Development of an iron-selective antioxidant probe with protective effects on neuronal function

Article abstract of DOI:10.1371/journal.pone.0189043

Iron accumulation, oxidative stress and calcium signaling dysregulation are common pathognomonic signs of several neurodegenerative diseases, including Parkinsońs and Alzheimer's diseases, Friedreich ataxia and Huntington's disease. Given their therapeutic potential, the identification of multifunctional compounds that suppress these damaging features is highly desirable. Here, we report the synthesis and characterization of N-(1, 3-dihy-droxy-2-(hydroxymethyl) propan-2-yl)-2-(7-hydroxy-2-oxo-2H-chromen-4-yl) acetamide, named CT51, which exhibited potent free radical neutralizing activity both in vitroand in cells. CT51 bound Fe²⁺ with high selectivity and Fe³⁺ with somewhat lower affinity. Cyclic voltammetric analysis revealed irreversible binding of Fe³⁺ to CT51, an important finding since stopping Fe²⁺/Fe³⁺ cycling in cells should prevent hydroxyl radical production resulting from the Fenton-Haber-Weiss cycle. When added to human neuroblastoma cells, CT51 freely permeated the cell membrane and distributed to both mitochondria and cytoplasm. Intracellularly, CT51 bound iron reversibly and protected against lipid peroxidation. Treatment of primary hippocampal neurons with CT51 reduced the sustained calcium release induced by an agonist of ryanodine receptor-calcium channels. These protective properties of CT51 on cellular function highlight its possible therapeutic use in diseases with significant oxidative, iron and calcium dysregulation.

Full text of DOI:10.1371/journal.pone.0189043

RESEARCH ARTICLE
Development of an iron-selective antioxidant
probe with protective effects on neuronal
function
1
¤
2
2
3
Olimpo Garc ´ı a-Beltr a´ n , Natalia P. Mena , Pabla Aguirre , Germ a´ n Barriga-Gonz a´ lez ,
4
5
1,6
1,7
Antonio Gald a´ mez , Edgar Nagles , Tatiana Adasme , Cecilia Hidalgo *,
2
Marco T. N u´ ñez *
1
2
Biomedical Neuroscience Institute (BNI), Faculty of Medicine, Universidad de Chile, Santiago, Chile,
Department of Biology, Faculty of Sciences, Universidad de Chile, Santiago, Chile, 3 Universidad
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
Metropolitana de Ciencias de la Educaci o´ n, Facultad de Ciencias B a´ sicas, Departamento de Qu ´ı mica,
Santiago, Chile, 4 Department of Chemistry, Faculty of Sciences, University of Chile, Santiago, Chile,
5 Facultad de Ciencias Naturales y Matem a´ ticas, Universidad de Ibagu e´ , Ibagu e´ , Colombia, 6 Integrative
Center for Applied Biology and Chemistry (CIBQA), Universidad Bernardo O’Higgins, Santiago, Chile,
7
Department of Neuroscience, CEMC and ICBM, Faculty of Medicine, Universidad de Chile, Santiago, Chile
¤
*
Current address: Facultad de Ciencias Naturales y Matem a´ ticas, Universidad de Ibagu e´ , Ibagu e´ , Colombia
chidalgo@med.uchile.cl (CH); mnunez@uchile.cl (MTN)
OPEN ACCESS
Abstract
Citation: Garc ´ı a-Beltr a´ n O, Mena NP, Aguirre P,
Barriga-Gonz a´ lez G, Gald a´ mez A, Nagles E, et al.
Iron accumulation, oxidative stress and calcium signaling dysregulation are common patho-
gnomonic signs of several neurodegenerative diseases, including Parkinson´s and Alzhei-
mer’s diseases, Friedreich ataxia and Huntington’s disease. Given their therapeutic
potential, the identification of multifunctional compounds that suppress these damaging fea-
tures is highly desirable. Here, we report the synthesis and characterization of N-(1,3-dihy-
droxy-2-(hydroxymethyl)propan-2-yl)-2-(7-hydroxy-2-oxo-2H-chromen-4-yl)acetamide,
named CT51, which exhibited potent free radical neutralizing activity both in vitro and in
(2017) Development of an iron-selective
antioxidant probe with protective effects on
neuronal function. PLoS ONE 12(12): e0189043.
https://doi.org/10.1371/journal.pone.0189043
Editor: Yoshiaki Tsuji, North Carolina State
University, UNITED STATES
Received: June 27, 2017
2+
3+
cells. CT51 bound Fe with high selectivity and Fe with somewhat lower affinity. Cyclic
Accepted: November 19, 2017
Published: December 11, 2017
3+
voltammetric analysis revealed irreversible binding of Fe to CT51, an important finding
2+
3+
since stopping Fe /Fe cycling in cells should prevent hydroxyl radical production resulting
from the Fenton-Haber-Weiss cycle. When added to human neuroblastoma cells, CT51
freely permeated the cell membrane and distributed to both mitochondria and cytoplasm.
Intracellularly, CT51 bound iron reversibly and protected against lipid peroxidation. Treat-
ment of primary hippocampal neurons with CT51 reduced the sustained calcium release
induced by an agonist of ryanodine receptor-calcium channels. These protective properties
of CT51 on cellular function highlight its possible therapeutic use in diseases with significant
oxidative, iron and calcium dysregulation.
