Coordination-triggered NO release from a dinitrosyl iron complex leads to anti-inflammatory activity

Article abstract of DOI:10.1039/c3sc53319k

Dinitrosyl iron complexes (DNICs) are widely considered NO storage and donor molecules in cells. However, what induces an NO release from iron in DNICs and the subsequent biological consequences remain elusive. The chemistry and biology of the NO release activity of DNICs are reported here. Changes in redox status or coordination number of discrete N-bound DNICs, respectively [Fe(TMEDA)(NO)₂] (1) and [Fe(TMEDA)(NO)₂I] (2), can generate a metastable {Fe(NO)₂}⁹ DNIC, [Fe(TMEDA)(NO) ₂]⁺, with ν_NO at 1769 and 1835 cm ^-1 and an EPR signal at g = 2.04, that spontaneously releases NO in solution. The NO release activity of 2 results in the up- and downregulation of heme oxygenase-1 (HO-1) and inducible nitric oxide synthase (iNOS), respectively, in murine RAW 264.7 macrophages. Furthermore, treatment with 2 leads to downregulation of pro-inflammatory cytokines, TNF-α and IL-6, and upregulation of the anti-inflammatory cytokine, IL-10. Taken together, these results demonstrate that the appropriate control of redox and coordination chemistry of DNICs could enable them to become anti-inflammatory agents, suggesting a potential new role for cellular DNICs. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c3sc53319k

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Coordination-triggered NO release from a
dinitrosyl iron complex leads to anti-inflammatory
activity†
Cite this: Chem. Sci., 2014, 5, 2374
a
b
b
a
*
*
Kelsey M. Skodje, Min-Young Kwon, Su Wol Chung and Eunsuk Kim
Dinitrosyl iron complexes (DNICs) are widely considered NO storage and donor molecules in cells.
However, what induces an NO release from iron in DNICs and the subsequent biological consequences
remain elusive. The chemistry and biology of the NO release activity of DNICs are reported here.
Changes in redox status or coordination number of discrete N-bound DNICs, respectively
[Fe(TMEDA)(NO)2] (1) and [Fe(TMEDA)(NO)2I] (2), can generate
a
metastable {Fe(NO)2}9 DNIC,
[Fe(TMEDA)(NO)2]+, with nNO at 1769 and 1835 cmꢀ1 and an EPR signal at g ¼ 2.04, that spontaneously
releases NO in solution. The NO release activity of 2 results in the up- and downregulation of heme
oxygenase-1 (HO-1) and inducible nitric oxide synthase (iNOS), respectively, in murine RAW 264.7
macrophages. Furthermore, treatment with 2 leads to downregulation of pro-inflammatory cytokines,
TNF-a and IL-6, and upregulation of the anti-inflammatory cytokine, IL-10. Taken together, these results
demonstrate that the appropriate control of redox and coordination chemistry of DNICs could enable
them to become anti-inflammatory agents, suggesting a potential new role for cellular DNICs.
Received 3rd December 2013
Accepted 19th February 2014
DOI: 10.1039/c3sc53319k
www.rsc.org/chemicalscience
of drugs useful in regulating diverse physiological functions
that are associated with nitric oxide.1 For example, adminis-
Introduction
tration of cysteine- or glutathione-bound DNICs resulted in
hypotensive effects in animal studies and clinical trials.8
Recognition of the biological importance of DNICs subse-
quently inspired chemists to study the chemical reactivity and
physical properties of synthetic DNICs. These investigations
have shown that the ligand environment and redox status of the
{Fe(NO)2} unit are important factors that may determine the
various forms of NO (i.e., NO, NO+, or NOꢀ) released from
DNICs.9 Despite a great deal of synthetic and reactivity studies,
the cellular response to these well characterized DNICs has not
been tested. In contrast, the water-soluble DNICs that are
known to exert vasodilation8,10 have not been isolated in a pure
form, which presents signicant challenges in correlating the
chemical properties of DNICs to their corresponding biological
effects. Because of this, we have chosen to use a synthetic DNIC
whose chemical reactivity can be studied both inside and
Dinitrosyl iron complexes (DNICs) are naturally occurring iron
species formed by reactions between nitric oxide (NO) and
cellular non-heme iron species such as iron–sulfur clusters and
labile iron pools (LIP).1 Although the biological fate of DNICs is
largely unknown, protein-bound DNICs have been observed
during the activation of several regulatory proteins2 while
glutathione-bound DNICs have been suggested to bind to
glutathione transferases (GSTs)3 and be transported via multi-
drug resistance-associated protein 1 (MRP1).4 The rst in vivo
DNICs were discovered in the 1960s,5 decades before the
importance of NO as a signaling molecule in humans was
noted. These DNICs were EPR active, cysteine bound and
formulated as [Fe(NO)2(SR)2]ꢀ, {Fe(NO)2}9 species in the Ene-
mark–Feltham notation,6 Chart 1. S-bound DNICs of this variety
are the most common, but both N- and O-bound ones have been
found in nature.7 Following the discovery of endogenous
DNICs, the biological effects of exogenously added DNICs have
been investigated, and suggest that DNICs could become a class
aDepartment of Chemistry, Brown University, 324 Brook Street, Providence, RI 02912,
USA. E-mail: eunsuk_kim@brown.edu; Fax: +1 401-863-9046; Tel: +1 401-863-3591
bDepartment of Biological Sciences, University of Ulsan 93 Daehak-ro, Nam-gu, Ulsan
680-749, Korea. E-mail: swchung@ulsan.ac.kr; Fax: +82 52-259-1694; Tel: +82 52-
259-2353
† Electronic supplementary information (ESI) available: Details of the chemical
and biological experimental procedures along with IR and EPR spectroscopic
data. See DOI: 10.1039/c3sc53319k
Chart 1 Dinitrosyl iron complexes.
