Anti-fibrotic activity of gold and platinum complexes – Au(I) compounds as a new class of anti-fibrotic agents

Article abstract of DOI:10.1016/j.jinorgbio.2020.111023

Molecular gold(I) and platinum(II) species were examined for the inhibition of liver fibrosis and the hepatitis C virus (HCV). Determination of inhibition efficiency was conducted via morphological analysis, cell viability, western blot analysis, and quantitative reverse transcription polymerase chain reaction (RT-PCR). Auranofin and Ph₃PAuCl demonstrated the greatest inhibition of liver fibrosis amongst the tested gold species in human hepatic stellate LX-2 cells. Western blot analysis indicated that auranofin and Ph₃PAuCl prevent signal transducer and activator of transcription 3 (STAT3) phosphorylation, which may be a key connection to fibrosis and inflammation. Auranofin and Ph₃PAuCl also reduced expression of HCV-nonstructural protein 3 (NS3) and HCV-NS5a proteins in a HCV subgenomic replicon system. These results demonstrate significant promise for the use of gold compounds in treating liver diseases such as HCV.

Full text of DOI:10.1016/j.jinorgbio.2020.111023

Journal of Inorganic Biochemistry 206 (2020) 111023
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Short communication
Anti-fibrotic activity of gold and platinum complexes – Au(I) compounds as
a new class of anti-fibrotic agents
a
a
b
Matthew Stratton , Akshaya Ramachandran , Ehxciquiel Jaeroume M. Camacho ,
c
a
b,⁎
Shivaputra Patil , Gulam Waris , Kyle A. Grice
a
Department of Microbiology and Immunology, Center for Cancer Cell Biology, Immunology and Infection, Chicago Medical School, Rosalind Franklin University of
Medicine and Science, North Chicago, IL 60064, USA
b
c
Department of Chemistry and Biochemistry, College of Science and Health, DePaul University, Chicago, IL 60614, USA
Pharmaceutical Sciences Department, College of Pharmacy, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Molecular gold(I) and platinum(II) species were examined for the inhibition of liver fibrosis and the hepatitis C
virus (HCV). Determination of inhibition efficiency was conducted via morphological analysis, cell viability,
western blot analysis, and quantitative reverse transcription polymerase chain reaction (RT-PCR). Auranofin and
Gold
Platinum
Hepatitis C virus
Liver fibrosis
Metal-based drugs
Anti-fibrotic compounds
Ph PAuCl demonstrated the greatest inhibition of liver fibrosis amongst the tested gold species in human hepatic
3
stellate LX-2 cells. Western blot analysis indicated that auranofin and Ph
3
PAuCl prevent signal transducer and
activator of transcription 3 (STAT3) phosphorylation, which may be a key connection to fibrosis and in-
flammation. Auranofin and Ph PAuCl also reduced expression of HCV-nonstructural protein 3 (NS3) and HCV-
3
NS5a proteins in a HCV subgenomic replicon system. These results demonstrate significant promise for the use of
gold compounds in treating liver diseases such as HCV.
1
. Introduction
Liver diseases such as those caused by the hepatitis B virus (HBV)
as progression of HCV-mediated liver disease. Human hepatic stellate
cells (HSCs) are a major fibrogenic cell type that contributes to liver
fibrosis [4,5]. Following hepatic injury, HSCs develop a myofibroblast-
like phenotype, and increase their expression of cytoskeletal proteins,
α-smooth muscle actin (α-SMA), and matrix proteins [5]. The activa-
tion of HSCs in response to liver injury is likely to occur by chemical
stimuli produced by neighboring cells such as hepatocytes, Kupffer
cells, circulating leukocytes, and/or sinusoidal endothelial cells [4,5].
These stimuli include reactive oxygen species, lipid peroxides, profi-
brogenic growth factors (transforming growth factor beta 1 (TGF-β1),
platelet-derived growth factor (PDGF)), and proinflammatory cytokines
(interleukin 1 beta (IL-1β), interleukin 18 (IL-18), interleukin 6 (IL-6),
and tumor necrosis factor alpha (TNF-α)). These cytokines and growth
factors are known to bind to their cell surface receptors to induce the
activation of cellular biomolecules such as protein kinases (c-Jun N-
terminal kinase (JNK), p38 mitogen-activated protein kinase
(p38MAPK) and extracellular signal-regulated kinase (ERK)) and tran-
scription factors such as activator protein 1 (AP-1), nuclear factor
kappa-light-chain-enhancer of activated B cells (NF-κB), and signal
transducer and activator of transcription 3 (STAT-3) in HSCs in re-
sponse to hepatic injury and inflammation [6,7]. These transcription
factors translocate into the nucleus of the activated cells to activate
and hepatitis C virus (HCV) represent a significant global health issue.
According to the World Health Organization (WHO), viral hepatitis
caused 1.34 million deaths in 2015 [1]. In particular, HCV merits more
attention for the development of new treatments. Persistent HCV in-
fection (when HCV infection lasts for > six months) in humans may
lead to chronic hepatitis in up to 60–80% of infected adults and can
progress to liver fibrosis, cirrhosis, and eventually hepatocellular car-
cinoma (HCC) [2]. Liver fibrosis is an early indicator of diseases on the
path to liver cirrhosis (a condition that results in over a million deaths
worldwide [3]) and HCC. Liver fibrosis is characterized by an accu-
mulation of extracellular matrix (ECM) molecules, such as collagens,
noncollagenous glycoproteins, elastin, and proteoglycan [4]. Interven-
tions that can prevent or reverse liver fibrosis may be valuable in
treating a wide array of liver diseases, including HCV infection.
During HCV-mediated disease pathogenesis, liver fibrosis is a re-
versible, wound-healing response to chronic liver injury. However, if
persistent, fibrosis can lead to cirrhosis and liver cancer [2]. Identifi-
cation of new anti-fibrotic compounds will provide opportunities for
novel therapeutic intervention for HCV-mediated liver fibrosis as well
⁎
Corresponding author.
E-mail address: kgrice1@depaul.edu (K.A. Grice).
https://doi.org/10.1016/j.jinorgbio.2020.111023
Received 25 November 2019; Received in revised form 6 February 2020; Accepted 9 February 2020
Available online 11 February 2020
0162-0134/ © 2020 Elsevier Inc. All rights reserved.

M. Stratton, et al.
Journal of Inorganic Biochemistry 206 (2020) 111023
undergoing chemotherapy treatments [22].
