Chemoproteomic Profiling Reveals Ethacrynic Acid Targets Adenine Nucleotide Translocases to Impair Mitochondrial Function

Article abstract of DOI:10.1021/acs.molpharmaceut.8b00250

Ethacrynic acid (EA) is a diuretic drug that is widely used to treat high-blood pressure and swelling caused by congestive heart failure or kidney failure. It acts through noncovalent inhibition of the Na⁺-K⁺-2Cl^- cotransporter in the thick ascending limb of Henle's loop. Chemically, EA contains a Michael acceptor group that can react covalently with nucleophilic residues in proteins; however, the proteome reactivity of EA remains unexplored. Herein, we took a quantitative chemoproteomic approach to globally profile EA's targets in cancer cells. We discovered that EA induces impaired mitochondrial function accompanied by increased ROS production. Our profiling revealed that EA targets functional proteins on mitochondrial membranes, including adenine nucleotide translocases (ANTs). Site-specific mapping identified that EA covalently modifies a functional cysteine in ANTs, a mutation of which resulted in the rescuing effect on EA-induced mitochondrial dysfunction. The newly discovered modes of action offer valuable information to repurpose EA for cancer treatment.

Full text of DOI:10.1021/acs.molpharmaceut.8b00250

Article
Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Chemoproteomic Profiling Reveals Ethacrynic Acid Targets Adenine
Nucleotide Translocases to Impair Mitochondrial Function
†
,‡,⊥
†,⊥
§
†
‡,§
,†,‡
Zi Ye,
Xiaoyun Zhang, Yuangang Zhu, Tong Song, Xiaowei Chen, Xiaoguang Lei,*
,
†,‡
and Chu Wang*
†Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic
Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University,
Beijing 100871, China
‡
Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China
§Institute of Molecular Medicine, Peking University, Beijing 100871, China
*
S Supporting Information
ABSTRACT: Ethacrynic acid (EA) is a diuretic drug that is widely used to treat high-
blood pressure and swelling caused by congestive heart failure or kidney failure. It acts
+
+
−
through noncovalent inhibition of the Na -K -2Cl cotransporter in the thick ascending
limb of Henle’s loop. Chemically, EA contains a Michael acceptor group that can react
covalently with nucleophilic residues in proteins; however, the proteome reactivity of
EA remains unexplored. Herein, we took a quantitative chemoproteomic approach to
globally profile EA’s targets in cancer cells. We discovered that EA induces impaired
mitochondrial function accompanied by increased ROS production. Our profiling
revealed that EA targets functional proteins on mitochondrial membranes, including
adenine nucleotide translocases (ANTs). Site-specific mapping identified that EA
covalently modifies a functional cysteine in ANTs, a mutation of which resulted in the
rescuing effect on EA-induced mitochondrial dysfunction. The newly discovered modes
of action offer valuable information to repurpose EA for cancer treatment.
KEYWORDS: ethacrynic acid, Michael acceptor, chemical proteomics, adenine nucleotide translocases, cysteine
INTRODUCTION
a chemotherapy enhancing agent and has been combined with
anticancer drugs to reduce drug resistance during cancer
■
Ethacrynic acid (EA) is a diuretic drug developed over 50 years
7
,8
1
treatment. For example, EA was shown to improve the
antitumor effects of arsenic trioxide (ATO) in myeloid
leukemia and lymphoma cells by inhibiting GSTP1-1 and
ago. Because it can produce a prompt and profound diuresis,
the drug has been widely employed in the treatment of high-
blood pressure and edematous states caused by congestive
heart failure, liver failure, or kidney failure. The diuretic effect
of EA is mainly by forming a conjugate with free cysteines in
vivo and acting on the Na -K -2Cl cotransporter in the thick
9
2
inducing cell apoptosis.
EA has also been reported in individual cases to modify
cysteine side chains in proteins and affect their functions. For
example, EA was shown to inhibit the kinase activity MAP2K6
in part by covalently modifying a nonconserved cysteine
+
+
−
ascending limb of Henle’s loop to block the re-absorption of
3
NaCl. Chemically, the conjugation with free cysteines occurs
10
residue. It was able to react with the p65 subunit of NF-kB at
at the α,β-unsaturated ketone group via a Michael addition that,
in principle, can also react with other nucleophilic thiol groups
on biomolecules (Figure 1A).
Indeed, EA has been known to form a covalent complex with
reduced glutathione (GSH) spontaneously via its cysteine side
chain, depletion of which leads to a mounting level of oxidative
stress that can sensitize certain tumor cells to death. In
addition, the EA-GSH adduct was demonstrated to impose a
strong inhibition on the activity of members of the glutathione
11
Cys38 and inhibit its binding with DNA. EA could also
directly bind to the LEF-1 protein and destabilize its complex
formation with β-catenin, which in turn suppresses the
12
recruitment of LEF1 to promoter regions on DNA. These
studies have provided clues to explain certain biological effects
of EA; however, the full spectrum of EA’s reactivity in
proteome still remains uncharacterized.
