A Modular Ionophore Platform for Liver-Directed Copper Supplementation in Cells and Animals

Article abstract of DOI:10.1021/jacs.8b08014

Copper deficiency is implicated in a variety of genetic, neurological, cardiovascular, and metabolic diseases. Current approaches for addressing copper deficiency rely on generic copper supplementation, which can potentially lead to detrimental off-target metal accumulation in unwanted tissues and subsequently trigger oxidative stress and damage cascades. Here we present a new modular platform for delivering metal ions in a tissue-specific manner and demonstrate liver-targeted copper supplementation as a proof of concept of this strategy. Specifically, we designed and synthesized an N-acetylgalactosamine-functionalized ionophore, Gal-Cu(gtsm), to serve as a copper-carrying Trojan Horse that targets liver-localized asialoglycoprotein receptors (ASGPRs) and releases copper only after being taken up by cells, where the reducing intracellular environment triggers copper release from the ionophore. We utilized a combination of bioluminescence imaging and inductively coupled plasma mass spectrometry assays to establish ASGPR-dependent copper accumulation with this reagent in both liver cell culture and mouse models with minimal toxicity. The modular nature of our synthetic approach presages that this platform can be expanded to deliver a broader range of metals to specific cells, tissues, and organs in a more directed manner to treat metal deficiency in disease.

Full text of DOI:10.1021/jacs.8b08014

Article
pubs.acs.org/JACS
Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
A Modular Ionophore Platform for Liver-Directed Copper
Supplementation in Cells and Animals
Timothy A. Su,† Diyala S. Shihadih,‡ Wendy Cao,† Tyler C. Detomasi,† Marie C. Heffern,†,#
Shang Jia,† Andreas Stahl,*,‡ and Christopher J. Chang*,†,§,∥,⊥
†Department of Chemistry, University of California, Berkeley, California 94720, United States
‡Department of Nutritional Sciences & Toxicology, University of California, Berkeley, California 94720, United States
§Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, United States
∥Helen Wills Neuroscience Institute, University of California, Berkeley, California 94720, United States
⊥Howard Hughes Medical Institute, University of California, Berkeley, California 94720, United States
S
* Supporting Information
ABSTRACT: Copper deficiency is implicated in a variety of
genetic, neurological, cardiovascular, and metabolic diseases.
Current approaches for addressing copper deficiency rely on
generic copper supplementation, which can potentially lead to
detrimental off-target metal accumulation in unwanted tissues
and subsequently trigger oxidative stress and damage cascades.
Here we present a new modular platform for delivering metal
ions in a tissue-specific manner and demonstrate liver-targeted
copper supplementation as a proof of concept of this strategy.
Specifically, we designed and synthesized an N-acetylgalactos-
amine-functionalized ionophore, Gal-Cu(gtsm), to serve as a copper-carrying “Trojan Horse” that targets liver-localized
asialoglycoprotein receptors (ASGPRs) and releases copper only after being taken up by cells, where the reducing intracellular
environment triggers copper release from the ionophore. We utilized a combination of bioluminescence imaging and inductively
coupled plasma mass spectrometry assays to establish ASGPR-dependent copper accumulation with this reagent in both liver
cell culture and mouse models with minimal toxicity. The modular nature of our synthetic approach presages that this platform
can be expanded to deliver a broader range of metals to specific cells, tissues, and organs in a more directed manner to treat
metal deficiency in disease.
and subsequent Cu+ release to copper-binding molecules and
INTRODUCTION
■
proteins (Figure 1A).
Copper is a required nutrient for all living organisms,1 enabling
Whereas ionophores offer improved cellular copper
fundamental life processes spanning respiration,2 antioxidant
supplementation relative to the inorganic copper salts typically
defense,3,4 neurotransmitter synthesis,5,6 metabolism,7,8 and
administered in vitamins, several inherent drawbacks arise from
cell signaling.9−12 Biological copper deficiency hinders these
the generic, untargeted nature of copper delivery. First,
because copper delivery occurs mostly through a passive
essential functions and correlates with various pathologies
including Menkes disease,13 familial amyotrophic lateral
transport mechanism, copper delivery into cells is not
sclerosis,14,15 neurodegenerative disorders,16−18 cardiovascular
regulated: for example, if copper supplementation exceeds
disease,19,20 and metabolic disorders.21−27 Current approaches
the buffering capacity of the cytosol, oxidative damage to
for addressing copper deficiency rely on metal supplementa-
tion, often through the administration of copper ionophores,
which are molecules that are distinct from copper chelators in
that they deliver and release metals into cells rather than
sequester and remove metals from cells.28,29 Conventional
copper ionophores include copper complexes with histidine,30
bisthiosemicarbazones,31−33 8-hydroxyquinolines,16,34 and
others.29 The small and hydrophobic nature of these
ionophores, which typically chelate copper in the Cu2+
oxidation state, enables their passive diffusion through the
cell membrane, after which the reducing intracellular environ-
ment acts as a redox trigger for reduction of the metal complex
biomolecules such as DNA, RNA, lipids, and proteins may
occur via Fenton-like chemistry.35 Indeed, copper ionophore
supplementation routinely leads to oxidative stress and
damage.36,37 Second, traditional untargeted copper delivery
approaches lack tissue specificity.38,39 As such, if the intent is to
treat copper deficiency in a specific organ of interest, such
indiscriminate copper delivery will lead to aberrant reactive
oxygen species (ROS) generation and oxidative damage in off-
target tissues (Figure 1A). To address these challenges, we
Received: July 27, 2018
© XXXX American Chemical Society
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Figure 1. Schematic diagram comparing delivery methods for ionophore-based copper supplementation. (A) Conventional ionophores increase
copper levels in many organs due to the nonspecific nature of passive diffusion. For example, Cu(gtsm) releases copper intracellularly following
reduction in the cytosolic medium. If too much copper is released such that it exceeds the cell’s copper-buffering capacity, excess Cu+ can generate
reactive oxygen species (ROS) through Fenton-like reactions to induce oxidative stress and damage to the cell. (B) Molecular structures for the
untargeted Cu(gtsm) and targeted Gal-Cu(gtsm) ionophores studied here. (C) The targeted ionophore-based metal supplementation (TIMS)
strategy presented here enables receptor-mediated metal accumulation with minimal off-target delivery, as shown by liver-selective copper
supplementation. The hydrophilicity of the targeted ionophore precludes passive diffusion; ionophore internalization only occurs upon ligand−
receptor recognition and endocytosis. Copper release is likely controlled by homeostatic cues at the level of the lysosome to enable regulated
copper delivery. (D) The bifunctional ionophore design for Gal-Cu(gtsm) uses a triethylene glycol linker to join the Cu(gtsm) moiety, which binds
copper selectively and releases it upon intracellular reduction, with a GalNAc targeting group that is a specific ligand for ASGPR proteins expressed
on the cell membranes of hepatocytes.
now present a new, versatile approach to site-specific metal
delivery, termed “targeted ionophore-based metal supplemen-
tation (TIMS)”, which relies on pairing metal-delivering
ionophores with targeting ligands that recognize specific cell
surface receptors localized to cells and tissues of interest.