Another example of a metal-based drug is auranofin (Riduara), an
anti-rheumatism drug based on gold(I) (Fig. 1) [23]. Gold has been used
as a medicine for thousands of years [24,25], and gold salts were used
as early as the 1930s to treat rheumatoid arthritis (RA) [26]. However,
it was not until relatively recently that discrete molecular gold com-
pounds have been used as medicine. Auranofin (Fig. 1), was approved
by the FDA for oral use in RA in 1985 [27], and molecular gold(I)
species are also potentially viable treatments for a variety of other
disease states. This resurgence in attention to molecular gold com-
pounds has resulted in significant progress towards using gold(I) spe-
cies, including auranofin, as anti-cancer [28], anti-parasitic [29], and
anti-fungal drugs [30]. Auranofin has recently been granted orphan
drug status by the FDA when it was found as a lead for treating ame-
biasis in humans [31,32].
Fig. 1. Examples of Metal-based drugs approved for use by the FDA.
collagen, glycoproteins, and extracellular matrix genes/proteins which
are critical for the induction of liver fibrosis in HCV infected cells. Type
I collagen, a key player in fibrosis in HSCs is known to be activated by
the TGF-β1-Smad signaling pathway. In the acute phase of liver disease,
fibrogenesis is usually counter balanced by fibrolysis, the removal of
excess ECM by proteolytic enzymes such as matrix metalloproteases
Given the success of Pt(II) species such as cisplatin and gold(I)
species such as Auranofin, and the fact that they are already effective in
vivo, their activities towards liver fibrosis models were examined. In
addition, we sought to gain evidence into their modes of action in a
HCV cell culture model. The platinum(II) and gold(I) compounds shown
in Fig. 2 were selected for our initial screen of metal complexes. They
included known drugs (cisplatin, carboplatin, sodium aurothiomalate
(
MMPs) [5,8]. With repeated injury by HCV-infection, fibrogenesis
prevails over fibrolysis, resulting in excess ECM deposition, i.e. liver
fibrosis.
Even though there are currently effective Direct-Acting Antivirals
(
DAAs) for HCV, patients treated with DAAs still have a risk of HCC [9],
(
mycrosin), and auranofin) as well as a few related molecules that have
as well as a risk of reinfection with HCV. Resistance to current DAA
therapies has already been noted in some HCV strains, and the possi-
bility exists that other HCV genotypes may develop resistance to current
therapies as well [10–12]. We have previously examined chromeno-
pyridine compounds as potential inhibitors of liver fibrosis and anti-
HCV activity [13,14]. While organic molecules offer a variety of
structures for the development of drugs, they are limited to the octet-
based structures of carbon. In comparison, metal complexes offer a
wide variety of geometries, structures, and reactivity that can be
exploited for drug development [15]. As such, the development of
metal-based medical compounds and their application for treating
challenging disease states is an area of increasing importance [16].
One of the most successful anti-cancer drugs, cisplatin (Fig. 1), is
platinum-based, and since its serendipitous discovery in the 1960s by
Rosenberg [17], there have been many advances in platinum-based
drugs such as the medical implementation of carboplatin, oxaplatin,
and similar complexes [18]. Newer generations of anti-cancer com-
pounds are based on platinum as well [19], and several other platinum
compounds have had promising results in research studies [20]. Cis-
platin is the first line of treatment in many forms of cancer such as
testicular cancer and non-small cell lung cancer [21]. There is also some
preliminary evidence that HCV replication is inhibited in patients
different hydrophilicities (K
2
PtCl
4
,
Ph PAuCl, and (PTA)AuCl;
3
PTA = 1,3,5-Triaza-7-phosphaadamantane). In this study, we used
human HSCs (LX-2 cells) to screen the compounds to determine their
anti-fibrotic activities and cells expressing a subgenomic replicon of
HCV to screen their effects against HCV. The results of our studies are
presented below in this communication.
2
. Experimental methods
2.1. General consideration
Platinum and gold compounds were purchased from commercial
suppliers (Sigma-Aldrich, Milwaukee, WI) and used as received, except
for Ph PAuCl [33] and (PTA)AuCl [34], which were synthesized similar
3
to literature reports. The synthesis of the compounds are reported here
1
as well for clarity, and spectroscopic data matched literature values. H
3
1
and P NMR spectroscopy was performed with a 300 MHz Bruker
spectrometer at 298 K. All manipulations were carried out on the
benchtop under standard conditions unless otherwise noted.
Fig. 2. Gold(I) and platinum(II) complexes examined in this study.
2

M. Stratton, et al.
Journal of Inorganic Biochemistry 206 (2020) 111023
was removed via rotary evaporation and the remaining solid was rinsed
with pentane and dried. Yield: 85.4 mg, 0.290 mmol, 56.9%.
2.3. Synthesis of (PTA)AuCl
A sample of PTA (107 mg, 0.681 mmol) was combined with of
SMe AuCl (187 mg, 0.633 mmol) in 10 mL of dichloromethane. The
2
material was sonicated for 30 min, then filtered and rinsed with pen-
tane, yielding 210 mg (0.539 mmol, 85% yield).
2.4. Density functional theory calculations
Density Functional Theory (DFT) calculations were performed using
the Gaussian 09 or the Gaussian 16 software package [35,36]. Input files
and output files were processed using GaussView [37]. All calculations
were performed with the B3LYP hybrid functional [38], which includes
Becke's exchange-correlation functional [39], the Lee-Yang-Parr 1988
correlation functional [40], and the Vosko, Wilk, and Nusair Local Spin
Density correlation [41]. Calculations for all compounds were per-
formed using the 6–311 + G(d,p) basis set for non-metal atoms [42],
and the LANL2DZ [43,44] basis set for gold. Solvation effects were
included with a Polarizable Continuum Model (PCM) using the integral
equation formalism variant (IEFPCM) and the default values assigned
for water [45]. Structures were optimized, followed by frequency cal-
culations to determine that they did not possess any imaginary fre-
quencies and were at a minimum on the potential energy surface. A
sample Gaussian input file and the final output geometries and energies
are given in the supplemental information.
2.5. Cell cultures
The HSC line LX-2 and human hepatoma cell line Huh7 were ob-
tained from S. Friedman (Mount Sinai Hospital, NY) and A. Siddiqui
(
UCSD, CA), respectively. K2040 is the Huh7 cell line, which contains
an adaptive mutation of the lysine residue at codon 2040 of the HCV
polyprotein. Cells were maintained as monolayer cultures in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% fetal bovine
serum, 100 units of penicillin/mL and 100 μg streptomycin sulfate/mL.