4
Chemical proteomic strategies, such as activity-based protein
profiling (ABPP), have become an enabling tool to discover
5
S-transferase (GST) family. This class of detoxifying enzymes
is often overexpressed in cancer cells because it is critically
needed to catalyze the conjugation of GSH with xenobiotic
substrates (e.g., chemotherapeutic agents) to maintain a healthy6
level of reactive oxygen species (ROS) for tumors to survive.
Based on these properties, EA is currently being investigated as
Received: March 9, 2018
Revised: May 2, 2018
Accepted: May 4, 2018
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.molpharmaceut.8b00250
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Figure 1. EA induces noncanonical cell death with the deformation of mitochondrial ridges and an increasing level of intracellular ROS.(A)
Structures of EA and its control analogue, dihydroethacrynic acid, EA-H . (B) EA but not EA-H imposes dose-dependent cytotoxicity to A375 cells
2
2
as evaluated using the MTT assay (n = 6). (C) EA induces death of A375 cells lacking canonical markers of apoptosis (cleaved PARP) or autophagy
(
LC3-II) as revealed by immunoblotting analysis. Staurosporine (1 μM) and rapamycin (0−50 μM) were used to induce apoptosis and autophagy,
respectively, and actin was used as a loading control. (D) EA induces cell death (left) with the deformation of mitochondrial ridges (right) in A375
cells as visualized by electron microscopy. (E) EA induces cell death with the loss of MMP (Δψm) (JC-1 and JC-9 mitochondrial potential sensors,
D22421), increased mitochondrial ROS (MitoSOX red mitochondrial superoxide indicator, for live-cell imaging, M36008), increased cytosolic ROS
(
DCFH-DA, 2′-7′dichlorofluorescin diacetate, D6883), and increased lipid peroxidation (BODIPY 493/503, D3922). A375 cells were exposed to
EA (150 μM) for 24 h and analyzed with different fluorescent probes by flow cytometry (n = 3). ** and *** denote p < 0.01 and p < 0.001 in a
student’s t test.
and interrogate functional proteins directly in complex
EXPERIMENTAL METHODS
■
1
3
proteomes.
heightened intrinsic reactivity for functional annotation,
ABPP-based methods have also been developed to map the
In addition to identifying residues with
Gel-Based ABPP. The A375 cells were lysed using a probe
14,15
sonicator in 0.1%NP40/PBS buffer, and the lysate was
fractionated by centrifugation at 100 000g for 45 min. The
whole proteome was transferred to a separate microfuge tube.
Protein concentrations for each sample were determined using
the BCA protein assay (Pierce BCA Protein Assay Kit, 23225)
and then normalized to 2 mg/mL in a volume of 100 μL. The
lysates were incubated with inhibitor (EA, 500 μM) or DMSO
at room temperature in the dark for 1 h. Then the lysates were
labeled by the EA-probe (100 μM) at room temperature in the
dark for 1 h and reduced with 4 mM NaBH4 for 1 h at room
temperature. The proteins were then precipitated with
methanol−chloroform (methanol/aqueous phase/chloroform,
4:3:1 (v/v/v)). The precipitated protein pellets were
resuspended with 0.4% SDS/PBS in a 100 μL volume. Click
chemistry was performed on each sample using final
concentrations of 50 μM rhodamine azide, 1 mM Tris(2-
carboxyethyl)phosphine (TCEP, Sigma-Aldrich), 100 μM
Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA,
Sigma-Aldrich), and 1 mM CuSO4 in a final volume of 100
μL. The samples were incubated at room temperature for 1 h,
followed by the addition of 25 μL 5× of the SDS loading buffer.
The samples were then loaded (20 μL) and resolved on a 10%
16
sites of modification by endogenous metabolites, natural
1
7
18,19
products, or anticancer drugs
containing reactive
moieties. In the current work, we applied several ABPP-based
techniques to profile protein targets and residue sites that are
sensitive to covalent modification by EA. We discovered that
many EA-hyperactive proteins were localized on mitochondrial
membranes, including the ADP/ATP translocases. Biochemical
experiments validated that one functional cysteine in ANTs was
covalently targeted by EA with outstanding sensitivity, the
mutation of which exhibited protecting effect against EA-
induced cytotoxicity, including the deformation of mitochon-
drial ridges, an elevated level of mitochondrial ROS, and the
loss of mitochondrial membrane potential (MMP). Our data
suggested that EA imposes cytotoxicity by impairing
mitochondrial functions, and these novel proteins and pathways
that are discovered to be targeted by EA will provide valuable
information for improving its safety profile as well as to
repurpose the drug for new clinical applications.
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SDS-PAGE gel. The gels were scanned by fluorescense and
stained by Coomassie brilliant blue to demonstrate equal
loading.
