalyzes the oxidation of phenols
and catechols into catechols and ortho-quinones, respec-
tively (Scheme 1).2 In mammals the transformations of Ty
neoglobisporus, TyB2),8 three TyM (from Agaricus bispo-
rus, TyM19,10 and TyM2,11,12 and Aspergillus oryzae, TyM3),13
and very recently one TyP (from Juglans regia)14 and one
polyphenol oxidase exhibiting tyrosinase activity towards
certain substrates (from Coreopsis grandiflora).15,16 More
precisely, the overall identity is low (10–30%) between
TyMs, TyBs and TyH. Around the active site (58% identity
within 6 Å from the metal center in TyM1 and TyB2), the
environments are similar (see Supporting Information),
despite some key features not shared among all Tys (e.g. a
conserved CuA histidine ligand cross-linked to a cysteine
residue in TyMs and TyP, a post-translational modifica-
tion absent in TyBs and TyH).17 While preliminary crystal-
lographic studies on TyH have been reported very recent-
ly, no structures are available to date at atomic resolu-
tion.18 However, a robust homology model built using
structural data from TyB2 and Ipomea batatas catechol
oxidase has recently been published.19
natural substrates, L-tyrosine and L-DOPA, into reactive
quinones are the first steps of melanogenesis.3 The result-
ing melanin pigments play a protecting role against UV
radiation and free radicals. Despite similar dicopper ac-
tive sites (see Supporting Information) and a common
function, i.e. phenol/catechol oxidation, significant differ-
ences were pointed out at cellular and structural levels
among the Tys from distinct sources, especially plant
(TyP), mushroom (TyM), bacterial (TyB) and human
(TyH).4 Indeed, while TyM and TyB are generally soluble
oligomeric enzymes present in the cytosol, TyH exists as a
highly glycosylated monomeric melanosomal transmem-
brane protein.5,6 Similarly, strong divergences are ob-
served upon sequence comparison, a discrepancy that has
been substantiated at a structural level in the last decade
by the resolution of crystallographic structures of two TyB
(from Bacillus megaterium, TyB17 and Streptomyces casta-
In human, melanin-related disorders are known to cause
cutaneous hyperpigmentation,20 skin lesions21 or mela-
noma resistance to conventional therapies.22 As Ty inhibi-
tion is a well-established strategy for controlling melanin
production in vivo, as demonstrated again recently,23 the
development of TyH inhibitors raises considerable inter-
est in dermocosmetics24 and cancer therapies25 fields.
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While the low homologies between the different forms of
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Ty exclude any extrapolation of inhibition studies per-
formed on TyM to TyH, an abundant literature is dedicat-
ed to TyM inhibitors for human applications.26 As a result,
only a handful of them are actually in use as depigmenta-
tion agents, such as hydroquinone, arbutin or kojic acid
(KA).27 Unfortunately, currently used inhibitors lack the
affinity and selectivity required for TyH-targeting applica-
tions and harmful toxicity has often been reported.4,28
Therefore, there is an urgent need for novel selective TyH
inhibitors that match efficacy and safety standards re-
quired for the development of products aimed to human
use.
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Over recent years, we reported that aurones (2-
benzylidenebenzofuran-3(2H)-ones), naturally occurring
flavonoids,29,30 act as inhibitors of melanin biosynthesis in
human melanocytes,31 as effectors of TyM,32,33 and can be
used as molecular probes for the investigation of the
binding-site structural homology between TyM and TyB.17
In one of these previous studies,32 we suggested that these
compounds bind to the Ty active site with their B-ring,
whose substituents determined the behavior of diverse
aurones toward both enzymes (i.e. inhibitor, activator or
substrate). Conversely, the A-ring hydroxylation pattern
of aurones affected the activity range and selectivity for
either TyM1 or TyB3 (TyB from Streptomyces antibioticus,
which belongs to the same Streptomyces genus and shares
82% identity with TyB2), sometimes considerably (au-
rones V in Figure 1). In parallel, we discovered and inves-
tigated the transition-state analogue HOPNO, a catechol-
mimicking, non oxidizable moiety (Scheme 1), as a potent
inhibitor of TyM1 (Kic = 1.8 µM).34 Embedding this small
moiety in an aurone backbone generated “hybrid” aurone
1a, which showed a TyM1 inhibition activity in the same
range than HOPNO (Figure 1).32
Figure 1. General structures of aurones previously identified
as effectors of TyM1 and TyB3. These compounds were either
substrates (B-ring in blue), activators (B-ring in green) or
inhibitors (B-ring in orange) of TyM1. For inhibitors, corre-
sponding IC50 values against TyM1 are indicated.17,32
actions of the most active hybrid aurone with TyH were
then rationalized by combining QM/MM dynamics and
noncovalent interaction (NCI) analysis, using the recent
homology model of TyH mentioned above.19 Comparisons
were made with the interactions of the HOPNO moiety
alone on TyH.