We demonstrate the viability of this TIMS approach through
the development of a targeted copper supplement that enables
selective delivery of copper to the liver in a nontoxic manner
owing to interest in addressing hepatic copper deficiency
connected to the pathogenesis of nonalcoholic fatty liver
disease (NAFLD),21−23,26,27 which is estimated to afflict 24%
of the global population (or 1.8 billion people).40 NAFLD
often leads to severe liver diseases and shows a strong
association with cardiovascular diseases, obesity, diabetes, and
cancer.41−43 Indeed, recent work from our laboratories utilized
a caged copper luciferin (CCL-1) probe to longitudinally
monitor copper levels over the course of NAFLD progression
in mice, revealing that hepatic copper deficiency manifests in a
diet-induced NAFLD model prior to the formal onset of fatty
liver disease. Along these lines, we prepared targeted
bifunctional ionophore Gal-Cu(gtsm) and benchmarked its
copper delivery properties in cell culture and mice against the
untargeted Cu(gtsm) ionophore (Figure 1B). We utilized ICP-
MS and bioluminescent imaging techniques to characterize
copper supplementation across tissues, observing that the
targeted Gal-Cu(gtsm) enables selective hepatic copper
supplementation. Furthermore, we utilized a variety of toxicity
and histological staining assays to establish that Cu delivery via
the TIMS compound is nontoxic, whereas untargeted delivery
via passive diffusion incurs significant damage to the liver as
well as off-target renal toxicity. Finally, we performed SDS-
PAGE and Western blot analysis to characterize expression of
copper storage and trafficking proteins, finding that both
copper import/export as well as storage proteins are elevated
with TIMS treatment. Taken together, this study provides a
new type of liver-specific copper supplement and establishes a
starting point for the broader use of TIMS reagents as tool
compounds to probe the functional roles that metals play in
specific cell, tissue, and/or organ types in normal physiology
and as directed therapeutic supplements to address metal-
deficiency-based pathologies.
RESULTS AND DISCUSSION
■
Design and Synthesis of a Liver-Targeted Copper
Supplement. Figure 1C outlines the TIMS strategy, where
(i) ligand−receptor recognition triggers endocytosis and
internalization of the receptor−ionophore complex, (ii) the
complex releases the metal in the endolysosomal pathway
through either intracellular reduction or degradation, and (iii)
the metal ion binds to metal storage or trafficking proteins,
which can then utilize the metal as needed. In support of this
approach, recent studies have shown that the lysosome is a key
organelle in dynamic metal regulation,44−46 and mediating
metal delivery through this organelle’s homeostatic cues may
offer a safer alternative for increasing bioavailable metal ion
pools compared to unregulated cytosolic metal release. We
note that for this strategy to be effective, the targeted
ionophore must be either large or hydrophilic enough to
hinder passive diffusion such that metal supplementation is
strictly receptor-mediated.
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a
Scheme 1. Synthetic Route for Accessing Gal-Cu(gtsm)
a
Conditions: (a) 2,2-dimethoxyacetaldehyde, MeOH, rt, quantitative yield; (b) LiBF4, MeCN, H2O, rt, 81% yield; (c) 4,4-dimethyl-3-
thiosemicarbazide, DMF, 4 Å MS, 60 °C, 38% yield; (d) HO-TEG-N3, TMSOTf, DCE, rt, 81% yield; (e) Pd/C, H2, MeOH, 92% yield; (f) i.
MeCN, 80 °C, 32% yield; ii. Cu(OAc)2, DMF, rt, 73% yield.
With these design principles in mind, we synthesized a
bifunctional ionophore that targets the asialoglycoprotein
receptor (ASPGR), a C-type lectin membrane receptor that
is specifically expressed in the liver at high levels (e.g., ca.
500 000 ASGPR copies per primary hepatocyte47), as shown in
Figure 1D. We utilized an N-acetylgalactosamine (GalNAc)
ligand as our targeting group, which binds ASGPR with a Kd
value of ca. 10−5 M−1.48 Liver targeting via ASGPR/galactose
recognition is a robust strategy that has been used to deliver
drugs,49,50 proteins,51 siRNA,52,53 and CRISPR-Cas9;54 related
work has employed metal chelators55,56 to address copper-
excess-based disorders such as Wilson’s disease. Although
multivalent47 galactose conjugates offer higher-affinity binding
over their monovalent48,57 counterparts, we decided on the
monovalent targeting group in our first-generation design
owing to improved atom economy for our payload, a small
organometallic complex, as well as for scalable synthesis for
longitudinal animal studies.
The design for our TIMS agent is conceptually related to the
approach taken for site-specific metal removal, with the key
difference being that we attach a targeting group to a metal
ionophore rather than a metal chelator.17,58−60 As such, we
paired a GalNAc targeting moiety with the bisthiosemicarba-
zone complex Cu(gtsm) (Figure 1D), which is a well-
established copper ionophore that has been explored as a
potential therapeutic for neurodegenerative disorders31,39,61,62
and cancer.36,63 The metal-binding and Cu release properties
of Cu(gtsm) complex are well-studied and characterized. The
gtsm ligand binds Cu2+ (Kd ≈ 10−18 M−1) much more tightly
than Cu+ (Kd ≈ 10−13 M−1)64 and releases Cu+ upon reduction
in the intracellular medium, where it can metalate copper-
binding small molecules and proteins.31,36,65 We reasoned that
the significant hydrophilicity conferred by the GalNAc subunit
may preclude the ionophore from passive diffusion and
encourage receptor-mediated uptake. We chose to link the
ionophore and targeting GalNAc ligand with a triethylene
glycol chain for enhanced hydrophilicity owing to literature
precedent.50
reacted oxazoline 570 with azidotriethylene glycol in the
presence of trimethylsilyl trifluoromethanesulfonate to access
the azide-protected peracetylated GalNAc 6. The azide and O-
acetyl groups were deprotected in a single palladium-catalyzed
hydrogenation reaction to give GalNAc amine 7. Trans-
amination between 4 and 7 gave apo-Gal-H2 gtsm, which we
then metalated with copper diacetate to give Gal-Cu(gtsm).
Log P measurements with a shake-flask octanol−water
partition experiment confirmed that Gal-Cu(gtsm) (log P =
−1.03 0.10) is more hydrophilic than Cu(gtsm) (log P =
1.39
0.14).66,71 Interestingly, we also found that Gal-
Cu(gtsm) forms colloids in aqueous buffer with a number-
weighted hydrodynamic diameter of 4.60 0.69 nm, whereas
Cu(gtsm) does not. Cu(gtsm) and Gal-Cu(gtsm) demonstrate
similar Cu−ligand charge transfer absorption band character-
istics (Figure S1). We performed further in vitro spectroscopic
studies to evaluate the stability of Gal-Cu(gtsm) under
physiologically relevant conditions. We found that the Gal-
Cu(gtsm) complex is stable in aqueous buffers ranging from
pH = 3 to 8 (Figure S2). The complex also retains copper
selectively in the presence of a 1000-fold excess of Zn2+ and
Fe2+(Figure S3), which are two other abundant intracellular
transition metal ions.72
ASGPR-Dependent Copper Delivery in Cell Culture.
With the two ionophores in hand, we characterized the
behavior of Cu(gtsm) and Gal-Cu(gtsm) in cell lines with high
ASGPR expression (HepG2) or no ASGPR expression (HEK
293T). We evaluated ionophore toxicity in these cell lines
using propidium iodide staining as a proxy for cell viability,
finding that that Cu(gtsm) treatment is toxic at an 8 μM dose
in HEK 293T cells, whereas Gal-Cu(gtsm) is nontoxic at a 500
μM dose in both cell lines (Figure S4). We then evaluated the
intracellular Cu delivery abilities of the two ionophores using
inductively coupled plasma-mass spectrometry (ICP-MS) to
analyze total copper levels. We find that Cu(gtsm) elicits dose-
dependent increases in cellular copper content for both cell
types, whereas Gal-Cu(gtsm) demonstrates a robust dose-
dependent copper delivery response only in the ASGPR-
expressing HepG2 cells (Figure S5).