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outside of a cell. Herein we report the chemistry and biology of is released, Scheme 1. The evolution of NO gas during the
discrete DNICs that can spontaneously release NO with external reaction was further conrmed by employing an NO trapping
stimuli such as changes in their redox status or coordination.
agent, iron(II) phthalocyanine (PcFe), Scheme 1. When the
headspace gas from the reaction mixture of 1 and FcPF6 was
transferred to a solution of PcFe, the formation of PcFe–NO was
at 1686 cmꢀ1
.
11
Results and discussion
observed in the IR spectrum with the known
n
NO
The assignment of nNO was further conrmed by employing
15NO labeled 1, from which the expected shi was observed at
We chose to investigate a pair of TMEDA based {Fe(NO)2}10 and
{Fe(NO)2}9 DNICs for this study (1 and 2, Chart 1), where
n
15NO
¼ 1654 cmꢀ1 (DnNO ¼ ꢀ32 cmꢀ1) for PcFe–15NO.
TMEDA
¼
N,N,N0,N0-tetramethylethylenediamine, because
We anticipated that removing the iodide ligand from [Fe(T-
these are known to be stable complexes in the absence of
external stimuli, which makes them an ideal system in search-
ing for the factors that trigger NO release. The {Fe(NO)2}10
DNIC, [Fe(TMEDA)(NO)2] (1), has two characteristic NO
stretching frequencies at 1629 and 1690 cmꢀ1 (blue dashed line,
Fig. 1A). We observed that oxidation of 1 by one equivalent of
ferrocenium hexauorophosphate (FcPF6) leads to complete
loss of its NO ligands by IR spectroscopy. During the reaction,
however, it was possible to detect a new intermediate species
formed by the reaction of 1 with FcPF6, with nNO at 1769 and
1835 cmꢀ1 (red solid line, Fig. 1A) before the complete loss of
NO. The reversible redox couple of 1 in the cyclic voltammo-
gram (E1/2 ¼ ꢀ527 mV vs. Fc/Fc+ in MeCN) suggests that a
cationic [Fe(TMEDA)(NO)2]+ species would be accessible by
oxidation. The EPR spectrum of the intermediate displays a
strong signal at g ¼ 2.04 (10 K, THF), supporting the formation
of an {Fe(NO)2}9 species. Thus, a metastable species generated
by chemical oxidation of 1 with FcPF6 is proposed to be a
cationic {Fe(NO)2}9 DNIC, [Fe(TMEDA)(NO)2]+, from which NO
MEDA)(NO)2I] (2), a ve-coordinate {Fe(NO)2}9 compound,
would generate the same metastable cationic {Fe(NO)2}9 species
that leads to NO release. To test this, we added one equivalent of
AgPF6 to an acetonitrile solution of 2. This resulted in precipi-
tation of AgI, loss of nNO at 1719 and 1777 cmꢀ1 from 2, and
trapping of NO(g) from the headspace by PcFe (Fig. 1B), as
expected.