Copyright: © 2017 Garc ´ı a-Beltr a´ n et al. This is an
open access article distributed under the terms of
the Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: This study was supported by the
Comisi o´ n Nacional de Investigaci o´ n Cient ´ı fica y
Tecnol o´ gica (CONICYT), Chile (http://spl.conicyt.cl/
auth/), which provided a postdoctoral research
grant (N˚ 3149451) to Dr. Olimpo Garc ´ı a-Beltr a´ n;
Fondo Nacional de Desarrollo Cient ´ı fico y
Introduction
2+
Iron is the most abundant essential trace element present in the human body. Both Fe and
Tecnol o´ gica (FONDECYT), Chile (http://www.
conicyt.cl/fondecyt/), which provided research
3+
Fe play vital roles in many biological processes, making iron a key element for plant and
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A new iron-selective antioxidant probe that safeguards cellular function
grants to Cecilia Hidalgo (N˚ 1149545), Marco T.
animal metabolic processes [1–4]. Iron, which is widely distributed in nature, via its capacity
to accept and donate electrons engages in one-electron exchange reactions with the potential
of producing reactive oxygen species (ROS) within cells [5]. Iron plays a central role in the bio-
sphere via its involvement in oxygen transport, electron transfer and enzymatic reactions [6].
In particular, iron is essential for hemoglobin synthesis in vertebrate red cells and thus exerts a
crucial function in the storage and transport of oxygen to tissues [7]. Through the Fenton and
N u´ ñez (N˚ 1130068) and Tatiana Adasme (N˚
1
1140580); Instituto de Neurociencia Biom e´ dica,
Iniciativa Cient ´ı fica Milenio, Chile (www.
iniciativamilenio.cl/), which provided research
funds to Cecilia Hidalgo (N˚ BNI P-09-015F).
Financial support to Prof. Marco T N u´ ñez was
provided by the Program for Research, Faculty of
Sciences (PAIFAC) and by U-APOYA 2236, VID,
Universidad de Chile. The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
2+
3+
Haber-Weiss reactions, reduced iron (Fe ) reacts with H O to generate Fe plus the highly
2
2
reactive hydroxyl free radical [8]. Under physiological conditions the intracellular medium
provides ample reducing power in the forms of ascorbate and reduced glutathione [9, 10],
3+
2+
which reduce Fe to the Fe state. These events, all of which exhibit negative ΔG values, gen-
erate redox cycling reactions in cells that result in the production of hydroxyl radical and the
consumption of reduced glutathione [8].
Competing interests: The authors have declared
that no competing interests exist.
The brain, an organ with significant energy demands, exhibits higher iron requirements
than other organs in the body [11]. Iron is an essential element for neuronal function [12].
Iron deficiency affects the activity of enzymes involved in neurotransmitter synthesis that pos-
sess iron as a prosthetic group, such as monoamine oxidases A and B, tryptophan hydroxylase
and tyrosine hydroxylase [12–14]. In humans and rodents, early postnatal iron deficiency
results in learning and memory impairments, which persist following iron repletion [15, 16].
Conversely, brain iron content gradually increases during normal aging and in neurodegener-
ative conditions such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) [17]. The
resulting increase in brain iron content is likely to cause increased ROS generation that can
lead to irreversible damage of lipids, proteins, and DNA [8, 18]. It follows, therefore, that opti-
mal iron levels are required for normal brain function since iron deficiency and iron excess
cause neuronal dysfunction or death.
At the cellular level, iron accumulation enhances ROS production in neuroblastoma cells,
which activates redox-sensitive signaling pathways [19]. Several studies in a variety of cells
types indicate that ROS affect the function of proteins engaged in calcium signaling, including
the highly redox-sensitive ryanodine receptor (RyR) channels that mediate calcium release
from the endoplasmic reticulum [20]. Accordingly, increases in neuronal iron content
enhance calcium signal generation, which if uncontrolled leads to neuronal cell death [12].
Previous studies have indicated that iron acquisition by primary hippocampal neurons gener-
ates calcium signals mediated by oxidative stimulation of RyR calcium release channels; the
ensuing iron-induced calcium signals stimulate phosphorylation and nuclear translocation of
extracellular signal-regulated kinases 1/2 (ERK1/2) [21], and promote significant neuronal
mitochondrial fragmentation [22]. In agreement with these results, iron overload promotes
mitochondrial fragmentation in mouse HT-22 hippocampal neurons via calcineurin-sensitive
signals [23] and increases intracellular free calcium concentration, which leads to calcineurin
activation via calcium-dependent pathways engaging calmodulin and calpain [24]. Hence,
non-physiological iron-induced calcium signals, which through calcineurin activation pro-
mote mitochondrial fragmentation, are a likely factor in iron-induced neurotoxicity.
Due to the central role of iron in biological processes, the generation of fluorescent probes
for iron determination has gained increased importance. Methodologies for the detection of
iron include physical, chemical and biological techniques such as atomic absorption spectros-
copy, colorimetry, spectrophotometry, voltammetry, and flow injection [25–30]. Fluorophores
with different chemical structures, including dansyl-based probes, rhodamine-based probes,
benzimidazole-based probes, imidazole-based probes, naphthyl-based probes, anthracene-
appended amino acid probes, coumarin-based probes, and N-azacrown carbazole probes, all
have been used for the detection of iron [31].
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A new iron-selective antioxidant probe that safeguards cellular function
As detailed above, iron accumulation occurs frequently in neurodegenerative diseases such
as AD and PD. Of note, iron chelation reduces the progression of neurological symptoms asso-
ciated with these pathological conditions [17, 32]. However, multifunctional iron-chelating
compounds with preference for a specific organelle are scarce since the vast majority partitions
to the cytoplasm. Accordingly, a key objective of our present work was to develop a new multi-
functional compound with protective properties against iron-induced oxidative cellular dam-
age. We placed particular emphasis on generating an agent that afforded protection against
mitochondrial dysfunction, oxidative damage and sustained calcium signal generation, three
conditions that are deleterious to cell function.
We report here the synthesis, chemical and biological characterization of a new polyhy-
droxyl coumarin, N-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)-2-(7-hydroxy-2-oxo-2H-
2+
chromen-4-yl)acetamide (CT51), as a highly selective Fe chelator and a potent antioxidant
agent that preferentially partitions to mitochondria.