K2040 cells stably expressing HCV subgenomic replicon (a gift from M.
Gale, University of Washington, Seattle, WA) were cultured in DMEM
supplemented with 10% fetal bovine serum and 200 μg of Geneticin
(
G418) per mL. The cells were cultured at 37 °C in a humidified at-
mosphere containing 5% CO
2
. For drug treatments, cells were serum
starved in Opti-MEM for 1 h followed by drug treatments for 6 h.
2.6. Antibodies and reagents
All the antibodies were purchased commercially and used according
to the manufacturer's protocols: mouse monoclonal anti-α-smooth
muscle Actin (Abcam, Cambridge, UK); rabbit polyclonal anti-COL1A1,
rabbit polyclonal anti-Phospho-Smad2 (Ser245/250/255), rabbit
monoclonal anti-Smad2 (86F7), mouse monoclonal anti-Phospho-Stat3
(
Tyr705) (M9C6), and rabbit polyclonal anti-Stat3 (Cell Signaling
Fig. 3. Effect of gold (A) and platinum (B) compounds on LX-2 cell morphology.
LX-2 cells were cultured on plastic plates in DMEM containing 10% FBS. LX-2
cells were serum starved for 1 h followed by treatments with the compounds at
different concentrations for 6 h. The pictures were taken under a phase contrast
light microscope. Untreated cells show a fibrous spindle-like shape, whereas the
healthy LX-2 morphology is more round, which can be seen in cells treated with
Auranofin.
Technology, Danvers, MA); mouse monoclonal anti-β-Tubulin
(
MilliporeSigma, Burlington, MA); mouse monoclonal anti-β-Actin
(Thermo Fisher Scientific, Waltham, MA); mouse monoclonal anti-
Hepatitis C NS3 and anti-Hepatitis C NS5a (Virogen, Watertown, MA).
2
.7. Cell viability assay
2
.2. Synthesis of Ph
3
PAuCl
(80 mg, 0.303 mmol) was combined with
LX-2, Huh7, and K2040 cells were grown in opaque 96-well plates.
The cells were then treated with the respective drug or carrier control
for 6 h, as indicated. After the drug treatments, cell viability was as-
sessed using the CellTiter-Glo Luminescent Cell Viability Assay kit
(Promega, Madison, WI) as per the manufacturer's protocol.
A sample of PPh
3
SMe AuCl (89 mg, 0.303 mmol) in a scintillation vial. Dichloromethane
2
(
10 mL) was added and the mixture was stirred for 15 min. The solvent
3

M. Stratton, et al.
Journal of Inorganic Biochemistry 206 (2020) 111023
Fig. 4. Hepatic stellate cell viability assay. Active LX-2 cells were treated with the gold (A) and platinum (B) compounds at indicated concentrations for 6 h and
subjected to the cell viability assay. All results are presented as means ± SD of data from three independent experiments, and statistical analysis was done by using
two-tailed, unpaired t-tests. *, P < .05; **, P < .01; ***, P < .001.
2.8. Western blot analysis
Tubulin was analyzed by Odyssey Infrared Imaging System software.
Cells were harvested and the cell pellets were suspended in 1:3 ratio
2.9. Quantitative real-time RT-PCR
of pellet and radioimmunoprecipitation assay (RIPA) lysis buffer
(
150 mM NaCl, 1 mM sodium orthovanadate, 1 mM sodium fluoride,
LX-2 cells were lysed in TRIzol Reagent (Thermo Fisher Scientific,
Waltham, MA) followed by RNA isolation according to the manufac-
turer's user guide. The RNA samples were then treated with RQ1 RNase-
Free DNase (Thermo Fisher Scientific, Waltham, MA) according to the
manufacturer's directions to remove any contaminating DNA. Next,
cDNA was synthesized by the addition of Oligo dT primers and dNTPs
(Thermo Fisher Scientific, Waltham, MA) followed by a 5 min incuba-
tion at 65 °C and subsequent snap chill on ice. Lastly, MultiScribe
Reverse Transcriptase, RNase inhibitor, 0.1 M DTT, 25 mM MgCl2, and
10× RT Buffer (Thermo Fisher Scientific, Waltham, MA) were added to
each sample, and the samples were then incubated in a MJ Mini
Gradient Thermal Cycler (Bio-Rad Laboratories, Hercules, CA) at 25 °C
for 10 min, 42 °C for 2 h, 70 °C for 15 min, and lastly held at 4 °C.
Gene specific primers were used to selectively amplify the target
genes using the ABI PRISM 7500 real-time PCR machine (Thermo
Fisher Scientific, Waltham, MA). Amplification reactions for LX-2
samples were then performed in a 25 μL mixture using the Power SYBR
Green PCR Master Mixture (Thermo Fisher Scientific, Waltham, MA),
the prepared cDNA, and gene specific primers (Thermo Fisher
Scientific, Waltham, MA). All reactions were performed in triplicates in
a 96-well plate. The expression levels of the target genes were
5
1
0 mM Tris, pH 7.5, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS,
mM ethylenediaminetetraacetic acid (EDTA), and 10 μL/mL protease
inhibitor cocktail) for 30 min on ice. The suspended cells were micro-
centrifuged at 12,000 rpm for 15 min. The cell supernatants were
harvested and the concentration of total protein in cell supernatants
was estimated by bicinchoninic acid (BCA) protein assay reagent
(
Pierce, Rockford, IL). Equal amounts of proteins were subjected to
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and then transferred to a nitrocellulose blotting membrane (Amersham,
GE Healthcare) in transfer buffer (25 mM Tris, 192 mM glycine, and
2
0% methanol). The membranes were incubated in blocking buffer (5%
Non-Fat Dry Milk in TBS with 0.1% Tween) for 1 h at room temperature
(
RT), probed with the indicated specific primary antibodies, washed
three times for 5 min with blocking buffer without milk, incubated with
species-specific secondary antibodies for 1 h at RT. After an additional
washing step, the immunoblots were visualized using either the
Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE) or
by enhanced chemiluminescence reaction (NEN Life Sciences Products,
Boston, MA) depending on which secondary antibody was used. The
percentage of reduction in protein band intensity normalized to β-
4

M. Stratton, et al.
Journal of Inorganic Biochemistry 206 (2020) 111023
independent experiments (n ≥ 3) to ensure reproducibility. The p-value
was calculated using two-tailed, unparied t-tests. In all tests, p < .05
was considered statistically significant.