Cu(gtsm) was synthesized using literature methods.66 For
Gal-Cu(gtsm), we employed a convergent synthetic approach
where we synthesized the ionophore and targeting group
fragments independently, then linked them together through a
transamination reaction in the penultimate step (Scheme
1).67,68 Briefly, we installed the glyoxal unit with an acetal
protecting group onto thiosemicarbazide 1, then unmasked
acetal 2 with lithium tetrafluoroborate to give iminoacetalde-
hyde 3.69 Condensation with 4,4-dimethyl-3-thiosemicarbazide
gave the asymmetrically substituted gtsm analogue 4. We
Figure 2A,B highlight these differences at an equimolar 4
μM copper dose. We treated HepG2 and HEK 293T cells with
either vehicle control, 4 μM Cu(gtsm), or 4 μM Gal-Cu(gtsm)
for three hours. Figure 2 displays 63Cu/31P ratios from ICP-MS
data as a metric for characterizing copper delivery for each
treatment condition, with the 31P signal normalizing for
number of cells based on cellular phosphate content. Figure 2A
shows that Gal-Cu(gtsm) leads to a 4-fold Cu increase relative
to the basal levels in HepG2 cells, while Cu(gtsm) treatment
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Cu(gtsm) between 4 and 37 °C, which suggests that Cu
delivery via Cu(gtsm) occurs primarily through a passive
diffusion mechanism (Figure 2D). As further support for an
ASGPR-mediated Cu delivery mechanism for Gal-Cu(gtsm),
we performed a competition experiment where we treated
HepG2 cells with vehicle control or ionophore in the presence
of D-galactose (1 M) for one hour. The presence or absence of
D-galactose does not alter Cu content upon vehicle or
Cu(gtsm) treatment in HepG2 cells; however, Cu delivery
via Gal-Cu(gtsm) is significantly attenuated in the presence of
D-galactose (Figure 2E). We also performed confocal
microscopy imaging of HepG2 cells treated with Cu(gtsm)
and Gal-Cu(gtsm) using the copper-sensing CF4 fluorescent
probe73 and observed increased fluorescence upon ionophore
treatment (Figure S6).
Although the untargeted Cu(gtsm) ionophore delivered
more total copper than Gal-Cu(gtsm) under equimolar doses
to both HEK 293T and HepG2 cells, the salient finding for
expanding the TIMS platform to animal studies was that
copper delivery via Gal-Cu(gtsm) occurred exclusively in the
ASGPR-expressing HepG2 cells. With these promising cell
culture results in hand, we sought to identify whether Gal-
Cu(gtsm) could selectively provide Cu supplementation to the
liver with minimal off-target delivery in the complex biological
milieu of live mice.
Evaluating Liver Target Specificity of Copper
Supplementation by Gal-Cu(gtsm) in Living Mice. We
chose to characterize liver-targeted copper supplementation in
mice because they are common model organisms for
elucidating the pathology of metabolic disorders such as
NAFLD. Our laboratory recently developed a copper caged
luciferin-1 (CCL-1) probe that reports on dynamic changes to
the loosely bound, labile Cu+ pool in transgenic bio-
luminescent mice (Figure 3A).27 Here we utilize CCL-1 to
characterize liver-directed copper supplementation in the
firefly luciferase-expressing FVB-luc+ mouse strain, where
luciferase expression enables detection of changes in copper
pools throughout the entire mouse. We also imaged
ionophore-treated mice with D-luciferin (D-Luc) to normalize
the CCL-1 signal and account for CCL-1 cleavage-independent
bioluminescent effects. Male FVB-luc+ mice (11−14 weeks
old) received intraperitoneal (i.p.) injections of vehicle control,
Gal-Cu(gtsm), or Cu(gtsm) at equivalent copper concen-
trations (0.75 mg Cu/kg mouse). We selected this copper dose
based on similar i.p. doses utilized by others to study the
therapeutic effects of Cu(gtsm) and its derivatives.63,74 After
six hours, the same mice were anesthetized with isoflurane and
given scapular subcutaneous (s.c.) injections of either CCL-1
or D-Luc (0.1 μmol) and imaged with an in vivo imaging
system (IVIS) every 5 min over a 40 min period.
Representative images of CCL-1-injected mice six hours
following vehicle or Gal-Cu(gtsm) treatment are provided in
Figure 3B. A full panel of images for CCL-1- and D-Luc-
injected mice both 6 and 24 h after ionophore treatment is
provided in Figures S7 and S8.
Figure 2. ICP-MS measurements reveal that Cu supplementation via
Gal-Cu(gtsm) is ASGPR-dependent. (A) ICP-MS studies comparing
Cu levels upon 4 μM Cu-ionophore treatment (0.2% DMSO in
serum-free DMEM) over a three-hour time course in HepG2
(ASGPR-expressing) cells. (B) ICP-MS studies upon 4 μM Cu-
ionophore treatment (0.2% DMSO in serum-free DMEM) over a
three-hour time course in HEK 293T (no ASGPR expression) cells.
(C−, D) HepG2 cells were treated with Gal-Cu(gtsm) (20 μM, C) or
Cu(gtsm) (4 μM, D) over a one-hour period at either 4 or 37 °C.
Data are plotted relative to Cu/P ratio for each ionophore at 4 °C.
(E) HepG2 cells were treated with or without D-galactose (1 M in
serum-free DMEM) as a competitive ASGPR ligand 15 min prior to
treatment with vehicle, Cu(gtsm) (4 μM), or Gal-Cu(gtsm) (20 μM)
over a one-hour period. Error bars = SEM (n = 6). Statistical analyses
were performed using a one-way ANOVA with Bonferroni’s multiple
comparisons test (A, B) or a two-tailed Student’s t test (C), where *P
≤ 0.05, ***P ≤ 0.001, and ****P ≤ 0.0001.
leads to a 45-fold increase in Cu content. In HEK 293T cells
(Figure 2B) that do not express ASGPR, Gal-Cu(gtsm)
treatment does not increase cellular copper beyond basal
levels, while Cu(gtsm) treatment leads to a 100-fold increase in
Cu levels. The data show that Cu(gtsm) delivers copper
indiscriminately to both cell types, likely via passive diffusion
since Cu(gtsm) is small, hydrophobic, and cell permeable.
Conversely, the hydrophilic Gal-Cu(gtsm) experiences limited
passive diffusion and does not deliver copper to HEK 293T
cells lacking ASGPRs, whereas copper delivery only occurs in
the ASGPR-expressing HepG2 cells upon Gal-Cu(gtsm)/
ASGPR recognition and internalization.