Results and discussion
I. Synthesis and properties of CT51
As illustrated in Fig 1, CT51 was synthesized from resorcinol, condensing it with citric acid
(
Pechmann) to obtain 2-(7-hydroxy-2-oxo-2H-chromen-4-yl) acetic acid (compound 1). To
synthesize CT51, compound 1 was esterified under Fischer-Speier conditions, giving ethyl 2-
7-hydroxy-2-oxo-2H-chromen-4-yl)acetate (compound 2), which was reacted with 2-amino-
-(hydroxymethyl)propane-1,3-diol (TRIS). The resulting CT51 product was characterized by
(
2
1
13
H-NMR spectroscopy (S1A Fig), C-NMR spectroscopy (S1B Fig), electrospray ionization
mass spectrum (S1C Fig) and X-ray crystallography (S1D Fig and S1 Table). The presence of
only 12 carbons instead of 13 atoms in the NMR spectrum stems from the fact that the signal
originated by the missing carbon is hidden by the DMSO signal. This carbon atom was
assigned to the methylene group, which acts as a bridge between the coumarin ring and the
amide group.
The CT51 molecule is water-soluble, most probably due to the three hydroxyl groups origi-
nating from the TRIS moiety, which also comprises a putative iron coordination site. A fourth
hydroxyl group is present in the phenol moiety attached to C7 in the benzopyrone ring,
which acts as a strong electron donor [33, 34], leading to polarity-dependent fluorescence
intensity that increases with increasing polarity of the medium with little spectral shift [35].
The absorption spectrum of CT51 displayed a maximum at 330 nm and a shoulder near
370 nm (S2A Fig). This result agrees with other absorption spectroscopic studies of
7-hydroxycoumarins, which exhibit absorption maxima in the range 320–400 nm, depending
on the nature of the substituent groups at C3 or C4. Regularly, these compounds have been
2 4
Fig 1. Synthetic route to produce CT51. Reagents and conditions: a) Citric acid, H SO , room temperature for 48 h; b)
H
2
SO , ethanol, reflux 6 h; c) 2-amine-2-hydroxymethyl-propane-1,3-diol, ethanol, reflux 48 h.
4
https://doi.org/10.1371/journal.pone.0189043.g001
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A new iron-selective antioxidant probe that safeguards cellular function
Ã
reported to have band groups of UV absorptions at 270–350 nm due to π–π transitions [36].
-1
-1
At 330 nm, CT51 displayed a molar extinction coefficient (ε) of 17,460 M cm . Fluorescence
studies revealed that CT51 had an emission band with a maximum at 460 nm (S2B Fig). The
emission quantum yield (F) of CT51 was F = 0.34, determined by using quinine sulfate as
standard; the lifetime (τ) was 5.95 ns, and the Stokes shift was 130 nm.
The cation selectivity of CT51 was detected by the quenching of fluorescence caused by the
2+
2
+
3+
2+
2+
2+
2+
2+
2+
2+
2+
2+
following cations: Hg , Fe , Fe , Co , Cu , Ca , Zn , Mn , Mg , Ni , Pb or Cd .
2+
3+
Only Fe , and to a lesser extent Fe , produced a significant quench of CT51 fluorescence
2+
(
Fig 2A). Increasing the concentration of Fe up to the 200 μM range partially reduced the
fluorescence of 5 μM CT51 (Fig 2B). The selectivity of CT51 for Fe and Fe makes it a possi-
ble candidate for iron chelation in living cells. A similar coumarin-TRIS compound (DAT-1)
2+ 3+
3+
was recently characterized as a Fe chelator [37]. At difference with CT51, DAT-1 did not
2+
demonstrate selectivity for Fe . We don’t know the causes for this discrepancy, but the use of
2
+
0
.2 M citrate, a mild Fe chelator, in the solutions used to test DAT-1 [37] possibly hindered
2
+
the interaction of Fe with DAT-1.
Cyclic voltammetry analysis represents a useful tool to study redox processes. Hence, to
confirm iron chelation, we performed voltammetric assays using a Screen Printed Carbon
2+
Electrode (SPCE) and a solution of 0.58 mM Fe in the absence (black line, pH 3.0) and in the
presence of 0.96 mM CT51 (red line, pH 6.8) (Fig 2C). These assays showed an oxidation cur-
2+
3+
rent for the reaction Fe $ Fe + e- at 0.18 V (reaction a), and a cathodic current for the
3+
2+
reaction Fe + e- $ Fe at 0.034 V (reaction b), with ΔV = 0.146 V, indicating a quasi-
2+
reversible process. When an optimal concentration of CT51 was added to the Fe -containing
2+
solution, a decrease in the Fe anodic current was noted. Also, a change in the potential at
3
+
0
.94 V emerged (reaction c), which may be due to CT51 oxidation in the complex with Fe .
3+
2+
Importantly, we did not observe a cathodic signal for the reaction Fe + e- $ Fe . Probably,
3+
2+
Fe generated from the oxidation of Fe forms a stable complex with CT51 and therefore it is
2+
2+
3+
3+
not reduced back to Fe , so that in the reaction Fe ! Fe + e-, Fe becomes irreversibly
2+
3+
oxidized. This is a relevant finding, since stopping Fe /Fe redox cycling in cells should pre-
vent the hydroxyl radical production resulting from the Fenton reaction [8]. Notwithstanding,
Fig 2. Cation selectivity of CT51. (A) Fluorescence emission spectra of CT51 (20 μM) recorded before and after addition of several different
2+
3+
2+
2+
2+
2+
2+
2+
2+
2+
2+
2+
chloride salts of Hg , Fe , Fe , Co , Cu , Ca , Zn , Mn , Mg , Ni , Pb or Cd ; all salt solutions (200 μM) were prepared in 20 mM HEPES
buffer, pH 7.4. (B) Emission spectra of CT51 (20 μM) were recorded (λex = 330 nm) upon addition of increasing concentrations (up to 200 μM) of Fe
(
NH
4
)
2
(SO4)2Á6H
2
O prepared in 20 mM HEPES buffer, pH 7.4. (C) Cyclic voltammetric analysis. The voltammograms represent the behavior of solutions
2+
containing 0.58 mM Fe without CT51 (black line), or in the presence of 0.96 mM CT51 (red line). The voltammograms were performed at a scan rate of
1
-1
00 mV s , using a potentiostat DropSens μStat 400.
https://doi.org/10.1371/journal.pone.0189043.g002
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A new iron-selective antioxidant probe that safeguards cellular function
3+
3+
undetermined cellular reactions could decompose the CT51-Fe complex making Fe avail-
able again.