3
. Results
3.1. Gold and platinum compounds inhibit hepatic stellate cell activation
All seven gold and platinum compounds – auranofin, sodium aur-
othiomalate, Ph
3
PAuCl, (PTA)AuCl, K
2
PtCl , cisplatin, and carboplatin
4
–
were examined for inhibition of fibrotic activity in LX-2 cells. In a
healthy liver, HSCs are in a quiescent state (observed with a round
shape), whereas they change into an activated state when the liver is
damaged [5]. LX-2 cells are known to take on their activated state when
cultured on uncoated, polystyrene culture dishes, mimicking their
morphology during liver fibrosis. For this reason, observing LX-2 cell
morphology in the presence of an experimental compound can be used
as a preliminary screen for anti-fibrotic activity, as an anti-fibrotic drug
should revert the activated LX-2 cells back to their quiescent state. LX-2
cells were serum starved for 1 h and subsequently treated with gold
compounds (Fig. 3A) or platinum compounds (Fig. 3B) at 2 μM and
5
μM for 6 h and observed for morphological changes via microscopy.
Control cells, untreated or vehicle (dimethyl sulfoxide, DMSO) treated
cells, were also observed. LX-2 cells treated with 2 μM and 5 μM of
auranofin and Ph PAuCl appeared more round, suggesting that they
3
may have reverted back to a quiescent state (Fig. 3A). However, we did
not observe any morphological change in LX-2 cells when treated with
the other gold or platinum compounds (Fig. 3A, B).
3.2. Gold and platinum compounds do not affect cellular viability
To verify that the observed morphological change in LX-2 cells
treated with auranofin and Ph PAuCl was indeed due to anti-fibrotic
3
activity and not a side effect of drug toxicity, cell viability in the pre-
sence of all seven gold (Fig. 4A) and platinum (Fig. 4B) compounds was
examined. The results showed that none of the compounds were sig-
nificantly toxic at 2 μM or 5 μM, confirming that the morphological
change was likely due to the HSCs reverting to a quiescent state (Fig. 4).
3.3. Reduction in fibrosis-associated markers
To determine if the observed morphological changes of the treated
cells correlated with other markers of fibrosis, western blotting was
performed (Fig. 5). HSCs were treated with gold and platinum com-
pounds as indicated above, and cellular lysates from control and drug-
treated LX-2 cells were analyzed by western blotting for cellular pro-
teins correlated with liver fibrosis (collagen-I, α-SMA) as well as pro-
teins known to play key roles in type I collagen expression (STAT3 [46],
SMAD2 [47]). β-tubulin was used as a loading control.
The first protein examined via immunoblot was collagen type I.
Collagen is one of the proteins most characteristically seen in liver fi-
brosis, and collagen type I has been reported to be associated with the
liver fibrosis stage of chronic HCV infection [48]. It has also been
shown previously that the end product of fibrosis is abnormal synthesis
and accumulation of type I collagen in the extracellular matrix, which is
produced by activated HSCs in the damaged liver [49]. As compared to
DMSO-treated HSCs, auranofin treatment of 2 μM and 5 μM resulted in
Fig. 5. Gold and platinum compounds reduce expression of fibrosis-associated
proteins in LX-2 cells. A. Effect of gold compounds on type-I collagen expres-
sion. B. Quantification of type-I collagen inhibition. Mean ± standard devia-
tion (n = 4). C. Effect of gold compounds on fibrosis-associated proteins (P-
STAT3, P-SMAD2, and αSMA). D. Effect of platinum compounds on fibrosis-
associated proteins (COL1A1, P-STAT3, P-SMAD2, and αSMA). Cellular lysates
from different drug treatment conditions were subjected to western blotting
using respective antibodies. β-tubulin was used as a protein loading control.
5
0% and 40% decreases in type I collagen expression, respectively
(
Fig. 5A). Ph PAuCl also reduced type I collagen expression as com-
3
pared to DMSO-treated HSCs, however the reduction was less dramatic
with only a 10% and 20% reduction at 2 μM and 5 μM, respectively
normalized to 18S gene expression.
(
Fig. 5A). Statistical analysis of four independent replicates indicated
that this reduction in type I collagen expression was statistically sig-
nificant in the case of auranofin at both 2 μM and 5 μM, but not so in
2.10. Statistical analysis
the case of Ph PAuCl (Fig. 5B). Interestingly, despite not previously
3
The results are expressed as means
±
SD of at least three
seeing a morphological change, HSCs treated with 5 μM Cisplatin and
5

M. Stratton, et al.
Journal of Inorganic Biochemistry 206 (2020) 111023
Fig. 6. Gold and platinum compounds affect fibrosis-linked mRNA. Total cellular RNA was extracted from LX-2 cells treated with the above compounds and subjected
to quantitative RT-PCR using Col1A1- αSMA-specific primers and SYBR Green. Relative mRNA expression was compared by setting the DMSO-treated control to an
arbitrary value of 1. All results are presented as means ± SD of data and are representative of three independent experiments. Statistical analysis was done by using
two-tailed, unpaired t-tests. *, P < .05; **, P < .01; ***, P < .001 compared with DMSO-treated cells.
Fig. 7. Hepatocyte cell viability assay. Huh7-K2040 cells were treated with the gold (A) and platinum (B) compounds at indicated concentrations for 6 h and
subjected to cell viability assay. All results are presented as means ± SD of data from three independent experiments, and statistical analysis was done by using two-
tailed, unpaired t-tests. *, P < .05; **, P < .01; ***, P < .001.
Carboplatin also saw modest reductions in type I collagen expression as
well (Fig. 5D).
two proteins which play key roles in its expression: STAT3 and SMAD2.
At both 2 μM and 5 μM concentrations, auranofin showed a complete
Given the observed reduction in type I collagen, we next examined
abrogation of STAT3 phosphorylation (Fig. 5C, lanes 3 &4).
6

M. Stratton, et al.
Journal of Inorganic Biochemistry 206 (2020) 111023
protein expression. Cisplatin at 5 μM inhibited expression of collagen-I,
phospho-STAT3, phospho-SMAD2, and α-SMA by approximately 30%
(
Fig. 5D, lane 6). Likewise, carboplatin at 5 μM inhibited expression of
collagen-I by approximately 40%, phospho-STAT3 by 60%, phospho-
SMAD2 by 40%, and α-SMA by 30% (Fig. 5D, lane 8). These results
suggest that a 2 μM concentration of auranofin and a 5 μM con-
centration of Ph PAuCl, cisplatin, or carboplatin were sufficient to
3
significantly reduce the expression of proteins correlated with fibrosis.