Protein Identification and Quantification. LC-MS/MS
data were analyzed by ProLuCID with a static modification of
cysteine (+57.0215 Da) and a variable oxidation of methionine
(+15.9949 Da). For RD-ABPP data, the isotopic modifications
(28.0313 and 34.0631 Da for light and heavy labeling,
respectively) were set as variable modifications on the N-
terminal of a peptide and lysines. For competitive isoTOP-
ABPP data, the isotopic modifications (464.28596 and
Profiling of EA-Modified Proteins by RD-ABPP. Two
milligrams of lysates of A375 cells or HeLa cells was treated
with either DMSO or EA (200 μM) for 1 h at room
temperature in the dark, respectively, and then labeled with the
EA-probe (40 μM) for another 1 h. The lysate was reduced
with 4 mM NaBH4 for 1 h at room temperature in the dark.
The proteins were then precipitated with methanol−chloro-
form as previously described. The precipitated protein pellets
were resuspended with 0.4% SDS/PBS in a 1 mL volume. Click
chemistry was performed with 100 μM azide-biotin tags for 1 h,
and the proteins were then precipitated with methanol−
chloroform. The precipitated protein pellets were resuspended
and dissolved in 1.2% SDS/PBS in a 1 mL volume. The
proteomes were boiled at 90 °C for 5 min, and after
centrifugation at 1400g for 1 min at room temperature, the
supernatant was diluted to 0.2% SDS/PBS. The streptavidin
beads (Pierce Streptavidin Agarose, 20353) were washed with
PBS 3 times, and the supernatant was enriched with beads for 3
h at 29 °C. The beads were washed with PBS 3 times and
4
70.29977 Da for light and heavy labeling respectively) were
set as variable modifications on cysteines. The searching results
were filtered by DTASelect, and peptides were also restricted to
fully tryptic with a defined peptide false positive rate of 1%. The
quantitation of the reductive demethylation-labeled protein was
16,21
done by an in-house software CIMAGE, accordingly.
The A375 cell was gifted from Prof. Qingsong Liu’s lab from
the Chinese Academy of Sciences, Hefei, China, and the HeLa
cell was gifted from Prof. Chengqi Yi’s lab from Peking
University. The cells were authenticated by the China Center
for Type Culture Collection, Wuhan, China. Details for other
experimental methods can be found in the Supporting
Information.
RESULTS
washed with H O 3 times. The enriched proteins were digested
■
2
EA Induces Noncanonical Cell Death with Mitochon-
drial Dysfunction. We first evaluated the cytotoxicity of EA
on two cancer cell lines, A375 and HeLa cells. MTT
experiments showed that EA was able to induce cell death at
by trypsin on-bead in 100 mM TEAB buffer and subjected to
16
reductive dimethylation labeling. Finally, heavy and light
labeled peptide samples were mixed, concentrated, separated by
20
the Fast-seq protocol, and analyzed on a Q Exactive Plus mass
spectrometer (Thermo Fisher). For identifying EA-hyper-
reactive proteins in proteomes, the cell lysate was treated
with 10, 20, or 40 μM EA-probe for 1 h at room temperature in
the dark.
5
7.26 ± 6.6 μM for A375 cells and 127.2 ± 13.1 μM for HeLa
cells, respectively (Figures 1B and S1A). A control compound,
dihydroethacrynic acid EA-H , which lacks the double bond at
2
the α,β position (Figure 1A), did not induce any obvious cell
death up to 240 μM, which suggests that the Michael acceptor
portion of EA is critical for its cytotoxicity (Figures 1B and
S1A). We next investigated the nature of the cell death induced
by EA. Staurosporine and rampamycin are classical inducers of
apoptosis and autophagy, which result in cleaved PARP and
Mapping EA-Reactive Cysteines by Competitive iso-
TOP-ABPP. The A375 cells were lysed using a probe sonicator
in a PBS buffer, and the lysate was fractionated by
centrifugation at 100 000g for 45 min at 4 °C. The whole
proteome was transferred to a separate microfuge tube. Protein
concentrations for each sample were determined using the BCA
protein assay (BioRad) and then normalized to 2 mg/mL in a
volume of 1 mL. The different concentrations of EA (10, 20, or
22,23
LC3-II as distinct markers, respectively.