The Stern-Volmer analysis yielded a linear representation (S3A Fig); based on this result we
2+
suggest that the metal-ligand (Fe -CT51) bond has 1:1 stoichiometry and that the quenching
is 100% static. This latter assumption was verified by determining the τ/2 value in the presence
and absence of the metal ion, as well as by determining the rate constant (kq) that displayed a
-
1
11
11
value (in s ) of 5.82 x 10 ± 0.4 x 10 ; kq values in this range are representative of the static
2+
quenching typical of chelators. The association constant of CT51-Fe was determined by the
Benesi–Hildebrand equation, which allows for the determination of the apparent association
2+
constant (Ka) value from the slope and intercept of the plot (S3B Fig). The Ka value for Fe
-1
binding to CT51 was 6.63 x 10 M . The constant slope of the Benesi–Hildebrand plot sup-
−4
+2
ports the above proposal of 1:1 stoichiometry for the complex CT51-Fe . The detection limit
2
+
−7
of this Fe fluorescent sensor was 1.1 x 10 M, which allows for the detection of the labile
−6
iron pool in the cell cytoplasm that presents values in the low 10 M range [38, 39].
Since antioxidant properties are a desired trait of neuroprotective agents, we evaluated next
through chemical and biological assays the putative antioxidant capacity of CT51. To explore
the antioxidant capacity of CT51, we determined the EPR spectra of 5,5-dimethyl-1-pyrroline-
N-oxide (DMPO) in the absence or presence of CT51. DMPO is a spin trap widely used to
study the levels of oxygen-based free radicals. Superoxide, hydroxyl and thiyl radicals form
adducts with DMPO that can be detected with EPR spectroscopy. Hydroxyl radicals were gen-
erated by the Fenton reaction in the presence of DMPO, or DMPO plus Trolox or CT51 as
hydroxyl radical competitors. As positive control for free radical quenching we used the anti-
oxidant agent 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), a gold stan-
dard for oxygen radical absorbance capacity [40]. The resulting EPR spectra indicated that
CT51 possesses considerable capacity for free radical quenching (Fig 3). The values of the per-
centage reduction of the signal, calculated using the double integral of the EPR spectra, were
DMPO: 0.0, Trolox: 84.8 and CT51: 99.8. In addition, voltammogram studies of CT51, deter-
mined at pH 6.8 or pH 3.0 (S4 Fig) showed that at pH 3.0, the oxidation current increased and
the potential shifted from 0.71 V, indicating involvement of the phenolic proton of CT51 dur-
ing the oxidation process. This proton has the capacity to neutralize ROS belonging to the free
radical category [41].
II. CT51 does not impair cell viability and protects cells from oxidative
stress
SH-SY5Y cells derived from a human neuroblastoma are used often as a robust in vitro model
for the evaluation of neurotoxicity induced by candidate drugs [42, 43]. To evaluate possible
neurotoxic effects of CT51, we incubated cultured SH-SY5Y human neuroblastoma cells with
CT51 and tested cell viability as a function of CT51 concentration. These assays revealed that
CT51 was not toxic in the 0.1–10 μM range although a change to a more rounded shape was
apparent (Fig 4A and 4B).
We tested next the antioxidant capacity of CT51 in SH-SY5Y human neuroblastoma cells.
The potential to modify lipid peroxidation was detected by the formation of 4-hydroxynonenal
(HNE)-protein adducts (Fig 5A). HNE is a highly reactive aldehyde resulting from lipid perox-
idation that quickly forms HNE-protein adducts [44]. Oxidative stress was induced by incuba-
tion of neuroblastoma cells with the mitochondrial complex I inhibitor rotenone [45].
Treatment with rotenone significantly increased the formation of HNE-protein adducts com-
pared with the control situation; this increase was completely abolished by 500 nM CT51,
while 250 nM CT51 afforded partial protection (Fig 5A). Complex I inhibition generates ROS
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A new iron-selective antioxidant probe that safeguards cellular function
Fig 3. CT51 is a free radical quencher. EPR spectra of the spin adduct DMPO recorded in the absence or
presence of CT51 (3 mM) or of the antioxidant agent Trolox (3 mM).
https://doi.org/10.1371/journal.pone.0189043.g003
that result in oxidative stress and decreased reduced glutathione (GSH) levels [46, 47]. Under
this consideration, we tested the putative capacity of CT51 to protect against GSH depletion
induced by complex I inhibition. Incubation of SH-SY5Y cells for 24-h with rotenone to
inhibit complex I caused a marked decrease in GSH content (Fig 5B). Co-treatment with
500 nM CT51 largely prevented this decrease, while co-treatment with 250 nM CT51 increased
GSH levels but this increase was not statistically significant (Fig 5B). We interpret these com-
bined results as a clear indication that CT51 exerts protective effects against the oxidative dam-
age induced by mitochondrial complex I inhibition. This CT51 property is common with that
of other substituted coumarins, which have the capacity to inhibit lipid peroxidation and to
scavenge hydroxyl radicals [48, 49].