3.4. Inhibition of fibrosis-associated transcription
To determine if the decrease in fibrosis-linked protein expression in
cells treated with auranofin, Ph PAuCl, cisplatin, and carboplatin was
3
due to a decrease in mRNA expression, total RNA was extracted from
untreated, DMSO-treated, and compound-treated LX-2 cells followed by
real-time quantitative RT-PCR. Compared to DMSO-treated cells, we
observed a significant decrease in the mRNA levels of both α-SMA and
type I collagen genes in LX-2 cells treated with auranofin at both 2 μM
and 5 μM (Fig. 6A), as well as those treated with Ph
cisplatin (Fig. 6B), or carboplatin (Fig. 6B) at 5 μM.
3
PAuCl (Fig. 6A),
LX-2 cells which were treated with auranofin at 2 μM and 5 μM
concentrations showed the most dramatic decrease in mRNA expression
of fibrosis-associated genes with type I collagen mRNA levels de-
creasing 80% and 98%, respectively and α-SMA mRNA levels de-
creasing 80% and 96%, respectively. While Ph PAuCl at 2 μM resulted
3
in no noticeable decrease in collagen-I mRNA expression and a < 20%
decrease in α-SMA expression, at 5 μM the same compound decreased
collagen-I and α-SMA expression by nearly 70% and 50%, respectively
(
Fig. 6A).
In LX-2 cells treated with cisplatin at 2 μM and 5 μM concentrations,
type I collagen mRNA expression decreased 10% and 50%, respectively.
Within the same treatment group, α-SMA mRNA expression decreased
2
5% and 40%, respectively. Likewise, in LX-2 cells treated with car-
boplatin at 2 μM and 5 μM concentrations, type I collagen mRNA ex-
pression decreased 30% and 60%, respectively whereas α-SMA mRNA
expression decreased 20% and 50% respectively (Fig. 6B).
3.5. Gold and platinum compounds inhibit HCV protein expression
All seven gold and platinum compounds were also examined for
inhibition of HCV nonstructural (NS) protein expression in Huh7-K2040
cells, a hepatocyte cell line containing the subgenomic HCV RNA re-
plicon [50]. First, Huh7-K2040 cell viability in the presence of all seven
gold (Fig. 7A) and platinum (Fig. 7B) compounds was assessed to en-
sure that all compounds were non-cytotoxic to human hepatocytes prior
to further analysis. The results indicated that, just as in LX-2 cells, the
compounds were not significantly toxic to Huh7-K2040 cells at 2 μM or
Fig. 8. Gold and platinum compounds reduce expression of HCV non-structural
proteins Effect of gold (A and B) and platinum (C) drugs on HCV non-structural
proteins (NS3 and NS5a) expression and fibrosis-associated STAT3 phosphor-
ylation (P-STAT3) by western blotting. The bands represent the expression of
HCV NS3, NS5a, P-STAT3, STAT3, and protein loading control, β-Tubulin under
various treatment conditions.
5
μM concentrations (Fig. 7).
To determine if the gold and platinum compounds inhibit HCV
protein expression, western blot was used to analyze cellular lysates
from Huh7 cells as well as untreated, DMSO-treated, and drug-treated
Huh7-K2040 cells. HCV non-structural proteins (NS3, NS5a) as well as
STAT3 were immunoblotted. We observed a significant decrease in NS3
and NS5a expression in Huh7-K2040 cells treated with auranofin,
Surprisingly, auranofin treatment also resulted in a dramatic increase in
SMAD2 phosphorylation at both 2 μM and 5 μM concentrations
(
Fig. 5C, lanes 3 & 4). Ph PAuCl showed a 60% and 90% inhibition of
3
STAT3 phosphorylation at concentrations of 2 μM and 5 μM, respec-
tively, and also resulted in an increase in SMAD2 phosphorylation, al-
beit to a lesser extent than auranofin (Fig. 5C, lanes 7 & 8). Ad-
ditionally, none of the gold compounds appeared to have a significant
impact on α-SMA protein levels (Fig. 5C).
Ph PAuCl, and cisplatin at a 5 μM concentration as compared to DMSO-
3
treated cells (Fig. 8). At 5 μM concentrations, auranofin showed nearly
0% inhibition of HCV-NS5a, over 70% inhibition of HCV-NS3, and a
complete abrogation of STAT3 phosphorylation (Fig. 8A, lane 5). Si-
6
milarly, a 5 μM concentration of Ph PAuCl showed a 50% inhibition of
3
We also observed a significant overall decrease in the expression of
fibrosis-associated proteins in cisplatin-treated and carboplatin-treated
cells as compared to their respective controls (Fig. 5D). While treatment
HCV-NS5a, 50% inhibition of HCV-NS3, and again a complete abro-
gation of STAT3 phosphorylation (Fig. 8B, lane 5). Likewise, cisplatin at
5
μM inhibited expression of HCV-NS5a, HCV-NS3, and STAT3 phos-
with K
2
PtCl did result in a noticeable decrease in STAT3 phosphor-
4
phorylation all by approximately 30–40% (Fig. 8C, lane 8). We also
observed a significant decrease in STAT3 phosphorylation in Huh7-
ylation, it did not have a significant impact on any other fibrosis-as-
sociated markers (Fig. 5D, lanes 3 & 4). However, both cisplatin and
carboplatin showed a dose-dependent effect on fibrosis-associated
K2040 cells treated with auranofin (Fig. 8A, lane 4), Ph PAuCl (Fig. 5B,
3
lane 4), and cisplatin (Fig. 8C, lane 6) at a 2 μM concentration as
7

M. Stratton, et al.
Journal of Inorganic Biochemistry 206 (2020) 111023
Fig. 9. Summary of the effects of Au(I) compounds. Auranofin and Ph
3
PAuCl inhibit fibrosis in LX-2 cells by preventing phosphorylation of STAT3, reducing mRNA
for collagen I, and therefore reduced collagen I. They also reduce HCV transcription.
compared to DMSO-treated cells. Collectively, these results indicate the
more compared to the other species. Overall, this suggests that gold(I)
of the type PR AuX are more effective than the other compounds tested.
The key findings of this study are highlighted in Fig. 9.