In support of this mechanistic proposal, we performed
temperature-dependent uptake studies at 4 and 37 °C. Gal-
Cu(gtsm) demonstrates much higher uptake at 37 °C than at 4
°C, which suggests that Gal-Cu(gtsm)-mediated Cu delivery is
an active uptake process (Figure 2C). In contrast, we did not
observe any significant differences in Cu delivery ability for
Figure 3C plots the ratio of CCL-1/D-Luc-integrated photon
fluxes in the liver region, six hours after ionophore
supplementation. We find high increases in liver CCL-1/D-
Luc signal ratio responses, suggesting increases in labile copper
levels for both the targeted Gal-Cu(gtsm) and Cu(gtsm)
compounds. There are two potential factors that may
contribute to the observed differences between cell culture
and live animal TIMS. First, there is a much higher expression
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Figure 3. In vivo bioluminescence imaging and ex vivo tissue ICP-MS analysis reveal a tissue-selective increase in hepatic copper stores after Gal-
Cu(gtsm) supplementation. (A) Schematic describing Cu+-dependent cleavage of CCL-1 to give bioluminescent signal. Free D-luciferin is released
upon binding between Cu and CCL-1, which then interacts with luciferase and emits a photon. (B) Representative images of mice injected s.c. with
CCL-1 6 h after vehicle or Gal-Cu(gtsm) i.p. administration. Images for Cu(gtsm)-treated mice are given in Figure S7. (C) Data plotted represent
CCL-1 total integrated photon flux as a ratio over D-Luc total integrated photon flux. Total integrated photon flux was collected 5−45 min
postinjection in a region of interest drawn over the liver. Liver signal ratio is normalized to vehicle control. Error bars = SEM (n = 5−7). Statistical
analysis performed with a one-way ANOVA with Bonferroni’s multiple comparisons test where *P ≤ 0.05. (D, E) Mice were injected i.p. with 0.75
mg equivalent Cu/kg mouse of Gal-Cu(gtsm) or Cu(gtsm) six hours prior to blood and tissue collection. Tissue copper levels relative to tissue wet
weight were determined using ICP-MS assays. Error bars = SEM (n = 5). Statistical analyses were performed with a two-tailed Student’s t test
where *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001.
of ASGPR in primary hepatocytes (ca. 500 000 ASGPR/cell)
than in HepG2 cells (ca. 76 000 ASGPR/cell).47,75 Second,
Cu(gtsm) delivery occurs through nonspecific passive
diffusion. In cell culture, this property results in high levels
of copper delivery. However, in animals, this property leads to
nonspecific copper delivery to off-target organs.
at 24 h (Figure S10), implying that the supplemented hepatic
copper pool is not redistributed from the liver to other organs
over time. We find that hepatic copper stores are cleared to
basal levels after 72 h (Figure S11).
In contrast, untargeted Cu(gtsm) ionophore treatment leads
to a 90% increase in liver copper content at the 6 h time point
(Figure 3E), with substantial off-target copper delivery to
organs including the intestines, kidney, heart, lungs, and brain.
At 24 h, there remains a statistically significant increase in liver,
heart, and lung copper relative to basal levels (Figure S10).
Although total hepatic copper levels remain substantially
elevated at 24 h for both Gal-Cu(gtsm) and Cu(gtsm),
interestingly, we find that the bioluminescent CCL-1/D-Luc
liver signal returns to basal levels after 24 h (Figure S8). These
observations suggest a potential difference in labile copper
status at 6 h compared to 24 h, where the delivered copper
becomes more tightly sequestered by metal storage proteins at
longer time points. Furthermore, we find that the admin-
istration of Gal-Cu(gtsm) or Cu(gtsm) offers minimal
perturbation to iron and zinc levels across tissues relative to
basal conditions (Figures S12 and S13). These data indicate
that appending an N-acetylgalactosamine moiety to the
Cu(gtsm) framework can enable copper supplementation
The greater propensity for off-target copper accumulation
with Cu(gtsm) is supported by ex vivo tissue metal analysis via
ICP-MS to evaluate total copper levels across organs after
Cu(gtsm) and Gal-Cu(gtsm) supplementation at a 0.75 mg
Cu/kg dose. We first performed ICP-MS analysis on the stock
solutions of Cu(gtsm) and Gal-Cu(gtsm) to confirm that we
are indeed administering equivalent copper doses between the
ionophores (Figure S9). We collected blood and harvested
organs at two different time points (6 h, 24 h) to study the
time dependence of copper content and distribution across
organs, then processed tissues for ICP-MS analysis. Gal-
Cu(gtsm) supplementation results in a 300% increase in liver
copper levels after 6 h, with no significant differences in Cu
levels in the other extracted organs compared to basal
conditions (Figure 3D). Half of this copper is excreted 24 h
following Gal-Cu(gtsm) supplementation, with a 150%
increase in liver copper relative to basal levels. Interestingly,
copper levels in serum and other organs remain at basal levels
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selectively to the liver in whole animal settings, likely through
an ASGPR-mediated pathway.
levels after Cu(gtsm) treatment (Figure 4B). Likewise, the
TBARS assay, which quantifies byproducts such as malondial-
dehyde generated from lipid peroxidation during oxidative
tissue damage, indicates there is significant oxidative-stress-
induced liver damage in Cu(gtsm)-treated mice compared to
Gal-Cu(gtsm) or vehicle-treated mice (Figure 4C). In both
assays, liver health recovers to some extent after 24 h, but there
are still significant elevations in ALT activity and TBARS levels
relative to basal conditions (Figure S14).
Evaluating Toxicity Differences between Targeted
Gal-Cu(gtsm) and Untargeted Cu(gtsm) Supplements.
Despite the fact that Gal-Cu(gtsm) delivers ca. 4 times more
hepatic copper than Cu(gtsm) at an equivalent dose, we find
that Gal-Cu(gtsm) treatment is nontoxic, while Cu(gtsm)
treatment incurs significant hepatocellular injury, as indicated
by liver histology and toxicity assays (Figure 4). Indeed,
Finally, we investigated off-target toxicity using blood urea
nitrogen (BUN) assays, which are commonly elevated under
conditions of kidney damage. Figure 4D shows elevated BUN
levels for Cu(gtsm), but not for Gal-Cu(gtsm), treatment,
which further supports the proposal that targeted copper
delivery via Gal-Cu(gtsm) does not promote damage due to
off-target copper accumulation. Indeed, previous studies by
Cater et al. found significant kidney necrosis occurring upon
long-term administration of Cu(gtsm).36 These studies
indicate that targeted copper supplementation via Gal-
Cu(gtsm) is nontoxic, whereas untargeted supplementation
via Cu(gtsm) incurs hepatic and renal damage.
Selective Upregulation of Copper Trafficking and
Storage Proteins as a Mechanism for Minimizing
Copper-Induced Toxicity. We speculate that the lack of
toxicity resulting from copper supplementation using Gal-
Cu(gtsm) compared to its generic nontargeted Cu(gtsm)
counterpart originates from their disparate mechanisms for
delivery, as hypothesized in Figure 1. In the case of Cu(gtsm)
and other conventional copper ionophores, the metal
complexes passively diffuse through the cell membrane and
release copper upon encountering the reducing cytosolic
medium.65,77 This process is unregulated, and the inability of
the cell to buffer such rapid excesses of cytosolic copper likely
promotes oxidative stress and damage via Fenton-like
chemistry and other redox-mediated pathways. Meanwhile, it
is well-established that ASGPR-mediated delivery occurs
through clathrin-mediated endocytosis, where ASGPR-ligand
recognition is followed by endolysosomal processing.47 In this
case, copper release from Gal-Cu(gtsm) is then compartmen-
talized and localized to endosomes and lysosomes, in contrast
to unregulated Cu+ release in the cytosol. Importantly, there is
mounting evidence that the lysosome is a central organelle for
copper storage and homeostasis from algae to mammals.44,45
Copper delivery via Gal-Cu(gtsm) may then conceivably occur
with far greater control, where the lysosome can recruit
copper-trafficking proteins in a regulated manner based on
homeostatic cues within the cell.44−46,78
To test this prediction, we performed Western blot analysis
on mouse liver lysates to investigate how these different routes
of copper supplementation may affect key players in copper
storage and trafficking. Figure 5 shows SDS/PAGE analysis of
liver lysates from mice treated with vehicle, Cu(gtsm), and
Gal-Cu(gtsm) after 6 or 24 h of treatment at equal protein
loading. Expanded blots for all proteins are provided in Figure
S15. We first blotted for ATP7B, the major copper export
protein in the liver, and found increased ATP7B expression at
both time points for Cu(gtsm) and Gal-Cu(gtsm). This
observation is consistent with the known role of ATP7B to
facilitate the removal of excess hepatic copper via biliary
excretion.46,79 Next, we investigated the expression patterns of
CTR1, a copper-trafficking protein that imports copper into
the cell from the extracellular matrix and transports copper
from the lysosome to the cytosol in concert with CTR2.45,78
Figure 4. Gal-Cu(gtsm) treatment is nontoxic despite delivering more
copper than Cu(gtsm). (A) Representative liver tissue slices from
H&E staining show significant hydropic degeneration (wispy/white
cytosolic areas surrounding nuclei) upon Cu(gtsm) treatment. Liver
sections were isolated six hours following vehicle or 0.75 mg Cu/kg
mouse ionophore i.p. injections. Scale bar = 100 μm. (B−D) Toxicity
assays were performed on serum (B, D) or liver lysate (C) collected
from mice treated with Cu(gtsm) or Gal-Cu(gtsm) at 0.75 mg Cu/kg
mouse after 6 h to evaluate liver (B, C) and kidney (D) toxicity. Error
bars = SEM (n = 5). Statistical analyses were performed with a one-
way ANOVA with Bonferroni’s multiple comparisons test where *P ≤
0.05, ****P ≤ 0.0001.