The question arose as to whether redox-active copper could also mediate rotenone-induced
oxidative damage. We tested this possibility by studying the effect of the high affinity, mem-
brane-permeant copper chelator bathocuproine [50]. As opposed to CT51, bathocuproine
(250–500 nM range) did not prevent the effect of rotenone on the formation of HNE-protein
adducts (S5 Fig). This result is consistent with previous reports showing that under physiologi-
cal conditions cellular copper chaperones keep free Cu ions in the zeptomolar range [51, 52].
This feature of copper cellular homeostasis contrasts with iron, which is present in the cellular
reactive pool in the micromolar range [38, 39].
Overall, the above results indicate that CT51 has strong antioxidant capacity by which it
inhibits in vitro the formation of DMPO-hydroxyl radical spin adducts while it protects cells
from lipid peroxidation and the associated decrease in GSH content. Probably, CT51 protects
cells from oxidative damage by two independent mechanisms, namely 1) by sequestering
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A new iron-selective antioxidant probe that safeguards cellular function
Fig 4. CT51 does not affect cell viability. Cell viability was determined in SH-SY5Y cells incubated for 48 h with varying concentrations of CT51. (A)
Representative images of the cell culture after 48 h of treatment with CT51. (B) Cell viability determined by the MTT assay. Values represent Mean ± SEM
from three independent experiments. Significance of differences was evaluated by one-way ANOVA followed by Tukey’s post-hoc test; ns, not significant.
https://doi.org/10.1371/journal.pone.0189043.g004
redox-active iron and 2) by neutralization of free radicals, although in turn, iron chelation
should have incidence over hydroxyl radical production.
III. Cellular distribution and iron-sensing properties of CT51
Multifunctional iron chelator/antioxidant compounds with preference for a specific cell organ-
elle are scarce, since the vast majority of membrane-permeant chelating reagents partitions to
the cytoplasm. Under this consideration, we determined the cellular distribution of CT51 in
SH-SY5Y human neuroblastoma cells. Using epifluorescence microscopy, we first determined
its intracellular distribution taking advantage of its intrinsic fluorescence. Initial observations
showed a punctate pattern reminiscent of mitochondria, so its putative mitochondrial distri-
bution was evaluated next (Fig 6). As shown in a representative experiment, mitochondria
labeled with Mito-Tracker distributed in a perinuclear pattern. The fluorescence due to CT51
(green) was also present into perinuclear organelles that co-localized with Mito-Tracker (Fig 6,
Merge, yellow). CT51 also distributed in the cytoplasm, an indication that in cells CT51 exhib-
its both mitochondrial and cytoplasmic distribution. Arguably, distribution of CT51 to the
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A new iron-selective antioxidant probe that safeguards cellular function
Fig 5. CT51 acts as a cellular antioxidant agent. (A) Immunofluorescence assays were performed to determine 4-HNE-protein adducts under basal
culture conditions (Control) and in cultures treated for 24 h with the oxidative stress inductor rotenone (3 μM), in the absence or presence of 250 or 500 nM
CT51. The figure shows images from a representative experiment out of 3 independent determinations. Rotenone treatment increased cell
immunostaining of 4-HNE-protein adducts (center-left hand panel); this increase was prevented by the presence of 250 or 500 nM CT51 (center-right and
right hand panels). To appreciate these differences better, the immunostaining intensity was transformed into a thermal scale with the ImageJ program
(right-hand color bar), in which fluorescence intensity increases from blue to red. The right-hand graph shows the quantification of fluorescence intensity
determined in 70–90 cells per experimental condition. Values represent Mean ± SD; n = 3. Significance of differences was evaluated by one-way ANOVA
followed by Tukey post-hoc test. ***: p < 0.001 compared to the rotenone condition; ns: not significant. Scale bar: 30 μm. (B) GSH levels determined in
cells treated for 24 h with the oxidative stress inductor rotenone (3 μM), in the absence or presence of 250 or 500 nM CT51. Values represent Mean ± SE;
n = 3. Significance of differences was evaluated by one-way ANOVA followed by Tukey post-hoc test; ***: p < 0.001, *: p < 0.05 compared to the control
##
condition. : p < 0.01 compared to the rotenone condition; ns: not significant.
https://doi.org/10.1371/journal.pone.0189043.g005
mitochondrion should decrease the risk of oxidative damage in this organelle, considering that
mitochondria contain redox-active iron and continuously generate ROS, a propitious environ-
ment for hydroxyl radical generation and the ensuing hydroxyl radical-mediated damage [53].
To determine the iron sensing properties of CT51 we measured its intracellular fluores-
cence upon modification of the labile iron pool in SH-SY5Y cells (Fig 7). Basal CT51 fluores-
2+
cence (trace a) increased after addition of the Fe chelator 2,2’-bipyridyl (bipy) (trace b), an
indication of CT51 de-quenching after losing iron to bipy. Next we tested reversibility, another
important feature of a good chemical probe, by the subsequent addition of ferrous ammonium
sulfate (FAS) (trace c). Addition of FAS decreased CT51 fluorescence, probably through the
2+
re-formation of the CT51-Fe complex.
IV. Effects of CT51 on calcium signals elicited by RyR channel activation
We tested next the effect of CT51 on another cellular component sensitive to oxidative stress,
namely RyR calcium release channels; these channels reside in the endoplasmic reticulum and
their activity is highly redox sensitive [54]. Many reports indicate that sustained increments of
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A new iron-selective antioxidant probe that safeguards cellular function
Fig 6. CT51 fluorescence in neuroblastoma cells. SH-SY5Y cells were loaded for 20 min with the mitochondrial tracer Mito-Tracker (A, red) plus 5 μM
CT51 (B) (green). The merge image (C) shows CT51 distribution in both mitochondria (yellow) and cytoplasm (green).
https://doi.org/10.1371/journal.pone.0189043.g006
intracellular calcium levels in several neurodegenerative disorders prompt cell death [55–57].