However, when
cells were incubated with EA, neither cleaved PARP nor LC3-II
was observed, excluding the possibility of canonical apoptosis
or autophagy (Figures 1C and S1B). To check if EA could
induce cell death through the necrosis pathway, we visualized
cell morphology using transmission electron microscopy
40 μM) or DMSO (control) were then added and incubated at
room temperature in the dark for 1 h. Each of the control and
EA-treated proteome samples was treated with 100 μM IA-
probe at room temperature in the dark for 1 h. Click chemistry
was performed by the addition of either 100 μM of the heavy-
TEV-tag (for control) or light-TEV-tag (for EA-treatment
sample), 1 mM TCEP, 100 μM TBTA, and 1 mM CuSO4 for 1
h at room temperature. After the click chemistry step, the
proteins were then precipitated with cooled methanol, and
heavy- and light-labeled protein samples were mixed. After the
(
TEM) (Figures 1D, S1C, S2, and S3). Under EA treatment
at 120 μM for 3 h, cells became rounded up, and a zoom-in
view showed that cell death was accompanied by mitochondria
deformation, where most of mitochondrial ridges disappeared
(
Figure 1D). This observation contradicts the expected
phenotype of necrosis, including cytoplasmic swelling and
24
plasma membrane rupture. Consistent with the observed
disappearance of mitochondrial ridges, EA led to a substantial
loss of MMP (Δψm) associated with increased mitochondrial
ROS, which confirmed that the normal function of mitochon-
dria was affected (Figures 1E and S1D). In addition to impaired
mitochondria, we also observed a significant increase in
cytosolic ROS and lipid peroxidation from the EA-treated
cells (Figures 1E and S1D). These results collectively suggested
that EA was able to induce a noncanonical type of cell death
other than apoptosis, necrosis, or autophagy with distinct
morphological changes on mitochondria.
Design and Synthesis of a Bioorthogonal EA-Probe.
To profile EA-reactive proteins in their native environment,
chemoproteomic probes provide a quite direct and powerful
approach for the target identification. Thus, the design and
100 μL streptavidin beads were washed with PBS 3 times, the
supernatant was enriched with beads for 3 h at 29 °C. The
enriched samples were subjected to sequential on-bead trypsin,
TEV digestion, and analyzed by LC-MS/MS on a Q-Exactive
plus mass spectrometer.
LC-MS/MS Analysis. The LC-MS/MS analysis was
performed on a Q-Exactive plus Orbitrap mass spectrometer
(
Thermo Fisher Scientific) coupled with an Ultimate 3000 LC
system. Briefly, the flow rate through the column was set to 0.3
μL/min, and the applied distal spray voltage was set to 2.8 kV.
MS/MS data collection was performed using one full scan
(
350−1800 M/Z), followed by data-dependent MS2 scans of
the 20 most abundant ions with dynamic exclusion enabled.
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Figure 2. Design and synthesis of a bioorthogonal EA-probe for profiling EA-reactive proteins. (A) Design and synthesis of a bioorthogonal EA-
probe (3) with the alkyne functional group. Reagents and conditions: (a) Ethyl bromoacetate, K CO , acetone, 50 °C, 3 h, and 98%; (b) 5-
2
3
bromovaleryl chloride, AlCl , CH Cl , 0 °C, 2 h, r.t., 48 h, and 60% brsm; (c) Ag O, acetone/H O (3/1), 60 °C, 48 h, and 60%; (d) DMP, CH Cl ,
3
2
2
2
2
2
2
1
h, and 80%; (e) dimethyl (1-diazo-2-oxopropyl)phosphonate, K CO , MeOH, 2 h, and 29%; and (f) 37 wt % HCHO, K CO , EtOH/H O (1/1),
2
3
2
3
2
overnight, and 46%. (B) The EA probe retains a similar cytotoxicity as EA as evaluated using the MTT assay (n = 6). (C) Concentration-dependent
labeling of the EA probe in proteomes can be stabilized by reduction with NaBH and completed by EA.
4
Figure 3. Chemproteomic profiling of EA-reactive proteins by RD-ABPP. (A) Scheme of profiling EA modified proteins by RD-ABPP with EA-
competitive probe labeling. (B) Ratio distribution of a representative RD-ABPP profiling experiment in which proteins with 0.9 ≤ R
≤ 1.1
probe vs probe
and R
≥ 2.0 are considered as EA-reactive proteins. (C) A total number of 277 EA-reactive proteins are commonly identified from two
DMSO vs EA
replicates of RD-ABPP experiments. (D) GO analysis of the EA-reactive proteins by cellular components. (E) GO analysis of the EA-reactive
proteins by molecular functions. (F) Extracted ion chromatograms of peptides from members of ANTs and VDACs families, which are localized on
mitochondrial membranes and involved in ADP/ATP transport. The EA probe labeling on these proteins can be completed by EA, indicating they
are EA-reactive targets. Heavy and light traces are colored in blue and red, respectively.
2
7
synthesis of a highly reactive chemical EA-probe is essential. We
considered to introduce a small-sized terminal alkyne to the EA
core, which allowed for a further enrichment or imaging step by
utilizing copper-catalyzed azide−alkyne cycloaddition
ability than that of the aromatic ring part and carboxyl group.
Thus, we designed the EA-probe with the alkyne group linking
to the alkyl chain (Figure 2A).