hematoxylin and eosin (H&E) staining on liver tissue slices
harvested from mice 6 h after ionophore treatment at 0.75 mg
Cu/kg mouse (Figure 4A) show that Gal-Cu(gtsm)-treated
liver slices largely resemble those of the vehicle-treated control
mice, whereas H&E histology reveals significant hepatocellular
damage upon Cu(gtsm) supplementation, with features
characteristic of hydropic degeneration that occurs upon
acute liver injury (Figure 4A).76
To further assess potential ionophore toxicity, we also
performed alanine transaminase (ALT) activity and liver
thiobarbituric acid reactive substances (TBARS) assays on
serum and liver lysate collected from mice treated with
Cu(gtsm) or Gal-Cu(gtsm) at the same Cu dose after 6 h
(Figure 4B,C). Serum ALT activity assays are commonly used
to evaluate hepatocellular injury and evaluate liver health, with
increased serum ALT activity indicating potential liver damage.
We find virtually the same serum ALT levels for Gal-Cu(gtsm)
compared to the vehicle control, but much higher serum ALT
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Figure 5. Western blots demonstrate how differences in copper delivery route affect copper storage and trafficking protein expression. SDS/PAGE
analysis of liver extracts from mice (n = 2, 3) sacrificed either 6 or 24 h after treatment with vehicle, Cu(gtsm), or Gal-Cu(gtsm) (0.75 mg Cu/kg
mouse). Tissues were probed for ATP7B, CTR1, and MT-I/II.
We find increased expression of both the monomer and
truncated forms of CTR1 at both 6 and 24 h time points for
Gal-Cu(gtsm) supplementation alone. Thiele and co-workers
have previously suggested that the truncated CTR1 form may
play an important role in copper transport from the lysosome
to the cytosol.78 Because we observe increased CTR1
expression only under conditions of endolysosomal Cu
delivery, we hypothesize that CTR1 plays a role in copper
transport from the lysosome to the cytosol. Finally, metal-
lothioneins (MTs) are key metal storage proteins in the
cytosol. MT-I/MT-II expression increases dramatically upon
Cu(gtsm) and Gal-Cu(gtsm) treatment after 6 and 24 h. Blots
for the copper chaperone for superoxide dismutase (CCS) did
not demonstrate any clear differences between treatment
conditions (Figure S15). Further studies are necessary to more
conclusively disentangle the relationship between Gal-Cu-
(gtsm) uptake and lysosomal homeostatic copper regulation
and will be the subject of future investigation.
interest. We envisage that the TIMS strategy will facilitate both
fundamental and therapeutic applications. The site-specific
nature of metal delivery may enable us to uncover and
elaborate upon the roles that metals play in cellular signaling
and function in specific organs of interest at the animal level.
As one possible example, Gal-Cu(gtsm) could be used as a tool
for studying the complex relationships between copper and
lipid signaling in the liver.25 Likewise, pairing copper
ionophores with other tissue-specific targeting groups80 should
further our understanding of the diverse roles that copper plays
in different tissue and organ systems.
From a therapeutic perspective, TIMS offers a potential
starting point to addressing metal deficiencies in a broad range
of diseases with minimal off-target effects. Studies are currently
underway in our laboratories to utilize Gal-Cu(gtsm) to study
the potential roles and impact of liver-targeted copper
supplementation in NAFLD and other metabolic disorders.
Gal-Cu(gtsm) may also find potential use in treating
hepatocellular carcinomas with minimal off-target effects, as
cancer cells demonstrate heightened sensitivity to copper-
induced toxicosis.77,81,82 Beyond the liver, one could use TIMS
to address diseases in organs such as adipose tissue, heart, and
brain. Indeed, our laboratory recently discovered that dynamic
copper binding to PDE3B activates lipolysis in adipose tissue.12
As such, this TIMS strategy offers a general approach to open
new areas for metals in medicine.
CONCLUDING REMARKS
■
In this report, we have introduced the concept of targeted
ionophore-based metal supplementation, a general strategy to
deliver metals in a site-specific manner within living organisms.
As an initial proof of concept demonstration of this approach,
we synthesized a hepatic copper delivery agent and validated
liver-directed copper supplementation in cell culture and mice
through in vivo bioluminescence imaging and ex vivo tissue
metal analysis via ICP-MS. We showed that the targeted Gal-
Cu(gtsm) ionophore brings far more copper to the liver and
minimal Cu to other organs compared to the generic
untargeted Cu(gtsm) ionophore. Moreover, receptor-mediated
metal supplementation proceeds in a nontoxic manner at the
investigated copper dose relative to Cu(gtsm). This work
establishes that the TIMS approach is a viable strategy for
delivering metal nutrients in a site-specific manner with
minimal off-target metal accumulation.
EXPERIMENTAL DETAILS
■
General Synthetic and Characterization Methods. Reactions
using air- or moisture-sensitive reagents were conducted in flame-
dried glassware under an inert atmosphere of N2. When dry solvent
was required, solvent was passed over activated alumina prior to use.
All commercially purchased chemicals were used as received without
further purification. 4,4-Dimethyl-3-thiosemicarbazide was purchased
from TCI America. All other chemicals and solvents were purchased
from Sigma-Aldrich. Cu(gtsm),66 CCL-1,27 HO-TEG-N3,83 and
oxazoline 5,70 were synthesized according to previously reported
procedures. Silica gel P60 (SiliCycle) was used for column
chromatography, and SiliCycle 60 F254 silica gel (precoated sheets,
0.25 mm thick) was used for analytical thin layer chromatography. 1H
and 13C NMR spectra were collected at 298 K in deuterated solvents
The modular nature of the targeted ionophore building
blocks should enable the delivery of other metals and metal-
based probes and therapeutics to other organs and sites of
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from Cambridge Isotope Laboratories (Cambridge, MA, USA) at 25
°C on Bruker AV-300, AVQ-400, AVB-400, AV-500, DRX-500, or
AV-600 instruments at the College of Chemistry NMR Facility at the
University of California, Berkeley. Chemical shifts for protons are
reported in parts per million downfield from tetramethylsilane and are
referenced to residual protium in the NMR solvent (CHCl3: δ 7.26;
CH3OH: δ 3.31, DMSO: δ 2.50). Chemical shifts for carbon are
reported in parts per million downfield from tetramethylsilane and are
referenced to the carbon resonances of the solvent (CDCl3: δ 77.16;
DMSO: δ 39.52). Data are represented as follows: chemical shift,
multiplicity (s = singlet, d = doublet, dd = doublet of doublets, t =
triplet, m = multiplet), coupling constants in hertz, and integration.