Considering that the antioxidant agent N-acetylcysteine (NAC) prevents the high and sus-
tained intracellular calcium increases generated by the RyR agonist 4-chloro-m-cresol
(4-CMC) [58], we tested whether CT51, through its antioxidant properties, had similar effects
as NAC. Addition of 4-CMC induced a persistent 2.4-fold increase of the calcium signal,
recorded in the soma of rat primary hippocampal neurons (Fig 8). Pre-incubation for 24 h
with 1 μM CT51 markedly decreased the calcium signal evoked by 4-CMC to 1.6-fold the
Fig 7. CT51 is a reversible intracellular iron sensor. The graph shows the fluorescence recorded in
SH-SY5Y cells incubated with CT51 (20 min, 10 μM) and detected in a fluorescence plate reader (λ Ex. 340
nm; λ Em. 460 nm). (a) Basal CT51 fluorescence in cells cultured for 24 h with 20 μM Fe. (b) CT51
2+
fluorescence recorded after addition of the Fe chelator bipy (20 μM). (c) CT51 fluorescence recorded after
addition of 80 μM FAS. Values represent Mean ± SEM; n = 3.
https://doi.org/10.1371/journal.pone.0189043.g007
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A new iron-selective antioxidant probe that safeguards cellular function
2+
2+
Fig 8. CT51 inhibits the sustained Ca increase induced by the RyR-agonist 4-CMC. Ca signals
elicited by the RyR agonist 4-CMC in rat primary hippocampal neurons incubated for 24 h in the absence
(open symbols) or the presence of 1 μM CT51 (solid symbols) were determined as a function of time. The
graph shows the fluorescence values expressed as F/F , where F represents the basal fluorescence
0
0
recorded before 4-CMC addition. Values represent Mean ± SE; n = 3.
https://doi.org/10.1371/journal.pone.0189043.g008
initial value (57% inhibition), whereas pre-incubation with 10 μM CT51 abolished the calcium
increase elicited by 4-CMC (data not shown). These results highlight CT51 as a suitable agent
to reduce the neuronal calcium dysregulation caused by oxidative conditions.
Conclusions
In summary, we report here the synthesis, chemical and biological characterization of the poly-
2+
3+
hydroxycoumarin CT51. In vitro, CT51 exhibited 1) selective Fe and Fe binding and 2) sig-
nificant capacity to quench free radicals. In neuroblastoma cells, CT51 1) bound iron
reversibly, 2) distributed to both mitochondria and cytoplasm and 3) protected against oxida-
tive damage, while in primary hippocampal neurons, CT51 reduced the long-lasting increase
in intracellular calcium levels produced by an agonist of redox-sensitive RyR channels. Based
on these combined results, we propose that CT51 has the potential to function both as selective
intracellular iron sensor and as protective agent against oxidative cellular damage. In particu-
lar, CT51 may protect neuronal cells in vivo against the coupled generation of iron-induced
anomalous calcium signals, which presumably play a significant role in the progression of
iron-mediated neuronal cell death and iron-related neurodegeneration. Hence, the results pre-
sented in this study may for the basis for the development of a novel iron-chelation therapy
against iron-associated neurodegenerative disorders.
Materials and methods
Reagents
Salts (FeNH (SO ) Á12 H O, CaCl , CuCl , CoCl , MgCl , MnCl , ZnCl , PbCl , CdCl ,
4
4 2
2
2
2
2
2
2
2
2
2
HgCl , solvents (analytical grade and spectroscopic grade) and other chemical reagents (ana-
2
lytical grade and spectroscopic grade) such as 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO),
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A new iron-selective antioxidant probe that safeguards cellular function
4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) and (±)-6-Hydroxy-2,5,7,8-tet-
ramethylchromane-2-carboxylic acid (Trolox) were purchased from Sigma-Aldrich; Hydrogen
peroxide 30% was purchased from Merck. Ferrous ammonium sulfate hexahydrate was pur-
chased from Mallinckrodt Baker. Stock solutions (200 μM) of the metal ions were prepared in
Milli-Q water with the salts Fe(NH ) (SO ) Á6H O, FeNH (SO ) Á12H O, CaCl , CuCl ,
4
2
4 2
2
4
4 2
2
2
2
CoCl , MgCl , MnCl , ZnCl , PbCl , CdCl , or HgCl .
2
2
2
2
2
2
2
Spectroscopy
1 13
H-NMR and C-NMR spectra were recorded on a Bruker multidimensional 400 MHz spec-
trometer, using the solvent or the TMS signal as an internal standard. All chemical shifts are
reported in the standard δ notation of parts per million. EPR spectra were recorded in the X
band (9.80 GHz) using a Bruker ECS 106 spectrometer with a rectangular cavity and 50 KHz
field modulation. Absorption spectra were recorded at 25˚C using a Perkin Elmer model
Lambda 11 spectrometer. Voltammograms were obtained using a potentiostat DropSens μStat
400. To perform this analysis, 7.5 mL of ultrapure water, 0.5 mL Phosphate-buffered saline,
pH 3.0 or 6.8, and 0.25 mL of 1.54 mM CT51 were added to the electrochemical cell. After a
-1
resting time of 3 s, the voltammograms were performed at a scan rate of 100 mV s .