The synthesis of the chemical probe 3 commenced with O-
alkylation of the commercially available 2,3-dichlorophenol 4
using ethyl bromoacetate under basic conditions, which
afforded ester 5 in a high yield (Figure 2A). We initially failed
to directly install the terminal alkyne to 5 with hex-5-ynoyl
2
5,26
(
(
CuAAC).
As previous structure−activity relationship
SAR) studies on proliferation of cancer cells have shown,
the length of the alkyl chain linking to the α,β-unsaturated
ketone unit shows much less influence on the anti-proliferative
D
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Figure 4. Identification of EA-hyperactive proteins by RD-ABPP. (A) Scheme of identifying EA-hyperreactive proteins by RD-ABPP with saturated
probe labeling. (B) Ratio distributions for ∼1200 proteins quantified from RD-ABPP experiments, where proteomes were labeled with three pairwise
EA-probe concentrations (10:40, 20:40, and 40:40 μM). The plot was sorted by the ratios quantified from the 10:40 μM pairwise RD-ABPP
experiment (n = 2). (C) A total number of 115 proteins with saturated RD-ABPP ratios from all of the three pairwise RD-ABPP profiling
experiments (0.8 ≤ R1 ≈ R
≈ R
≤ 1.2) were defined as EA-hyper-reactive proteins. (D) GO analysis of the EA-hyperactive proteins by
0:40
20:40
40:40
molecular functions. (E) Quantified ratios for members from the ANTs and VDACs family (n = 2).
chloride through Friedel−Crafts acylation in the presence of
quantitative profiling of EA-reactive proteins using the EA-
probe by combining competitive ABPP with stable isotope
reductive dimethylation (RD-ABPP). Cell lysates were first
pre-incubated with 200 μM of EA or DMSO control,
respectively, and labeled by 40 μM of the EA-probe for 1 h.
AlCl . We next tried 5-bromovaleryl chloride and successfully
3
16
transformed 5 to bromide 6. Treatment of 6 with Ag O
2
smoothly generated alcohol 7, which was further transformed
to 8 by Dess−Martin oxidation in a good yield. With the
treatment of the Ohira−Bestmann reagent, aldehyde 8 was
smoothly transformed to the terminal alkyne 9. Finally, aldol
condensation of 9 in the presence of formaldehyde and K CO ,
Probe-labeled proteins were reduced with NaBH , conjugated
4
with biotin-azide, and enriched by streptavidin. After on-bead
trypsin digestion, the released tryptic peptides were isotopically
labeled by light (for the EA-treated sample) or heavy (for the
DMSO-treated sample) formaldehydes. The dimethylated
samples were combined and analyzed by liquid chromatog-
raphy (LC)-MS/MS (Figure 3A). For each identified peptide, a
heavy/light ratio was quantified, and for a given protein, a
quantitative ratio, R, was obtained by calculating the median
value of the ratios of all of the quantified peptides from the
same protein. We designated the proteins with R ≥ 2.0 as
potential EA-reactive targets (Figure 3B). To further remove
false-positive targets, we required that the EA-reactive proteins
should display a R ratio between 0.9 and 1.1 when the samples
were prepared without extra EA competition. We performed
two replicated experiments and designated the overlapping 277
targets as the final list of EA-reactive proteins (Figure 3C and
Table S1).
We perfomed the gene ontology (GO) analysis on these EA-
reactive proteins, regarding their cellular component and
molecular functions (Figure 3D,E). The top two clusters in
terms of molecular function are ATP:ADP antiporter activity
and adenine transmembrane transporter activity, which include
members from ADP/ATP translocase (ANT) and voltage-
dependent anion channels (VDAC) families localized on the
2
3
followed by ester hydrolysis, successfully generated the EA-
probe 3.
The MTT experiments showed that the EA-probe
maintained a similar cytotoxicity as EA with an IC50 at 54.4
±
7.4 μM (Figure 2B). We further evaluated the labeling
efficiency of the EA-probe in cell lysates by in-gel fluorescence.
Cell lysates were pre-incubated with a series of concentrations
of EA-probe for 1 h and then reduced with NaBH for another
4
hour in the dark. The probe-labeled proteins were conjugated
with a rhodamine-azide reporter group through CuAAC and
visualized on SDS-PAGE by in-gel fluorescence. We observed a
concentration-dependent EA-probe labeling, which could be
enhanced by the incubation with NaBH (Figure 2C). This
4
observation is consistent with the premise that NaBH could
4
reduce and stabilize the Michael adduct of the EA-probe
labeling. Furthermore, the EA-probe labeling could be
effectively completed by 5-fold more of EA (Figure 2C),
suggesting that they modify an overlapping set of cellular
proteins, and the EA-probe could be used as a suitable
surrogate to profile targets of EA in proteomes.