The poor ability of K PtCl
inhibitory potential of these drugs against HCV protein expression.
3
2
4
and sodium aurothiomalate in inhibiting
4
. Discussion of biological activities of metal complexes
fibrosis and HCV proteins may be due to their ionic nature, which might
prevent them from entering the cells or allow them to be excreted from
the cells faster than the other species. While (PTA)AuCl is not proto-
nated under physiological pH, it does possess three hydrogen-bonding
amine groups, which makes it very water-soluble. As such, it may also
be less favorable for it to cross cell membranes and access the relevant
The results above indicate that gold(I) and platinum(II) complexes
hold promise for treating liver fibrosis and HCV. In particular, aur-
anofin and Ph PAuCl both reverted LX-2 cells back to their quiescent
3
morphology (Fig. 3). In comparison, the other two gold compounds and
the 3 platinum compounds did not show this activity. Auranofin and
targets in the cell. The aryl and alkyl phosphine groups on Ph PAuCl
3
Ph PAuCl also were much more effective than the other compounds at
3
and auranofin make them more hydrophobic/less hydrophilic than the
inhibiting type I collagen expression and phosphorylation of STAT3
other species, which might be a factor in their activity. Density
than the other compounds in LX-2 cells. Interestingly, auranofin and
Functional Theory calculations of PEt
3
AuCl, PPh
3
AuCl, and (PTA)AuCl
Ph PAuCl both had a much more significant impact on type I collagen
3
also indicate that the PeAu bond is much stronger for PEt
3
than for the
and α-SMA mRNA expression than that of their corresponding proteins.
This may be due to the slow turnover rate of these proteins as well as
collagen's excretion from cells into the extra cellular matrix. Type I
collagen in particular has been shown to last upwards of 90 days in the
extracellular matrix of liver tissue [51]. So while these compounds
appear to be inhibiting the translation of new protein, the previously
formed protein may have yet to be degraded within our 6 h timepoint.
As a result, we would expect type I collagen protein expression to more
closely mirror mRNA levels as the treatment window is widened be-
yond 6 h.
other phosphines, which may be key in keeping the molecular gold
compound from being metabolized and excreted from the cell. The
calculated PEt AuCl PeAu bond strength in water was 46 kcal/mol as
3
compared to 38 and 40 kcal/mol for PPh
3
and PTA (see Supporting
Information).
Whereas the platinum-based drugs have well-studied cellular be-
havior [55], auranofin's cellular metabolism and complete mechanism
of action in RA has not been definitively determined. Indeed, gold
compounds have been found to affect a wide variety of targets and
pathways [56]. There is evidence that indicates that it blocks IL-6 sig-
naling by inhibiting the phosphorylation of STAT3 by Janus kinase
We also observed an unexpected increase in SMAD2 phosphoryla-
tion in HSCs treated with auranofin and Ph PAuCl even though phos-
3
(
JAK) [57]. Auranofin also inhibits STAT3 phosphorylation and
phorylation of STAT3 was inhibited. One possible explanation for this
observation stems from literature reports that found that SMAD2 ac-
tually protects against hepatic fibrogenesis [52]. Specifically, over-
expression of SMAD2 aided in the resolution of hepatic fibrosis by in-
creasing apoptosis in activated LX-2 HSCs in addition to reducing the
expression of α-SMA and type I collagen [53]. Another research group
similarly found that, in the case of renal fibrosis, SMAD2 protects
against TGF-β–mediated fibrosis by counteracting TGF-β/Smad3 sig-
naling and that overexpression of SMAD2 attenuated type I collagen
matrix expression in tubular epithelial cells [54]. Given these findings,
our unexpected increase in SMAD2 expression and phosphorylation in
downregulates NF-κB in multiple myeloma cells [58], and inhibits
STAT3 and telomerase activation in breast cancer cells [59]. In our own
results reported above, gold(I) compounds inhibit STAT3 phosphor-
ylation in both LX-2 and K2040 cells, confirming that this is an effect of
auranofin and similar gold(I) compounds. A connection between STAT3
phosphorylation and RA has been described, and is consistent with it
being a target for the treatment of RA [60,61]. In addition, gold(I)
compounds have been found to inhibit protein tyrosine phosphatases
(
PTPs) [62–64], which play a role in regulating the JAK/STAT3
pathway [65,66]. There are other systems that are known to be in-
hibited by Au(I), such as cathepsins [67,68], which may play a role in
auranofin- and Ph
further highlighting the protective effect of these gold complexes
against liver fibrosis. Treatment with auranofin and Ph PAuCl also
impaired the expression of HCV NS3 and NS5a non-structural proteins
3
PAuCl-treated cells is consistent with the literature,
the anti-fibrotic activity of auranofin and Ph PAuCl. Another commonly
3
invoked target for Au(I) is the thioredoxin reductase system [69,70].
The thioredoxin and cathepsin systems contain sulfur groups that are
3
8

M. Stratton, et al.
Journal of Inorganic Biochemistry 206 (2020) 111023
attacked by the thiophilic gold species. The relationships between the
antifibrotic and anti-HCV behavior observed here and the specific
pathways that are affected by gold(I) species are yet to be elucidated,
and more mechanistic work is needed.
[17] B. Rosenberg, L. Van Camp, T. Krigas, Nature 205 (1965) 698–699.
[
[
[
18] E. Wong, C.M. Giandomenico, Chem. Rev. 99 (1999) 2451–2466.
19] D. Lebwohl, R. Canetta, Eur. J. Cancer 34 (1998) 1522–1534.
20] T.C. Johnstone, J.J. Wilson, S.J. Lippard, Inorg. Chem. 52 (2013) 12234–12249.
[21] D.A. Fennell, Y. Summers, J. Cadranel, T. Benepal, D.C. Christoph, R. Lal, M. Das,
F. Maxwell, C. Visseren-Grul, D. Ferry, Cancer Treat. Rev. 44 (2016) 42–50.
[
[
22] J. Hosry, G. Angelidakis, A. Kaseb, Y. Jiang, H.A. Torres, Clin. Infect. Dis. (2018)
5
. Conclusions and future directions
1
635–1636.
23] B.M. Sutton, Gold Bull. 19 (1986) 15–16.
In conclusion, gold(I) complexes, specifically auranofin and
[24] G.J. Higby, Gold Bull. 15 (1982) 130–140.
[
[
[
25] Z. Huaizhi, N. Yuantao, Gold Bull. 34 (2001) 24–29.