Synthesis of CT51
Resorcinol was condensed with citric acid (Pechmann) [59] obtaining the compound 2-
(7-hydroxy-2-oxo-2H-chromen-4-yl)acetate (compound 1). Subsequently, compound 1 was
esterified according to Fischer-Speier [60], generating 2-(7-hydroxy-2-oxo-2H-chromen-4-yl)
acetate (compound 2), which was reacted with 2-amino-2-(hydroxymethyl) propane-1, 3-diol
(TRIS) to obtain, by analogy to a published procedure (Jones et al, 1985), N-(1,3-dihydroxy-2-
(hydroxymethyl)propan-2-yl)-2-(7-hydroxy-2-oxo-2H-chromen-4-yl)acetamide (CT51). The
CT51 molecule, isolated by column chromatography using as mobile phase ethyl acetate:
MeOH 9:1 (0.9 g, 70%), was crystallized in methanol. The melting point of solid CT51, deter-
1
mined on a Reichert-Jung Galen III equipped with a thermocouple, was 145–147˚C. H-NMR
spectroscopy of CT51 (400 MHz, DMSO-d ) yielded: 7.65 (s, 1H), 7.62 (d, 1H, J = 8.6), 6.76
6
(
dd, 1H, J = 8.6, 2.0), 6.69 (d, 1H, J = 2,0), 6.17 (s, 1H), 4.7 (br, 3H), 3.7 (s, 2H), 3.55 (s, 6H).
1
3
C-NMR spectroscopy of CT51 (100 MHz, DMSO-d ) yielded: δ 60.8, 63.0, 102.7, 111.8,
6
113.5, 127.3, 152.1, 155.5, 160.8, 162.0, and 169.3. The m/z observed value was 346.0897, and
the calculated value for C H NO Na was 346.0903.
15
17
7
Deactivation of fluorescence
Deactivation of fluorescence was analyzed by the Stern-Volmer equation [59], [61], defined in
S1 Appendix.
Benesi-Hildebrand plot analysis
2+
Fluorescence intensity data for the CT51-Fe complex were plotted according to the Benesi-
Hildebrand equation [62], defined in S1 Appendix.
Calculation of fluorescence quantum yield
The fluorescence quantum yield (F) was determined using quinine sulfate dissolved in 0.5 M
H SO (Fr = 0.546) as standard [63], and was calculated using the equation defined in
S1 Appendix.
2
4
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A new iron-selective antioxidant probe that safeguards cellular function
Free radical generation and EPR spectroscopy
A competition assay was performed to determine and compare the antioxidant capacities of
the vitamin E analogue Trolox, a known free radical quencher [64], with those of compound
CT51 synthesized in this work. For this purpose, hydroxyl radical was first generated through
2+
the Fenton reaction using an aqueous Fe solution (1.0 mM aqueous ferrous ammonium sul-
fate hexahydrate), combined with an aqueous solution of hydrogen peroxide (1%) in HEPES
buffer, pH 7.3. A control was performed by trapping the hydroxyl radical with DMPO
(50 mM) and a total of 12 scans per spectra were acquired [65]; this control was assigned a
100% value for the hydroxyl radical signal. Next, the effects of Trolox or CT51 on the above
signal were determined. EPR spectra were recorded in the X band (9.80 GHz) using a Bruker
ECS 106 spectrometer with a rectangular cavity and 50 KHz field modulation. All spectra were
in the same scale and the most resolved line was selected to obtain the area under the curve
through the double integration of the selected signal.
SH-SY5Y cells and primary hippocampal neuronal cultures
Human neuroblastoma SH-SY5Y cells (CRL-2266, American Type Culture Collection, Rock-
ville, MD) were cultured at low humidity, at 37˚C and 5% CO , in Dulbecco’s modified essen-
2
tial medium (DMEM)-F12 medium supplemented with 10% fetal bovine serum, non-essential
amino acids, antibiotic-antimycotic mixture, and 20 mM HEPES buffer, pH 7.2. The medium
was replaced every 2 days. Cells were used at day 5 of culture. Primary rat hippocampal cul-
tures were prepared from eighteen-day old embryos obtained from pregnant Sprague-Dawley
rats as previously described [58]. Briefly, brains were removed and placed in a dish containing
Hank’s-glucose solution. Hippocampi were dissected and neurons were gently mechanically
dissociated in Hank’s-glucose solution, centrifuged and suspended in DMEM supplemented
with 10% horse serum. Dissociated hippocampal neurons were plated on polylysine-treated
glass coverslips. After 40 minutes, DMEM was replaced by Neurobasal medium supplemented
with B-27. Cells were incubated for 14 days in vitro (DIV) at 37˚C in a humidified 5% CO2
atmosphere prior to experimental manipulations. All experimental protocols used in this work
complied with the “Guiding Principles for Research Involving Animals and Human Beings” of
the American Physiological Society and were approved by the Bioethics Committee on Animal
Research, Faculty of Medicine, Universidad de Chile.
Mitochondrial localization of CT51
SH-SY5Y cells grown in 12-well plates for 5 days were incubated for 15 min with the mito-
chondrial probe Mito-Tracker Orange (0.5 μM) (Thermo-Fisher Scientific) and CT51
(25 μM). After washing the probes, the fluorescence intensity of Mito-tracker and CT51 was
detected using a Zeiss Axiovert epifluorescence inverted microscope. Co-staining between
CT51 and Mitotracker was determined with the ImageJ program.
Immunodetection of HNE-protein adducts
HNE immunodetection was performed as described [66]. Briefly, SH-SY5Y cells grown in
glass coverslips were treated for 24 h with the oxidative stress inductor rotenone (3 μM) in
+1
the absence or presence of 250 or 500 nM CT51, or of 250 or 500 nM of the Cu chelator bath-
ocuproine. Protein-HNE adducts were detected with mouse monoclonal anti-4-HNE antibody
(J-2, Abcam, dilution 1:500) as a primary antibody and Alexa-488-conjugated anti-mouse as
secondary antibody. Fluorescence intensity of HNE adducts was detected in a Zeiss Axiovert
epifluorescence inverted microscope.
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A new iron-selective antioxidant probe that safeguards cellular function
GSH determination
Total GSH content was quantified using the enzymatic recycling method adapted to micro-
plate readers as described [67, 68].