Mass Spectrometry (MS)-Based Quantitative Profiling
of EA-Reactive Proteins. We next performed MS-based
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Figure 5. Site-specific mapping of EA-reactive cysteines in proteomes by competitive isoTOP-ABPP. (A) Scheme of identifying EA-reactive cysteines
by competitive isoTOP-ABPP using a cysteine-reactive IA-probe. (B) Ratio distributions of proteins quantified from competitive isoTOP-ABPP
experiments, where IA-probe labeling of cell lysates were competed with 10, 20, or 40 μM of EA. Cys257 in ANT1/3 and ANT2 was quantified with
outstanding ratios, suggesting it is sensitive to EA modification. (0 < R10 μM < R20 μM < R40 μM ≤ 15). (C) Representative MS1 profiles for EA-
adducted peptides from ANTs peptides containing Cys257, whose IA-probe labeling showed a strong competition with EA. Heavy (untreated) and
light (EA-treated) traces are colored in blue and red, respectively. (D) Sequence alignment of all four members of ANTs in the human genome, with
the four highly conserved cysteine residues highlighted in red. (E) Validation of covalent modification of ANT3 by EA with immunoblotting. ANT
was overexpressed in HeLa cells with a 6xHis tag, and the cell lysates were first pre-incubated with 200 μM of EA or DMSO, respectively, and labeled
by 40 μM of EA-probe for 1 h. The probe-labeled proteins are enriched by streptavidin and immunoblotted with an anti-6xHis antibody (Output) (n
=
3). (F) Biochemical validation of EA-cysteine interactions in ANTs by immunoblotting. HeLa cells were stably overexpressed with wild-type (WT)
ANT3 or a C257A mutant with a 6xHis tag and labeled with 40 μM of EA-probe. The probe-labeled proteins are enriched by streptavidin and
immunoblotted with an anti-6xHis antibody (Output) (n = 3). In (E) and (F), equal loading is validated by immunoblotting of the input samples
before enrichment (Input), and *** denotes p < 0.001 in a student’s t test.
mitochondrial membrane (Figure 3F). The adenine transporter
works by exchanging ADP and ATP across the inner
mitochondrial membrane, resulting in a net export of ATP
from the mitochondrial matrix and an import of ADP into the
matrix. Under normal conditions, ATP and ADP bearing high
negative charges cannot freely cross the inner mitochondrial
membrane, and ANTs function couples the transport of the
two molecules. Human genome encodes four ANTs (ANT1−
a similar approach hoping to detect a subset of EA-hyper-
reactive cysteines directly. However, we only identified a very
few number of EA-adducted peptides (data not shown), which
reflects the challenge of analyzing Michael adducts by mass
spectrometry with collision-induced dissociation (CID) frag-
mentation. We instead applied this strategy at the protein level
and performed three parallel quantitative RD-ABPP experi-
ments using the EA-probe at pairwise concentrations of 10:40,
20:40, and 40:40 μM, respectively (Figure 4A). A light/heavy
ratio R closer to 1.0 indicates saturated labeling of a protein at a
lower probe concentration, suggesting that it is an EA-hyper-
2
8
4
) and our quantitative profiling identified ANT1, ANT2, and
2
9
ANT3, all of which showed clear EA-competitive labeling by
the probe (Figure 3F). VDACs are a class of porin ion channel
proteins located on the outer mitochondrial membrane, and
they form a general diffusion pore for small hydrophilic
molecules. The genomes of humans, as well as other higher
eukaryotes, encode three different VDACs (VDAC1−3), and
we identified VDAC2 and VDAC3 as EA-reactive proteins
reactive target. We applied the filter of 0.8 ≤ R
≈ R
≈
1
0:40
20:40
R
≤ 1.2 and identified 115 targets, which showed nearly
40:40
3
0
identical ratios around 1.0 at all three conditions tested (Figure
4B,C and Table S2). GO analysis of these EA-hyperactive
proteins again ranked the adenine transmembrane transporter
activity as the top functional cluster, among which ANT1, 2,
and 3 were quantified with ratios of 0.92, 0.92, and 0.91,
respectively, at the 10:40 μM labeling condition (Figure 4D,E).
VDAC2 and VDAC3 were also quantified albeit with much
31
(Figure 3F). A previous ABPP study has quantified intrinsic
cysteine reactivity by comparing the extent of labeling at
different probe concentrations and identified a subset of hyper-
32
reactive cysteines in native proteomes. We initially attempted
F
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Figure 6. Rescue of EA-induced cell death and mitochondrial dysfunction by the C257A ANT mutant. (A) Stable overexpression of wild-type ANT3
caused significant resistance to EA-induced cytotoxicity as evaluated by the MTT assay. Stable overexpression of the C257A mutant caused further
resistance (n = 6). (B) Stable overexpression of the C257A mutant resulted in elevated mitochondrial ROS that are not sensitive to EA treatment
anymore. EA-induced mitochondrial ROS production was assessed by flow cytometry using mitoSOX. (C) Stable overexpression of the C257A
mutant rescued the EA-induced loss of mitochondrial transmembrane potential (Δψm) as assessed by flow cytometry using JC-1 and JC-9
mitochondrial potential sensors. (D) Stable overexpression of the C257A mutant did not rescue the EA-induced increase of cytosolic ROS as
assessed by flow cytometry using DCFH-DA. (E) Stable overexpression of the C257A mutant rescued the EA-induced deformation of mitochondrial
ridges as visualized by TEM (scale bars 500 nm). In (B−E), cells were treated with DMSO or 120 μM of EA for 24 h. In (B−D), * p < 0.05; ** p <
0.01; *** p < 0.001; N.S, not significant; and n = 3, student’s t test. Error bars represent mean ± SD.