Low-resolution electrospray mass spectral analyses were performed
using an LC-MS (Advion Expression-L Compact MS, ESI source).
High-resolution mass spectral analyses (ESI-MS) were carried out at
the QB3 Mass Spectrometry Facility at the University of California,
Berkeley.
(E)-2-(2,2-Dimethoxyethylidene)-N-methylhydrazine-1-car-
bothioamide (2). Synthesis of 2 was adapted from previously
reported procedures.69 4-Methyl-3-thiosemicarbazide (1.00 equiv,
10.35 g, 98.4 mmol) was added to a 1 L round-bottom flask equipped
with a stir bar followed by methanol (500 mL). 2,2-Dimethox-
yacetaldehyde (60% wt solution in water) (1.01 equiv, 15.0 mL, 99.43
mmol) was added via syringe, and the resulting solution was stirred
overnight, after which a significant white precipitate developed.
Volatiles were removed in vacuo to yield 2 as a white solid (18.9 g,
quantitative yield). 1H NMR (400 MHz, CDCl3): δ 9.20 (s, 1H), 7.37
(s, 1H), 7.04 (d, J = 4.9 Hz, 1H), 4.80 (d, J = 4.9 Hz, 1H), 3.39 (s,
6H), 3.20 (d, J = 4.9 Hz, 3H). 13C NMR (151 MHz, DMSO-d6): δ
178.28, 140.78, 102.08, 53.44, 30.88. ESI-MS(+): calcd for
C6H13N3O2SNa [M + Na]+ m/z 214.1; found 214.6.
(E)-N-Methyl-2-(2-oxoethylidene)hydrazine-1-carbothioa-
mide (3). Acetal 2 (1.00 equiv, 5.63 g, 29.4 mmol) and lithium
tetrafluoroborate (2.00 equiv, 5.52 g, 58.8 mmol) was added to a 2 L
round-bottom flask equipped with a stir bar. Acetonitrile (750 mL)
was added to the flask, followed by water (15 mL). The reaction
mixture was stirred at room temperature for 6 h, during which the
reaction mixture transformed from a neon yellow to orange color. The
reaction mixture was concentrated, then diluted with ethyl acetate
(300 mL). The organic layer was washed with saturated NaHCO3 (50
mL), water (50 mL), and brine (50 mL), dried over Na2SO4, filtered,
and then concentrated in vacuo. The resulting brown solid was
sonicated and extracted with hexane/ethyl acetate, 1:1, v/v (400 mL),
then filtered. The filtrate was concentrated in vacuo to obtain 3 as an
orange powder (3.41 g, 81% yield). 1H NMR (600 MHz, DMSO-d6):
δ 12.24 (s, 1H), 9.46 (d, J = 7.8 Hz, 1H), 9.00 (d, J = 5.7 Hz, 1H),
7.44 (dd, J = 7.8, 1.1 Hz, 1H), 3.01 (d, J = 4.6 Hz, 3H). 13C NMR
(151 MHz, DMSO): δ 191.30, 178.33, 138.58, 39.52, 31.17. ESI-MS
could not be obtained.
TEG-N3 (1.10 equiv, 1.93 g, 11.00 mmol), and dichloroethene
(DCE) (60 mL) were added to a 200 mL round-bottom flask
followed by a scoop of activated 4 Å molecular sieves. After stirring
for 5 min, trimethylsilyl trifluoromethanesulfonate (0.50 equiv, 0.91
mL, 5.01 mmol) was added via syringe to the reaction mixture and
stirred overnight. The reaction mixture was filtered, and the filtrate
was washed with a saturated sodium bicarbonate solution. The
organic layer was dried with Na2SO4, filtered, concentrated in vacuo,
and then purified with silica gel chromatography (ethyl acetate to 9/1
ethyl acetate/methanol) to yield 6 as a pale orange oil (3.65 g, 72%
yield). 1H NMR (600 MHz, CDCl3): δ 6.14 (d, J = 9.3 Hz, 1H), 5.32
(dd, J = 3.4, 1.1 Hz, 1H), 5.05 (dd, J = 11.2, 3.4 Hz, 1H), 4.78 (d, J =
8.6 Hz, 1H), 4.22 (dt, J = 11.2, 8.9 Hz, 1H), 4.19−4.10 (m, 2H),
3.93−3.83 (m, 3H), 3.76−3.65 (m, 4H), 3.63 (dd, J = 7.1, 3.2 Hz,
4H), 3.51−3.39 (m, 2H), 2.15 (s, 3H), 2.04 (s, 3H), 1.99 (s, 3H),
1.98 (s, 3H). 13C NMR (151 MHz, CDCl3): δ 170.54, 170.42, 170.39,
170.29, 102.15, 71.56, 70.82, 70.64, 70.56, 70.32, 69.72, 68.51, 66.65,
61.50, 50.73, 50.47, 23.12, 20.68, 20.62, 20.61. ESI-MS(+): calcd for
C20H32N4NaO11 [M + Na]+ m/z 527.2, found 527.7.
N-((2R,3R,4R,5R,6R)-2-(2-(2-(2-Aminoethoxy)ethoxy)-
ethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-
pyran-3-yl)acetamide (7). Compound 6 (1.00 equiv, 1.36 g, 2.70
mmol) was added to a 100 mL round-bottom flask equipped with a
stir bar and dissolved in methanol (15 mL). Palladium (10%) on
activated carbon (∼50 mg) was slowly added to the flask, which was
subsequently placed under vacuum and backfilled with hydrogen gas
three times. The reaction mixture was stirred under a hydrogen gas
balloon for 20 h. The reaction mixture was filtered over Celite, which
was then rinsed with methanol (40 mL). The filtrate was concentrated
to yield 7 as a pale yellow semisolid (0.88 g, 92% yield), which was
then carried forward to the next step. 1H NMR (500 MHz, CD3OD):
δ 4.39 (d, J = 8.4 Hz, 1H), 4.01−3.87 (m, 2H), 3.83 (d, J = 3.4 Hz,
1H), 3.80−3.54 (m, 12H), 3.49 (t, J = 6.2 Hz, 1H), 2.91 (t, J = 5.3
Hz, 1H), 1.98 (s, 3H). 13C NMR (151 MHz, MeOD): δ 170.38,
103.55, 76.62, 73.00, 71.32, 71.21, 71.00, 70.81, 69.92, 69.64, 62.52,
53.94, 49.58, 23.16. ESI-MS(+): calcd for C14H29N2O8 [M + H]+ m/z
353.2, found 353.4.