We report herein the synthesis of three hybrid aurones,
whose A-ring substitution patterns were selected reminis-
cently of our previous results on human melanocytes,31
along with their biological evaluation through in vitro
assays using 1. purified recombinant TyH (from Homo
As a whole, our recent studies highlighted a remarkable
versatility of aurones as Ty-interacting agents, allowing us
to gather valuable information on the relation between
their substitution pattern and their activities.17,31,32 The B-
Scheme 1. Inhibition and catalysis pathways: putative interactions between HOPNO derivatives and the met
form of Ty.
sapiens), and 2. human MNT-1 melanoma cells. The inter-
ring of aurones, as it interacts directly with the active site,
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Table 1. Protonation constants, inhibition constants (Ki) on purified TyH, IC50 on human MNT-1 melanoma
cells, and cytotoxicity values (IC50) on MNT1 cells for compounds 1a–c.
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2
3
4
5
6
7
8
Purified
TyH
MNT-1
lysate
MNT-1
whole cells
MNT-1
cytotoxicity
Protonation constants
Compound
pK4-OH
pK6-OH
pKN-OH
Ki (µM)
IC50 (µM)
16.6 ± 0.3
IC50 (µM)
85.3 ± 0.6
IC50 (µM)
>500
6.71
± 0.05
5.40
± 0.08
0.35
± 0.04
1a
1b
–
7.57
± 0.07
5.6
± 0.1
1.02
± 0.04
–
30 ± 2
34 ± 3
120 ± 10
119 ± 1
80 ± 20
>500
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7.2
± 0.1
8.3
± 0.1
5.8
± 0.1
1.2
± 0.2
1c
6.07
± 0.02a
128
± 2
HOPNO
–
–
–
–
1,300 ± 100
2,800 ± 800
150 ± 20
>200
350
± 70b
15,000 ±
2,000
KA
–
>80,000
aSee ref. 35. bSee ref. 4.
completely determined their general behavior toward
TyM1 and TyB3. Indeed, aurones I and II act as alternative
substrates, aurones III and IV as activators (for TyM1) or
weak inhibitors (for TyB3), and aurones V as mixed inhib-
itors (Figure 1). We also demonstrated the influence of
the poorly conserved second and third coordination
spheres of the dicopper active site as strong discriminat-
ing features. Indeed, the differences in terms of activity
among variously A-ring substituted aurones for a single
Ty type reached up to 100-fold. The same range of varia-
bility was observed upon measuring the activity of a given
aurone against Tys from different sources.
(see Supporting Information).39 We then comparatively
studied hybrid aurones and HOPNO on isolated TyH. For
this study, we have used a recombinant TyH produced in
insect cells that was very recently validated through the
evaluation of a number of known substrates and inhibi-
tors.4 For this enzyme we have previously determined the
Michaelis-Menten kinetic parameters for L-DOPA oxida-
tion, obtaining a Km value of 0.34 ± 0.03 mM and a kcat
Scheme 2. Synthesis of hybrid aurones 1.a
In addition, a significant improvement was provided by
the HOPNO moiety in terms of TyM1 inhibition activity
(e.g. IC50 = 1.5 µM for 1a versus >1,000 µM for the analo-
gous 6-hydroxyaurone V).17,32 To further support the po-
tential of this group, we defined the ionization state in-
volved in the binding. Protonation constant values for 1a–
c have thus been determined by spectrophotometric titra-
tions in water/DMSO (90/10, w/w, see Supporting Infor-
mation). Protonation constants corresponding to the
HOPNO moiety (log KN-OH, range 5.4–5.8) embedded on
aurones indicate that at physiological pH, HOPNO moie-
ty in 1a–c exists exclusively in an anionic form, thereby
facilitating the binding on dicopper center (Table 1). The-
se values are lower than that of free HOPNO (6.07),35 and
lower than the protonation constants for hydroxyl groups
at position 4’ of aurones I (R4 = H or OH, R6 = OH) and II
(R4 = R6 = OH), in the range 8.3–8.936 (corroborated by
classical values found for 4’-hydroxy groups of flavonoids
in the literature),37,38 indicating that these moieties are
fully protonated at physiological pH. These data rein-
forced the potential of HOPNO contribution vs. phenolic
derivatives, for interacting with the copper ions.