lower ratios (0.65 and 0.51, respectively) (Figure 4E and Table
S2). These results revealed members of the ANT family as EA-
hyper-reactive proteins.
Sequence alignment of three EA-reactive ANTs (ANT1−3)
showed that they contain four highly conserved cysteines:
Cys57, Cys129, Cys160, and Cys257 (Figure 5D). To verify
that Cys257 is a direct target site of EA, we constructed cell
lines with stable overexpression of wild-type ANT3 and its
C257A mutant, respectively, and quantitative proteome
profiling measured that the exogenously overexpressed ANT3
is about 3-fold more abundant than the endogenous one
Site-Specific Mapping of EA-Reactive Cysteines in
ANTs. Given that we failed to identify the EA-probe adducted
peptides directly, we resorted to a competitive isoTOP-ABPP
platform to map EA-reactive cysteines in proteomes. The
method was originally developed to quantify the cysteine site of
21
(
Figure S4 and Table S4). We immunoprecipitated the EA-
modification by endogenous lipid-derived electrophiles and
probe-modified ANT3 variants from these cells by streptavidin
for Western blot analysis. The results showed that the EA-
probe labeling on the wild-type ANT3 was effectively
completed by extra EA (Figure 5E), and the labeling on
ANT3-C257A was significantly reduced but not completely
abolished as compared to that on wild-type ANT3 (Figure 5F),
suggesting that Cys257 is one of the sites of action through
which EA modulates the function of ANTs.
recently expanded to discover druggable cysteines by small-
33
molecule electrophilic fragments in proteomes. In such an
experiment, cellular proteomes were divided equally into two
aliquots, with one treated with EA (experimental sample) and
the other with DMSO (control sample). Both aliquots were
then labeled with a cysteine-reactive alkynylated iodoacetamide
probe (IA-probe) and conjugated by CuAAC with an
isotopically coded light and heavy azide-biotin tags, respec-
tively, each of which contains a tobacco etch virus (TEV)
cleavage sequence. The light and heavy samples were combined
and subjected to a tandem orthogonal proteolysis (TOP)-
Rescue of EA-Induced Mitochondrial Dysfunction by
the ANT Mutant. The ANT families are a class of important
transporter proteins that are located on the inner mitochondrial
membrane with important functions. A variety of diseases are
associated with dysfunctional human ANTs, many of which
have common features of mitochondrial structural abnormal-
3
4
ABPP protocol, from which the IA-probe-labeled cysteines
are identified, and their extent of labeling quantified. EA-
sensitive cysteines were quantified by measuring the MS
chromatographic peak ratios, R, for heavy (DMSO-treated)
over light (EA-treated) samples, with higher R values reflecting
greater sensitivity to EA modification (Figure 5A). We
performed the competitive isoTOP-ABPP experiments with
29
ities. Given EA can induce mitochondrial deformation with
loss of mitochondrial membrane potential, we postulated that
EA modifications on ANTs might be the trigger for the
observed mitochondrial deformation.
To test this hypothesis, we first evaluated the ability of the
C257A mutant to counteract the cytotoxicity imposed by EA.
Cells with a stable expression of GFP, wild-type ANT3, or the
C257A mutant were incubated with various concentrations of
EA for 24 h, and cell survivals were monitored by MTT assays.
The results showed that overexpression of the wild-type ANT3
caused significant resistance to the cytotoxicity of EA, with IC50
1
1
0, 20, and 40 μM of EA and collectively quantified more than
20 cysteines, whose ratios increased with increasing
concentrations of EA as competitors (Figure 5B and Table
S3). Among these EA-sensitive cysteines is Cys257 from
ANT1/3 and ANT2, both of which exhibited nearly complete
competition at 40 μM of EA (Figure 5C).
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Molecular Pharmaceutics
Article
shifting from 94.31 ± 5.75 to 116.2 ± 4.95 μM (Figure 6A).
Consistent with the fact that EA directly targets Cys257 in
ANTs, cells with stable overexpression of C257A exhibited
further resistance to the cytotoxicity induced with EA treatment
in cancer cells (Figure 6A).
EA-adducted peptides by LC-MS/MS, especially that alter-
native fragmentation methods (e.g., electron-transfer dissocia-
tion) should be tested. This would also allow for the
identification of sites of EA modifications on nucleophilic
residues other than cysteines, such as lysines and histidines.