Intracellular iron sensing by CT51
SH-SY5Y cells grown in 12-well plates for 5 days were incubated with CT51 (20 min, 10 μM).
After washing of CT51, basal fluorescence (λ Ex. 340 nm; λ Em. 460 nm) was recorded in a
2+
BioTek Gen5 microplate fluorescence reader. To de-quench CT51 fluorescence, the Fe che-
lator bipy (20 μM) that competes with CT51 for iron, was added to the cells. CT51 fluorescence
was subsequently re-quenched by addition of 80 μM ferrous ammonium sulfate (FAS).
Changes in intracellular calcium levels
Primary hippocampal neurons were pre-incubated for 12 h with 1 μM CT51 and were then
loaded with the fluorescent calcium probe Fluo-4 AM [58]. Fluorescence changes as a function
of time were followed in a Zeiss 710 confocal fluorescence microscope.
Supporting information
1
13
S1 Fig. Spectral properties of CT51. (A) H-NMR spectrum. (B) C-NMR spectrum. (C)
ESI-MS analysis of CT51. (D) Crystal structure of CT51. Crystallographic data (excluding
structure factors) for the structural analysis have been deposited in the Cambridge Crystallo-
graphic Data Centre, CCDC 897001. These data can be obtained free of charge from the Cam-
bridge Crystallographic Data Centre; Postal Address: CCDC, 12 Union Road, Cambridge
CB21EZ, UK, Phone: (44) 01223 762910, Fax: (44) 01223 336033, e-mail: deposit@ccdc.cam.
ac.uk.
(PDF)
S2 Fig. Absorption and emission spectra of CT51. (A) Absorption spectra. (B) Emission
spectra.
(PDF)
S3 Fig. Stern-Volmer and Benesi-Hildebrand analysis. (A) Stern-Volmer relation for CT51
2+
in the presence of Fe . Deactivation of fluorescence was analyzed by the Stern-Volmer equa-
2+
tion. (B) Benesi-Hildebrand plot for CT51 with added Fe . Fluorescence intensity data for the
2+
CT51-Fe complex were plotted according to the Benesi-Hildebrand equation.
PDF)
(
S4 Fig. Voltammograms of CT51 at pH 6.8 and pH 3. Voltammograms of CT51 (0.96 mM)
determined at pH 6.8 (red line) or pH 3.0 (black line). To perform the cyclic voltammetry anal-
ysis, 7.5 mL of ultrapure water, 0.5 mL Phosphate-buffered saline, pH 3.0 or 6.8, and 0.25 mL
of 1.54 mM CT51 were added to the electrochemical cell. After a resting time of 3 s, the vol-
-1
tammograms were performed at a scan rate of 100 mV s .
PDF)
(
S5 Fig. Cu is not involved in rotenone-induced 4-HNE-protein adduct formation. (A)
Immunofluorescence of 4-HNE-protein adducts formed under basal culture conditions (Con-
trol) and in cultures treated for 24 h with the oxidative stress inductor rotenone (3 μM), in the
+1
absence or presence of 250 or 500 nM of the Cu chelator bathocuproine (BC) or CT51. The
figure shows images from representative experiments out of 2 independent determinations.
Rotenone treatment increased cell immunostaining of 4-HNE-protein adducts. This increase
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13 / 18

A new iron-selective antioxidant probe that safeguards cellular function
was partially prevented by 250 nM CT51 and completely prevented by 500 nM CT51 but not
by 250 or 500 nM bathocuproine. To appreciate these differences better, the immunostaining
intensity was transformed into a thermal scale with the ImageJ program (right-hand color
bar), in which fluorescence intensity increases from blue to red to white. (B) Quantification of
fluorescence intensity determined in 25–45 cells per experimental condition. Values represent
Mean ± SEM. Significance of differences was evaluated by one-way ANOVA followed by
Tukey’s post-hoc test; ns: not significant.
(PDF)
S1 Appendix. Stern-Volmer equation (A), Benesi-Hildebrand equation (B) and equation
used to calculate the fluorescence quantum yield (C).
(
PDF)
S1 Table. Crystal data and details of CT51 structure determination.
PDF)
(
Author Contributions
Conceptualization: Olimpo Garc ´ı a-Beltr a´ n, Cecilia Hidalgo, Marco T. N u´ ñez.
Data curation: Natalia P. Mena, Pabla Aguirre, Germ a´ n Barriga-Gonz a´ lez, Antonio Gald a´ -
mez, Edgar Nagles, Tatiana Adasme, Cecilia Hidalgo, Marco T. N u´ ñez.
Formal analysis: Cecilia Hidalgo, Marco T. N u´ ñez.
Funding acquisition: Cecilia Hidalgo, Marco T. N u´ ñez.
Investigation: Natalia P. Mena, Pabla Aguirre, Germ a´ n Barriga-Gonz a´ lez, Antonio Gald a´ mez,
Edgar Nagles, Cecilia Hidalgo, Marco T. N u´ ñez.
Methodology: Pabla Aguirre, Antonio Gald a´ mez, Edgar Nagles, Tatiana Adasme, Cecilia
Hidalgo, Marco T. N u´ ñez.
Project administration: Cecilia Hidalgo, Marco T. N u´ ñez.
Supervision: Olimpo Garc ´ı a-Beltr a´ n, Marco T. N u´ ñez.
Validation: Olimpo Garc ´ı a-Beltr a´ n, Cecilia Hidalgo, Marco T. N u´ ñez.
Visualization: Cecilia Hidalgo, Marco T. N u´ ñez.
Writing – original draft: Olimpo Garc ´ı a-Beltr a´ n, Cecilia Hidalgo, Marco T. N u´ ñez.
Writing – review & editing: Olimpo Garc ´ı a-Beltr a´ n, Cecilia Hidalgo, Marco T. N u´ ñez.
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