The observed properties of [Fe(TMEDA)(NO)2I] (2) subse-
quently led us to investigate its biological activity towards living
cells as a pro-drug candidate for an NO releasing agent. We
reasoned that 2 is a stable and inactive molecule that can be
easily administered to the cell and then it can be converted to its
active form, a cationic four-coordinate {Fe(NO)2}9 species that
spontaneously releases NO, through ligand dissociation inside
the cell. Because the chloride concentration inside a cell is
signicantly lower than outside, it is likely that the labile iodide
ligand would dissociate upon entering the cell to form a meta-
stable cationic DNIC, leading to NO release. A very similar
activity is well known for the anticancer agent cisplatin,
[Pt(NH3)2Cl2], which becomes an effective DNA damaging agent
by losing its Clꢀ ligand once inside the cell.12 In the case of 2,
physiological effects due to NO release would be expected upon
treatment of cells with 2. Accordingly, we monitored changes in
expression levels of heme oxygenase-1 (HO-1) and inducible
nitric oxide synthase (iNOS) upon treatment with 2, because NO
is known to be a potent inducer of HO-1 in macrophages and
vascular cells13 and a negative feedback regulator of iNOS
activity and expression.14
The effects of [Fe(TMEDA)(NO)2I] (2) on the protein expres-
sion levels of HO-1 and iNOS were investigated in a murine
Fig. 1 (A) IR spectra (KBr) of [Fe(TMEDA)(NO)2] (1) (blue dashed line),
[Fe(TMEDA)(NO)2I] (2) (black dotted line), and the metastable inter-
mediate generated from 1/FcPF6 (red solid line) and (B) IR spectra (KBr)
of iron(II) phthalocyanine before (purple dashed line) and after (green Scheme 1 Oxidation of 1 and removal of iodide from 2 result in the
solid line) exposure to headspace from the 2/AgPF6 reaction.
release of NO, which is then captured from the headspace by PcFe.
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macrophage cell line. RAW 264.7 cells were treated with 2,
lipopolysaccharide (LPS), or a combination of 2 and LPS in a
time- and dose-dependent manner (Fig. 2), where LPS is a
bacterial virulence factor with known pro-inammatory prop-
erties.15 The treatment of 2 resulted in an increase of the HO-1
level in the absence or the presence of LPS (Fig. 2A and B). On
the contrary, the LPS-induced iNOS protein levels were mark-
edly decreased by treatment with 2 (Fig. 2A and B). These results
are reminiscent of what was observed from cells treated with a
well known NO donor, NONOate,13d,14a–c implying that 2 likely
acts as an NO donor inside the cell. In order to further support a
hypothesis that the changes in HO-1 and iNOS levels were due
Fig. 3 Levels of mRNA for HO-1 (A) and iNOS (B) in RAW 264.7 cells
assessed by quantitative real-time RT-PCR. Total RNA was extracted
after administration of 2 (500 mM), LPS (100 ng mLꢀ1), or a combination
of LPS + 2 at 12 hours. Expression levels of HO-1 and iNOS mRNA are
divided by expression of the control gene, b-actin, and shown as a fold
to the NO release from 2, the effects of a NO-free control induction of the vehicle (DMSO). The asterisk indicates significant
upregulation of HO-1 mRNA expression by the treatment of LPS with 2
vs. LPS alone (P < 0.05). The dagger indicates significant down-
regulation of iNOS mRNA expression by the treatment of LPS with 2 vs.
LPS alone (P < 0.05). For all of the real-time PCR experiments, values
compound, [Fe(TMEDA)Cl2]2 (3), on the HO-1 and iNOS
expression levels were also examined, in which no change was
observed (Fig. 2C).16
The effects of [Fe(TMEDA)(NO)2I] (2) on HO-1 and iNOS at
are presented as mean ꢁ SD, n ¼ 3.
the gene expression level were subsequently studied by quan-
titative real-time RT-PCR in RAW 264.7 cells. Upon treatment of
2 (500 mM) in the presence of LPS (100 ng mLꢀ1), mRNA
examine such effects, the levels of well known pro-inammatory
expression of HO-1 was increased by 280.2 ꢁ 7.6% while that of
cytokines such as TNF-a and IL-6, and an anti-inammatory
iNOS was decreased by 34.9 ꢁ 2.6% (Fig. 3). In the absence of
cytokine, IL-10, were assessed upon treatment of 2. RAW 264.7
cells were stimulated with 2 (500 mM), LPS (100 ng mLꢀ1), or a
combination of 2 and LPS for 6 hours, aer which supernatants
LPS, however, only the basal expression of HO-1 and iNOS
mRNA were observed upon treatment of 2 (Fig. 3), which
suggests that 2 may have post-transcriptional regulation effects
were harvested and measured for TNF-a, IL-6, and IL-10 by
on HO-1 expression in the absence of LPS.