apo-Gal-H2 gtsm. Compound 4 (1.00 equiv, 0.56 g, 2.27 mmol),
compound 7 (1.10 equiv, 0.88 g, 2.50 mmol), and acetonitrile (100
mL) were added to a 250 mL round-bottom flask equipped with a
reflux condenser and stir bar. The reaction mixture was refluxed at 82
°C overnight, in which the reaction mixture turned from a light
orange to brown color. The reaction mixture was cooled to room
temperature, and Celite (10 g) was added to the flask. The contents
were then concentrated in vacuo. The crude material loaded onto
Celite was subjected to flash silica gel chromatography (100%
dichloromethane (DCM) to 9/1 DCM/MeOH to 4/1 DCM/MeOH
gradient) to afford apo-Gal-H2 gtsm as a beige solid (436 mg, 32%
yield). 1H NMR (400 MHz, DMSO-d6): δ 11.80 (d, J = 18.1 Hz, 2H),
8.51 (d, J = 4.7 Hz, 1H), 8.43 (t, J = 5.8 Hz, 1H), 7.73 (s, 2H), 7.61
(d, J = 9.0 Hz, 1H), 4.62−4.55 (m, 3H), 4.50 (d, J = 4.3 Hz, 1H),
4.28 (d, J = 8.4 Hz, 1H), 3.78 (dt, J = 10.1, 4.2 Hz, 1H), 3.74−3.62
(m, 5H), 3.61−3.38 (m, 15H), 3.32−3.23 (m, 1H), 2.96 (d, J = 4.5
Hz, 3H), 1.80 (s, 3H). 13C NMR (101 MHz, DMSO-d6): δ 177.57,
177.05, 169.63, 140.51, 140.00, 101.40, 75.36, 71.66, 69.81, 69.66,
68.30, 67.67, 67.60, 60.55, 52.03, 43.15, 39.52, 30.99, 23.17. ESI-
MS(−): calcd for C19H34N7O8S2 [M − H]− m/z 552.2; found 552.4.
Gal-Cu(gtsm). Compound 8 (1.00 equiv, 0.24 g, 0.43 mmol) and
copper acetate (1.02 equiv, 87 mg, 0.44 mmol) were added to a 25
mL round-bottom flask equipped with a stir bar. Anhydrous DMF
(4.7 mL) was added, and the reaction mixture immediately adopted a
crimson red color. The resulting solution was stirred at room
temperature overnight. The reaction mixture was concentrated in
vacuo, and ethanol (2.5 mL) was added to precipitate a dark red solid,
which was then frit filtered and rinsed with ethanol (2.5 mL) and
diethyl ether (2 × 5 mL), giving Gal-Cu(gtsm) as a dark red powder
(192 mg, 73% yield). High-resolution ESI-MS(+): calcd for
C19H34CuN7O8S2 [M + H]+ m/z 615.1201, found 615.1202.
(E)-N,N-Dimethyl-2-((E)-2-(2-(methylcarbamothioyl)-
hydrazinylidene)ethylidene)hydrazine-1 Carbothioamide (4).
Compound 3 (1.00 equiv, 1.70 g, 11.71 mmol), 4,4-dimethyl-3-
thiosemicarbazide (1.05 equiv, 1.47 g, 12.30 mmol), activated 4 Å
MS, and DMF (50 mL) were added to a 100 mL round-bottom flask
equipped with a stir bar and condenser. The reaction mixture was
stirred at 60 °C overnight under nitrogen to yield a dark brown
solution. The reaction mixture was filtered to remove sieve dust and
other particulates, then distilled to remove dimethylformamide
(DMF). The resulting crude brown solid was sonicated and washed
with ethanol (3 × 20 mL) and methanol (1 × 3 mL) to yield 4 as a
beige powder (1.08 g, 38% yield). Subsequent filtrate precipitation at
−20 °C yielded additional material albeit of reduced purity. 1H NMR
(400 MHz, DMSO-d6): δ 11.67 (s, 1H), 11.18 (s, 1H), 8.42 (d, J =
4.8 Hz, 1H), 7.88 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 8.5 Hz, 1H), 3.25
(s, 6H), 2.97 (d, J = 4.5 Hz, 3H). 13C NMR (101 MHz, DMSO): δ
180.11, 177.54, 142.26, 140.66, 41.94, 39.52, 30.94. ESI-MS(−): calcd
for C7H13N6S2 [M − H]− m/z 245.1; found 245.2.
(2R,3R,4R,5R,6R)-5-Acetamido-2-(acetoxymethyl)-6-(2-(2-
(2-azidoethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl-
diacetate (6). Compound 5 (1.00 equiv, 3.3 g, 10.02 mmol), HO-
Cell Culture Procedures. Cells were maintained by the UC
Berkeley Tissue Culture Facility. HEK 293T and HepG2 cells were
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maintained as a monolayer in exponential growth at 37 °C in a 5%
CO2 atm. HEK 293T cells were maintained in Dulbecco’s modified
Eagle medium (DMEM, Gibco) supplemented with 10% fetal bovine
serum (FBS, Hyclone) and Glutamax (Gibco). HepG2 cells were
maintained in low-glucose DMEM with L-glutamine and sodium
pyruvate (Gibco). One day before ionophore treatment, cells were
passaged and plated in DMEM with Glutamax supplemented with
10% FBS on either poly D-lysine-coated (HEK 293T) or gelatin-
coated (HepG2) sterile six-well Corning polystyrene plates. Cells
were grown to 60−80% confluency prior to ionophore treatment.
Cellular Ionophore Treatment and ICP-MS Assays. Cells were
washed twice with serum-free DMEM. Stock solutions of Cu(gtsm)
or Gal-Cu(gtsm) (2 mM) were diluted in serum-free DMEM to a
final ionophore concentration of 4 μM. A 2 mL amount of vehicle
(0.2% DMSO in DMEM), 4 μM Cu(gtsm), or 4 μM Gal-Cu(gtsm)
was then added to the six-well plates and incubated for three hours.
For the galactose competition experiment, cells were washed twice
with serum-free DMEM, then incubated with 2 mL of serum-free
DMEM either with or without 1 M D-galactose (Sigma-Aldrich) for
15 min. A 4 μL amount of DMSO, 2 mM Cu(gtsm), or 10 mM Gal-
Cu(gtsm) was diluted with 300 μL of cell media, which was then
mixed back into each well. Cells were then incubated for one hour.
Cells were then rinsed twice with ice-cold ethylenediamine
tetraacetate (EDTA) (1 mM in 50 mM HEPES buffer, pH = 7.4)
to remove cell-surface-bound copper and rinsed twice with ice-cold 50
mM HEPES buffer (pH = 7.4), followed by the addition of 215 μL of
concentrated nitric acid (BDH Aristar Ultra). The plates were sealed
with Parafilm and incubated on a shaker overnight. Samples (150 μL)
were further diluted in 2 mL of 2% nitric acid (made freshly from
concentrated nitric acid and Milli-Q water) in 15 mL tubes (Sarstedt)
and analyzed on a Thermo Fisher iCAP Qc ICP mass spectrometer in
kinetic energy discrimination (KED) mode against a standard curve of
known copper and phosphorus concentrations (CMS-5, Inorganic
Ventures), with Ga (20 μg/L, Inorganic Ventures) as an internal
standard. Each experiment was carried out twice, and each condition
was repeated in at least triplicate.
Xenogen IVIS Spectrum and imaged for 40 min under 2% isoflurane
anesthesia to characterize ionophore-treated liver signal response.
Tissue Harvesting and Serum Isolation. FVB-luc+ mice were
heavily anesthetized, and blood was collected via cardiac puncture.
Mice were immediately euthanized by cervical dislocation. Tissues
were harvested, rinsed twice with DPBS, snap-frozen under liquid
nitrogen, placed on dry ice in cryotubes, and stored at −80 °C until
analysis. Serum was isolated by allowing blood samples to coagulate
for 1 h at room temperature, centrifuging at 1500g for 15 min at 4 °C,
then collecting the serum supernatant. Samples were aliquoted, snap-
frozen, and stored at −80 °C until analysis.