a(a) 6-methoxy-3-pyridinecarboxaldehyde, KOH, MeOH
or EtOH, H2O, 65–80°C, 2–6 h; (b) Ac2O, 140°C, 5–16 h; (c)
urea–H2O2 complex, TFAA, CH2Cl2, rt, 5–16 h; (d) HCl, H2O,
MeOH, 100°C, 16–40 h.
All these elements provided the rationale to design and
produce the reported HOPNO-embedded aurones. The
synthesis of aurones 1a–1c was performed according to
Scheme 2 from benzofuran-3(2H)-one derivatives 2a–2c
value of 38.1 ± 0.7 sec-1.4 The effect of aurones 1a–c on the
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L-DOPA by TyH was studied. First, we veri-
fied whether TyH inhibition was concentration-
dependent. The kinetic behavior of TyH during the oxida-
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oxidation of
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2
3
tion of L-DOPA was then investigated generating sub-
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strate velocities curves in the presence of various concen-
trations of inhibitors. The analysis of the inhibition curves
indicates a competitive mode of inhibition, consistent
with a direct link to the active site. The inhibition con-
stants were then determined by nonlinear regression and
curve fitting (see Supporting Information).
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Compounds 1b and 1c shared a similar inhibition potency
(Kic = 1.02 and 1.2 µM respectively, Table 1), whereas ana-
logue 1a was found 3.5 times more active. This possibly
indicates a detrimental effect of the presence of a hydrox-
yl group at position 4 of aurones. Interestingly, the evalu-
ation of the HOPNO moiety in the same conditions pro-
vided very different results. Indeed, considering TyH
inhibition, HOPNO was found as a very weak inhibitor,
with a Ki of 128 µM, approximately 350 times higher than
the value measured for compound 1a, whereas it was
previously found as a rather potent inhibitor of TyM134
and TyB340 (Ki = 1.8 µM and 7.7 µM respectively). These
data emphasize the crucial role of the whole aurone
backbone, and not only of the embedded HOPNO moie-
ty, in activities reported herein. Additionally, a correla-
tion could be observed between pKN-OH values and Ki
values against TyH, the inhibition potency of tested com-
pounds (including HOPNO) enhancing as deprotonation
of the N–OH moiety increases.
In order to understand the reactivity of aurones with TyH
and more precisely the role of the aurone backbone, theo-
retical studies were conducted. We focused our attention
on the most active hybrid aurone 1a and comparisons
were done with the interactions of the HOPNO moiety on
TyH. QM/MM dynamics followed by a NCI analysis were
performed. For the initial position, we made the hypothe-
sis that 1a and HOPNO would adopt the monodentate
binding mode (Scheme 1) obtained in our previous work
on TyB240 in which the oxygen atom of the nitrosyl group
of HOPNO is fixed only on one copper atom (see Sup-
porting Information).
Figure 2. Average position of HOPNO–TyH (A) and 1a–TyH
(B) complexes after QM/MM dynamics. NCI surfaces (in
green) represent molecular interactions and are generated
with an electron density gradient value of 0.35 au (atomic
units).
as Ile368, Arg308, Lys306 and His304, and allowing a
robust fit of 1a inside the active site pocket. These obser-
vations are in good agreement with the aforementioned,
experimentally measured Ki values.