26] J. Forestier, Lancet 224 (1934) 646–648.
Ph PAuCl, are effective at inhibiting fibrosis in LX-2 cells as compared
3
to other gold(I) and platinum(II) species. They do not induce cell death
at their active concentrations, but do reduce type I collagen formation
in LX-2 cells, and also significantly inhibit phosphorylation of STAT3 in
both LX-2 and K2040 cells. In the HCV-infected cells, they inhibit for-
mation of HCV protein, suggesting possible inhibitory effects on HCV
translation/replication. These compounds are therefore promising
starting points for the development of drugs targeting liver fibrosis and
HCV infection. Further work is needed to elucidate the mechanisms of
action that lead to fibrosis and HCV inhibition, including the specific
targets that the gold is interacting with. In addition, modification of the
supporting phosphine ligand or use of other ligands such as N-hetero-
cyclic carbenes on the Au(I) species may allow for structure-activity
relationships to be developed, leading to improved metal-based drugs
for liver fibrosis and HCV.
27] R.C. Blodgett, M.A. Heuer, R.G. Pietrusko, Semin. Arthritis Rheum. 13 (1984)
255–273.
[
[
[
28] I. Ott, Coord. Chem. Rev. 253 (2009) 1670–1681.
29] M. Navarro, Coord. Chem. Rev. 253 (2009) 1619–1626.
30] N.P. Wiederhold, T.F. Patterson, A. Srinivasan, A.K. Chaturvedi, A.W. Fothergill,
F.L. Wormley, A.K. Ramasubramanian, J.L. Lopez-Ribot, Virulence 8 (2017)
1
38–142.
[
31] A. Debnath, D. Parsonage, R.M. Andrade, C. He, E.R. Cobo, K. Hirata, S. Chen,
G. García-Rivera, E. Orozco, M.B. Martínez, Nat. Med. 18 (2012) 956.
[32] E.V. Capparelli, R. Bricker-Ford, M.J. Rogers, J.H. McKerrow, S.L. Reed,
Antimicrob. Agents Chemother. 61 (2017) (e01947-e01916).
[
33] F.A. Vollenbroek, J.P. Van den Berg, J.W.A. Van der Velden, J.J. Bour, Inorg. Chem.
1
9 (1980) 2685–2688.
[34] Z. Assefa, B.G. McBurnett, R.J. Staples, J.P. Fackler, B. Assmann, K. Angermaier,
H. Schmidbaur, Inorg. Chem. 34 (1995) 75–83.
[
35] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman,
G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato,
X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada,
M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda,
O. Kitao, H. Nakai, T. Vreven, J.A. Montgomery, J.E. Peralta, F. Ogliaro,
M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi,
J. Normand, K. Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi,
M. Cossi, N. Rega, J.M. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken,
C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin,
R. Cammi, C. Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski,
G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich, A.D. Daniels, O. Farkas,
J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J, Fox, Wallingford, CT, 2009.
36] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman,
G. Scalmani, V. Barone, G.A. Petersson, H. Nakatsuji, X. Li, M. Caricato,
A.V. Marenich, J. Bloino, B.G. Janesko, R. Gomperts, B. Mennucci, H.P. Hratchian,
J.V. Ortiz, A.F. Izmaylov, J.L. Sonnenberg, F. Williams, F. Ding, F. Lipparini,
J. Egidi, B. Goings, A. Peng, T. Petrone, D. Henderson, V.G. Ranasinghe,
J. Zakrzewski, N. Gao, G. Rega, W. Zheng, M. Liang, M. Hada, K. Ehara, R. Toyota,
J. Fukuda, M. Hasegawa, T. Ishida, Y. Nakajima, O. Honda, H. Kitao, T. Nakai,
K. Vreven, J.A. Throssell, J.E. Montgomery Jr., F. Peralta, M.J. Ogliaro,
J.J. Bearpark, E.N. Heyd, K.N. Brothers, V.N. Kudin, T.A. Staroverov, R. Keith,
J. Kobayashi, K. Normand, A.P. Raghavachari, J.C. Rendell, S.S. Burant, J. Iyengar,
M. Tomasi, J.M. Cossi, M. Millam, C. Klene, R. Adamo, J.W. Cammi, R.L. Ochterski,
K. Martin, O. Morokuma, J.B. Farkas, D.J. Foresman, Fox, Wallingford, CT, (2016).
37] R.D. Dennington, T.A. Keith, J.M. Millam, Gaussian Inc, (2008).
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
[
Acknowledgements
We thank Dr. Caitlin Karver for valuable discussions about the
biological chemistry of gold(I). Initial work on this project was sup-
ported by DePaul-Rosalind Franklin University of Medicine and Science
Collaborative Pilot Grant program. This work was later partially sup-
ported by the National Institutes of Health grant (DK106244) and the
Rosalind Franklin University of Medicine & Science, H.M. Blight Cancer
Research Fund to G.W.
[
[
38] P. Stephens, F. Devlin, C. Chabalowski, M.J. Frisch, J. Phys. Chem. 98 (1994)
Appendix A. Supplementary data
1
1623–11627.
[
39] A.D. Becke, J. Chem. Phys. 98 (1993) 5648–5652.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jinorgbio.2020.111023.
[40] C. Lee, W. Yang, R.G. Parr, Phys. Rev. B 37 (1988) 785.
[
[
[
41] S.H. Vosko, L. Wilk, M. Nusair, Can. J. Phys. 58 (1980) 1200–1211.
42] M.J. Frisch, J.A. Pople, J.S. Binkley, J. Chem. Phys. 80 (1984) 3265–3269.
43] P.J. Hay, W.R. Wadt, J. Chem. Phys. 82 (1985) 270–283.
References
[44] T.H. Dunning, P.J. Hay, H.F. Schaefer (Ed.), Methods of Electronic Structure
Theory, Boston, MA, 1977, pp. 1–27.
[
[
[
45] J. Tomasi, B. Mennucci, R. Cammi, Chem. Rev. 105 (2005) 2999–3094.
46] D.E. Levy, C. Lee, J. Clin. Invest. 109 (2002) 1143–1148.
[
1] W.H, Organization, Global Hepatitis Report, 2017 World Health Organization,
Geneva, 2017.
47] F. Xu, C. Liu, D. Zhou, L. Zhang, Journal of Histochemistry & Cytochemistry 64
[
[
2] A.M. Di Bisceglie, Hepatology 26 (1997) 34S–38S.
(
2016) 157–167.