Tissue Copper Analysis with ICP-MS. Portions of 20−100 mg
of the harvested tissues were digested in concentrated nitric acid (100
mg tissue/mL HNO3, BDH Aristar Ultra) at 95 °C for 2 h in 1.5 mL
tubes (Sarstedt) with small holes poked in the caps with an 18G
needle. After overnight incubation at room temperature, samples were
diluted into freshly prepared 2% nitric acid and doped with a gallium
internal standard (Inorganic Ventures, diluted from 1 ppm in 2%
nitric acid to a 20 ppb final concentration). The copper content was
determined by measuring 63Cu using a Thermo Fisher iCAP-Qc ICP-
MS in KED mode. Measurements were normalized to a standard
curve of known copper concentrations doped with 20 ppb Ga. The
standard curve was diluted from CMS-5 (Inorganic Ventures).
Liver Tissue Histology. FVB-luc+ mice were injected i.p. with
vehicle (50 μL 1:1 DMSO/DPBS), Cu(gtsm), or Gal-Cu(gtsm) at a
0.75 mg Cu/kg mouse dose. After six hours, mice were euthanized
with CO2 asphyxiation followed by cervical dislocation, and liver
tissue was extracted. Liver sections were fixed in a 10% formalin in
PBS solution and sent to Histowiz Inc. (Brooklyn, NY, USA) for
further processing and hematoxylin and eosin staining.
Liver Tissue Lysis. Frozen mouse livers were minced into 50 mg
samples on dry ice and homogenized in ice-cold RIPA buffer (25 mM
Tris·HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium
deoxycholate, 0.1% sodium dodecyl sulfate (SDS)) containing
protease inhibitor cocktail with EDTA (Roche) at 100 mg/mL
using a hand-held mechanical homogenizer. Homogenates were
incubated on ice for 30 min and centrifuged at 12000g for 20 min at 4
°C. The soluble protein lysates were collected from underneath the
upper lipid layer with a pipet and transferred to new tubes. Protein
concentration was determined using a detergent-compatible Bradford
Assay (Pierce).
Toxicity Assays of Liver Lysate and Serum. Liver lysate
(TBARS assay via TCA method, Cayman Chemical) and serum
(BUN assay, Invitrogen; ALT assay, Cayman Chemical) samples were
processed according to the manufacturers’ instructions; sample
concentration was selected based on initial dilution screening to fall
within a linear range of the standard curve. All samples were analyzed
in triplicate.
Western Blot Analysis. Protein lysates were denatured in
NuPAGE lithium dodecyl sulfate sample buffer (Invitrogen)
containing 10% v/v β-mercaptoethanol as a reducing agent. The
samples (20 μg for CCS, CTR1, ATP7B; 50 μg for MT) were
resolved by SDS-PAGE using 15-well or 17-well NuPAGE 4−12%
Bis-Tris gels (Invitrogen) with MES SDS running buffer (Invitrogen)
with 10 μL of loading sample. Proteins were transferred to a
polyvinylidene difluoride membrane (BioRad, Munich, Germany)
with the use of the Trans-Blot Turbo transfer system (BioRad,
Munich, Germany). The membranes were blocked in 5% nonfat dry
milk in TBST buffer (10 mM Tris, pH 7.5, 100 mM NaCl, 0.1%
Tween-20) for 1 h at room temperature. After blocking, the
membranes were incubated at 4 °C overnight with primary antibodies
diluted with TBST buffer containing 5% bovine serum albumin
(BSA). The anti-ATP7B (NB100-360, Novus Biologics) and anti-
metallothionein (sc-11377, Santa Cruz Biotechnology) were used at
1:250 dilution. The anti-CTR1 antibody (13086, Cell Signaling
Technology) was used at 1:1000 dilution. The anti-CCS (sc-20141,
Santa Cruz Biotechnology) was used at 1:500 dilution. The
membranes were washed three times for 5 min in TBST and
incubated for 1 h at room temperature with horseradish peroxidase
(HRP)-conjugated anti-rabbit IgG secondary antibody (sc-2004,
Animals. FVB-luc+ (FVB-Tg (CAG-luc,-GFP)L2G85Chco/J)
mice were obtained from our in-house breeding colony. Mice were
group housed on a 12:12 h light−dark cycle at 22 °C with free access
to food and water. All animal studies were approved by and
performed according to the guidelines of the Animal Care and Use
Committee of the University of California, Berkeley.
General Animal Imaging Methods and Data Analysis. A
Xenogen IVIS Spectrum instrument (Caliper Life Sciences) was used
for bioluminescence imaging in all animal experiments, and image
analysis was performed using the Living Image software. The total
photon flux for each animal was determined by drawing a region of
interest around the liver and integrating photon flux over the total
imaging period (area under the curve). We selected our liver region of
interest based on comparing the bioluminescence data in vivo and ex
vivo, as described in our previous studies with the CCL-1 probe.27
The same region of interest around the liver was applied to analyzing
both the CCL-1 and D-Luc data. The data plotted in Figure 3A
represent the ratio of CCL-1 integrated photon flux to basal D-luc
integrated photon flux. Mice were anesthetized prior to injection and
during imaging via inhalation of isoflurane. Isoflurane was purchased
from Phoenix Pharmaceuticals, Inc. DMSO was purchased from
Sigma-Aldrich, Dulbecco’s phosphate-buffered saline (DPBS) was
purchased from Gibco, and medical-grade oxygen was purchased from
Praxair.
In Vivo Imaging with CCL-1. FVB-luc+ mice were given i.p.
injections of vehicle (50 μL 1:1 DMSO/DPBS), 3.46 mg/kg
Cu(gtsm), or 7.26 mg/kg Gal-Cu(gtsm) under anesthesia with
isoflurane. The ionophore concentrations were chosen to give an
equivalent copper dose of 0.75 mg Cu/kg mouse at 0.6 mg Cu/mL
vehicle. Six hours later, the same mice were anesthetized and
subjected to scapular s.c. injection of CCL-1 (0.1 μmol in 50 μL
DMSO/150 μL DPBS) or D-luciferin (0.1 μmol in 50 μL DMSO/150
μL DPBS). Five minutes after s.c. injection, mice were transferred to a
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Santa Cruz Biotechnology) at a 1:2000 dilution in TBST containing
5% BSA. The membranes were washed five times for 5 min in TBST,
then visualized using enhanced chemiluminescence (Western Lighting
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.8b08014.
Additional experimental details (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
*astahl@berkeley.edu
*chrischang@berkeley.edu
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ORCID
Timothy A. Su: 0000-0001-5934-3292
Marie C. Heffern: 0000-0001-7501-2741
Christopher J. Chang: 0000-0001-5732-9497
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#Department of Chemistry, University of California, Davis,
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Alison Killilea and Carissa Tasto at UC Berkeley’s
Cell Culture Facility for expert technical assistance. We thank
National Institutes of Health (NIH) (Grants GM79465 and
ES4705 to C.J.C. and Grant R01CA221916 to A.S.) for
support. T.A.S. thanks the NIH Ruth L. Kirschstein National
Research Service Award (Grant F32 GM122248) for support
and Dr. Lakshmi Krishnamoorthy for helpful discussions. W.C.
was supported by the UC Berkeley Amgen Scholars Program.
M.C.H. was supported by the University of California
President’s Postdoctoral Program. T.D. was partially supported
by NIH Chemical Biology Training Grant T32 GM066698.
C.J.C. is an Investigator with the Howard Hughes Medical
Institute.
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