The NCI method calculates an index based on the elec-
tron density and the electron density gradient.41 This
index presents singularities at low density when a weak
interaction appears between two fragments. The strength
of these interactions can also be calculated and isosurfac-
es can be plotted to visualize the domains of noncovalent
interactions. Thus the blue surfaces represent strong and
attractive interactions (as hydrogen bonds) whereas the
green ones indicate weak interactions (as van der Waals
interactions). Figure 2 shows an average position with
NCI surfaces from the TyH–HOPNO and TyH–1a com-
plexes extracted from their QM-MM dynamics trajecto-
ries. If HOPNO appeared as firmly linked to one of the
two copper centers in a monodentate mode, only few
interactions were observed with the surrounding residues
of the second coordination sphere. On the contrary, com-
pound 1a revealed an extended interaction pattern involv-
ing hot spot residues relatively far from the coppers, such
Finally, the ability of aurones 1a–c to suppress melanin
biosynthesis in a human integrated cellular model (hu-
man melanoma MNT-1 cells) was evaluated. Results from
cell lysates confirmed the potency of hybrid aurones to
prevent melanogenesis in a human complex cytoplasmic
environment, with an order of inhibition potency similar
to that obtained from the isolated TyH inhibition assay
(Table 1). Indeed, compound 1a reached a lower IC50 (IC50
= 16.6 µM), compared to compound 1b, and compound 1c,
which obtained IC50 values about two times higher (IC50
=
30 µM and 34 µM respectively). The contribution of the
aurone scaffold remained very significant, as HOPNO
alone had a very low activity on lysates (IC50 = 1,300 µM).
The “hybrids” revealed a limited capacity to hinder mel-
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recombinant tyrosinase: a testing bench for new drugs to
anogenesis in MNT-1 whole cells. The recorded IC50 values
1
2
3
4
5
6
7
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treat melanin-related disorders” (Progetto di Ricerca di
Ateneo CPDA110789/11) from the University of Padova to E.B.
ranged from 85.3 µM (for 1a) to 120 µM (for 1b), with a
concomitant low cytotoxicity of the compounds at such
concentrations, except for 1b (Table 1). These values were
thus found 3.5–5 times higher than those from lysates.
These results suggest a scarce ability of hybrid com-
pounds 1a–c to cross MNT-1 cell membranes that may
originate from the partial zwitterionic nature of the
HOPNO group, as this moiety is probably experiencing a
tautomeric equilibrium between the N-hydroxy-2-
pyridinone and 2-hydroxypyridine-N-oxide forms. Never-
theless, HOPNO-based compounds have already demon-
strated very good ability to act in cell-based and physio-
logical contexts.42,43 Finally it is worth noting that, at the
tested conditions, the reference KA was found almost
completely inactive both on lysate and whole cells.
ABBREVIATIONS
DOPA,
3,4-dihydroxyphenylalanine;
HOPNO,
2-
hydroxypyridine-N-oxide; KA, kojic acid; NCI, noncovalent
interaction; TFAA, trifluoroacetic anhydride.
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In conclusion, a series of HOPNO-embedded aurones
have been synthesized and evaluated to determine their
inhibition potency against TyH, and to elucidate the add-
ed value of the aurone backbone compared to the
HOPNO moiety alone. If the described aurones are excel-
lent inhibitors of TyH, a considerable gap exists in terms
of inhibition potency between these compounds and the
HOPNO moiety, indicating an undeniable beneficial ef-
fect of the aurone backbone, confirmed by theoretical
calculation. Finally, it is worth noting that, to the best of
our knowledge, compound 1a is the most active TyH in-
hibitor ever reported (Ki = 0.35 µM). Its lower efficiency in
suppressing melanogenesis in MNT-1 human whole cells
remains to be addressed. We thus shed light on the great
potential of these compounds for human-directed appli-
cations, and as tools for deciphering tyrosinase role in
pathologies such as melanoma.25
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Synthesis and characterizations of compounds, TyH inhibi-
tion assays, MNT-1 cellular assays, cytotoxicity assays, deter-
mination of protonation constants, computational methods
and QM/MM calculations (PDF).
AUTHOR INFORMATION
Corresponding Author
*E-mail: Romain.Haudecoeur@univ-grenoble-alpes.fr.
Author Contributions
§These authors contributed equally.
ACKNOWLEDGMENT
The authors are grateful to ANR (Agence Nationale pour la
Recherche) for financial support (Labex Arcane ANR-11-
LABX-0003-01 and Blanc program 2Cu-TargMelanin ANR-09-
BLAN-0028-01/02/03), ICMG (Institut de Chimie Moléculaire
de Grenoble) for computer, analysis and characterization
facilities, and the European COST Program in the framework
of which this work was carried out (COST action CM1003
WG 2). This work has been financed by the grant “Human
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