ELISA. The results (Fig. 4) show that the LPS-induced pro-
inammatory cytokines (TNF-a and IL-6) were downregulated in
the presence of 2, whereas the production of anti-inammatory
It is well known that cytoprotective HO-1 plays a critical role
in defending the body against oxidant-induced injury during
inammatory processes,17 whereas the activation of iNOS can
cytokine, IL-10, was upregulated by 2. These data strongly
lead to organ destruction in inammatory diseases.18 Therefore,
suggest that 2 leads to an anti-inammatory response in
macrophages.
one can expect that [Fe(TMEDA)(NO)2I] (2) would have regula-
tory effects on inammation in macrophages. In order to
Nitric oxide (NO) is a signalling molecule involved in
cardiovascular function, neural signalling, immunodefence,
and apoptosis.19 Loss of endogenous NO has harmful effects
that include vasoconstriction, greater smooth cell proliferation,
and increased platelet and inammatory cell activity and
adherence at sites of endothelial damage.19,20 Because of the
great physiological importance of NO and the associated
medical needs, synthetic NO donors have emerged as a class of
Fig. 2 The time- and dose-dependent expression of HO-1 and iNOS
protein levels in RAW 264.7 cells assessed by Western blot analyses.
Dimethyl sulfoxide (DMSO) was used as a vehicle to solubilise the test
compounds. (A) Time course of HO-1 and iNOS levels after adminis-
tration of lipopolysaccharide (LPS) (100 ng mLꢀ1) or a combination of
LPS (100 ng mLꢀ1) and [Fe(TMEDA)(NO)2I] (2) (500 mM). Lane V shows Fig. 4 The effects of [Fe(TMEDA)(NO)2I] (2) on cytokine production
protein levels in a vehicle-treated cell control; (B) dose-dependent measured by ELISA. RAW 264.7 cells were stimulated with 2 (500 mM)
HO-1 and iNOS levels after exposure to varying concentrations of 2 (0, LPS (100 ng mLꢀ1), or a combination of LPS and 2 for 6 hours. Dimethyl
10, 100, and 500 mM) in the absence or the presence of LPS (100 ng sulfoxide (DMSO) was used as a vehicle for 2. Supernatants were then
mLꢀ1) at 12 hours and (C) dose-dependent HO-1 and iNOS levels after analyzed for TNF-a (A), IL-6 (B), and IL-10 (C). The asterisks indicate
exposure to varying concentrations of [Fe(TMEDA)Cl2]2 (3) (0, 10, 100, significant downregulation of TNF-a and IL-6 production by the
and 500 mM) in the absence or the presence of LPS (100 ng mLꢀ1) at 12 treatment of LPS with 2 vs. LPS alone (P < 0.05). The dagger indicates
hours. The housekeeping protein, glyceraldehyde 3-phosphate significant upregulation of IL-10 by the treatment of LPS with 2 vs. LPS
dehydrogenase (GAPDH), was used as a loading control. These alone (P < 0.05). Values are mean ꢁ SD, n ¼ 6. Results are represen-
experiments were performed three independent times.
tative for three independent experiments.
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drug useful in regulating these functions.20 The present study
shows that controlling the coordination properties of DNICs
can be a useful strategy to create a new class of therapeutic NO
donors. A neutral ve-coordinate {Fe(NO)2}9 DNIC possessing a
labile anionic ligand can be used as a pro-drug that becomes
active upon entering the cell by forming a cationic DNIC that
readily releases NO. One may expect that the NO releasing
ability of DNICs can be systematically tailored via coordination
chemistry. Possible ne-tuning of these compounds would
involve changing the electronics and bite angle of the chelates
to modulate the reaction rate, and using hydrophilic substitu-
ents on the periphery of the chelate to increase the solubility of
the compounds in an aqueous medium. Some such efforts are
currently being made in our laboratory.
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Conclusions
We have demonstrated previously unknown NO donor activity
of two synthetic DNICs, [Fe(TMEDA)(NO)2] (1) and [Fe(TME-
DA)(NO)2I] (2), as well as anti-inammatory activity of a DNIC
for the rst time. Chemical oxidation of 1 or removal of the
iodide ligand from 2 lead to the formation of a putative 4-
coordinate {Fe(NO)2}9 DNIC that spontaneously releases NO in
solution. When complex 2 is administered to cells, it becomes a
potent regulator for HO-1 and iNOS expression in macrophages
and causes anti-inammatory effects by downregulating pro-
inammatory cytokines (TNF-a and IL-6) and upregulating an
anti-inammatory cytokine (IL-10) in macrophages. Taken
together, these results show that DNICs offer promise in
developing a new class of anti-inammatory agents. Further-
more, the current nding regarding the biological effects of 2
sheds light on the possibility of a new physiological role for
naturally occurring DNICs. It is conceivable that cellular DNICs
may act as anti-inammatory agents as part of the defense
mechanism against oxidative–nitrosative stress.
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Acknowledgements
We thank Dr Ralph T. Weber and the Bruker BioSpin laboratory
for their help with the EPR instrumentation and experiments.
This research was supported by the US National Science Foun-
dation (CHE-1254733 to EK) and the Korean government
(2012M3A9C3048686 to SWC).
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