3] A.A. Mokdad, A.D. Lopez, S. Shahraz, R. Lozano, A.H. Mokdad, J. Stanaway,
C.J. Murray, M. Naghavi, BMC Med. 12 (2014) 145.
[
[
48] A.M. Attallah, T.E. Mosa, M.M. Omran, M.M. Abo-Zeid, I. El-Dosoky, Y.M. Shaker,
J. Immunoass. Immunochem. 28 (2007) 155–168.
[
[
[
[
4] A.Y. Hui, S.L. Friedman, Expert Rev. Mol. Med. 5 (2003) 1–23.
5] S.L. Friedman, J. Biol. Chem. 275 (2000) 2247–2250.
6] Y. Zhang, X. Yao, APMIS 120 (2012) 101–107.
49] S. Koilan, D. Hamilton, N. Baburyan, M.K. Padala, K.T. Weber, R.V. Guntaka,
Oligonucleotides 20 (2010) 231–237.
[
[
50] M. Ikeda, M. Yi, K. Li, S.M. Lemon, J. Virol. 76 (2002) 2997–3006.
51] E. Gineyts, P.A. Cloos, O. Borel, L. Grimaud, P.D. Delmas, P. Garnero, Biochem. J.
7] A. Wang, F. Zhang, H. Xu, M. Xu, Y. Cao, C. Wang, Y. Xu, M. Su, M. Zhang,
Y. Zhuge, Mol. Immunol. 87 (2017) 67–75.
3
45 (2000) 481–485.
[
[
8] R.C. Benyon, M.J. Arthur, Proc, Semin. Liver Dis. 21 (2001) 373–384.
9] G.N. Ioannou, P.K. Green, L.A. Beste, E.J. Mun, K.F. Kerr, K. Berry, Journal of
Hepatology, Elsevier 69 (2018) 1088–1098.
[
[
[
52] L. Zhang, C. Liu, X. Meng, C. Huang, F. Xu, J. Li, Mol. Cell. Biochem. 400 (2015)
1
7–28.
53] F. Xu, D. Zhou, X. Meng, X. Wang, C. Liu, C. Huang, J. Li, L. Zhang, Int.
Immunopharmacol. 32 (2016) 76–86.
[
[
[
10] J. Vermehren, C. Sarrazin, Best Pract. Res. Clin. Gastroenterol. 26 (2012) 487–503.
11] C. Sarrazin, S. Zeuzem, Gastroenterology 138 (2010) 447–462.
54] X.M. Meng, X.R. Huang, A.C.K. Chung, W. Qin, X. Shao, P. Igarashi, W. Ju,
E.P. Bottinger, H.Y. Lan, J. Am. Soc. Nephrol. 21 (2010) 1477–1487.
55] D. Wang, S.J. Lippard, Nat. Rev. Drug Discov. 4 (2005) 307.
56] J. Madeira, D. Gibson, W. Kean, A. Klegeris, Inflammopharmacology 20 (2012)
12] E. Lontok, P. Harrington, A. Howe, T. Kieffer, J. Lennerstrand, O. Lenz, F. McPhee,
H. Mo, N. Parkin, T. Pilot-Matias, V. Miller, Hepatology 62 (2015) 1623–1632.
13] R. Patil, A. Ghosh, P. Sun Cao, R.D. Sommer, K.A. Grice, G. Waris, S. Patil, Biorg.
Med. Chem. Lett. 27 (2017) 1129–1135.
[
[
[
[
2
97–306.
14] K.A. Grice, R. Patil, A. Ghosh, J.D. Paner, M.A. Guerrero, E.J.M. Camacho, P. Sun
Cao, A.H. Niyazi, S. Zainab, R.D. Sommer, G. Waris, S. Patil, New J. Chem. 42
[
[
[
57] N.H. Kim, M.Y. Lee, S.J. Park, J.S. Choi, M.K. Oh, I.S. Kim, Immunology 122 (2007)
07–614.
58] A. Nakaya, M. Sagawa, A. Muto, H. Uchida, Y. Ikeda, M. Kizaki, Leuk. Res. 35
2011) 243–249.
59] N.-H. Kim, H.J. Park, M.-K. Oh, I.-S. Kim, BMB Rep. 46 (2013) 59.
6
(
2018) 1151–1158.
[
[
15] T.W. Hambley, Dalton Trans. (2007) 4929–4937.
(
16] K.D. Mjos, C. Orvig, Chem. Rev. 114 (2014) 4540–4563.
9

M. Stratton, et al.
Journal of Inorganic Biochemistry 206 (2020) 111023
[
[
[
60] A. Krause, N. Scaletta, J.-D. Ji, L.B. Ivashkiv, J. Immunol. 169 (2002) 6610–6616.
61] M. Katoh, M. Katoh, Int. J. Mol. Med. 19 (2007) 273–278.
[67] S.S. Gunatilleke, C.A.F. de Oliveira, J.A. McCammon, A.M. Barrios, JBIC, J. Biol.
Inorg. Chem. 13 (2008) 555–561.
62] M.R. Karver, D. Krishnamurthy, R.A. Kulkarni, N. Bottini, A.M. Barrios, J. Med.
Chem. 52 (2009) 6912–6918.
[68] S.S. Gunatilleke, A.M. Barrios, J. Inorg. Biochem. 102 (2008) 555–563.
[69] V. Gandin, A.P. Fernandes, M.P. Rigobello, B. Dani, F. Sorrentino, F. Tisato,
M. Björnstedt, A. Bindoli, A. Sturaro, R. Rella, C. Marzano, Biochem. Pharmacol. 79
(2010) 90–101.
[
[
63] M.R. Karver, D. Krishnamurthy, N. Bottini, A.M. Barrios, J. Inorg. Biochem. 104
(
2010) 268–273.
64] D. Krishnamurthy, M.R. Karver, E. Fiorillo, V. Orrú, S.M. Stanford, N. Bottini,
A.M. Barrios, J. Med. Chem. 51 (2008) 4790–4795. American Chemical Society.
65] D. Xu, C.-K. Qu, Front Biosci 13 (2008) 4925.
[70] F. Angelucci, A.A. Sayed, D.L. Williams, G. Boumis, M. Brunori,
D. Dimastrogiovanni, A.E. Miele, F. Pauly, A. Bellelli, J. Biol. Chem. 284 (2009)
28977–28985.
[
[
66] R. Morris, N.J. Kershaw, J.J. Babon, Protein Sci. 27 (1984-2009) 2018.
10