GO analysis on the EA-hyperactive proteins ranked the top
functional cluster as being involved in ADP/ATP transport
process, among which the protein family of ANTs caught our
attention. This class of adenine transporters works by
exchanging ADP and ATP across the inner mitochondrial
membrane, resulting in the net export of ATP from the
mitochondrial matrix and the import of ADP into the matrix.
Our competitive isoTOP-ABPP experiments mapped one
highly conserved Cys257 in the ANT family that is covalently
modified by EA with outstanding sensitivity. ANT has another
three conserved cysteines besides Cys257. Since the EA-probe
labeling on ANT3-C257A was not completely abolished
We next evaluated the effects on mitochondrial ROS and
MMP by the wild-type and mutant ANTs. We found that
overexpression of the C257A mutant of ANT3 resulted in
elevated mitochondrial ROS, which was not further increased
by the treatment with EA (Figure 6B). We also found that cells
with C257A overexpression were insensitive toward EA-
induced MMP loss (Figure 6C). Consistently, visualization of
these cells by TEM showed that overexpression of the C257A
mutant was able to rescue the deformation of mitochondrial
ridges induced by EA (Figures 6E and S5). Interestingly,
overexpression of the C257A mutant of ANT3 did not prevent
EA-induced elevation of cytosolic ROS (Figure 6D).
Collectively, these results suggested that EA targets the
functional Cys257 in ANTs to impair mitochondrial function.
(
Figure 5F), the results suggested these three cysteines could
also be modified by EA, which reflects a limitation of the
competitive isoTOP-ABPP to quantify the EA modification
indirectly (e.g., the IA-probe labeling on these three cysteines
could not be detected by LC-MS/MS).
DISCUSSION
■
As an effective loop diuretic drug, EA has been widely used in
clinical practice for treating high-blood pressure and edematous
states from heart, liver, and kidney failure. Its known side effects
Mutation of Cys257 was able to render the protecting effects
against EA-induced cytotoxicity and mitochondrial dysfunction
partially. Interestingly, Cys257 seems to play a more profound
role in mediating the impact of EA on the loss of MMP, the
elevation in mitochondrial ROS, and the deformation of
mitochondria but not the increased cytosolic ROS. Notably, the
C257A mutation can cause elevated mitochondrial ROS and
decreased MMP alone without EA treatment, and the stress
may initiate certain defense mechanisms in cells to adapt them
for resistance to the additional stress imposed by EA.
Structurally, Cys257 is predicted to be located on the matrix
35,36
include hearing loss and jaundice.
In both A375 and HeLa
cells, we found that EA could induce a noncanonical type of cell
death aside from apoptosis, autophagy, and necrosis, with the
deformation of mitochondrial ridges, increased cytosolic/
mitochondrial ROS production and lipid peroxidation, as well
as the loss of mitochondrial membrane potential. It remains
investigated whether the side effects of EA could be attributed
to the apparent dysfunction of mitochondria.
Chemically, the α,β-unsaturated ketone group in EA can
react with nucleophilic thiol groups via Michael addition, which
include free cysteine, GSH, as well as cysteine side chains in
proteins. The conjugate of EA-cysteine is believed to be the
active form in vivo to block NKCC for the diuretic effect. The
complex of EA-GSH has been shown as a potent inhibitor for
GSTs, a family of detoxification enzymes that are often
overexpressed in tumor cells. The dual GSH-depleting and
GST-inhibiting effect of EA makes it a promising chemotherapy
enhancing agent. Protein targets of EA have been reported in
individual cases, including MAP2K6, LEF-1, and NF-kB;
however, the full proteome reactivity of EA remains poorly
explored.
In the current work, we performed a quantitative chemical
proteomic profiling of target proteins and cysteine sites that are
covalently modified by EA. We designed and synthesized a
bioorthogonal EA-probe that retains cytotoxicity similar to EA,
and when it was applied in combination with RD-ABPP, we
quantified 277 potential EA-reactive proteins, including a subset
showing hyper-sensitivity toward EA modifications. Many of
these EA-hyper-reactive proteins are localized on mitochondrial
membranes, which is consistent with the observed phenotypical
abnormality of mitochondria induced by EA.
37
side of ANT, and EA modification on it might perturb the
mitochondrial function locally. A high-resolution experimental
structure of ANT will be warranted to help decipher the role of
Cys257.
While we chosen to focus on ANTs for functional
characterization, survey on the RD-ABPP and competitive
isoTOP-ABPP data revealed other novel targets of EA, which
were unconsciously neglected before. For example, four out of
the six members of the peroxiredoxins (PRDXs) family were
identified as EA-reactive proteins (Figure S6). This class of
widespread and highly expressed cysteine-based peroxidases
functions to rapidly detoxify excessive hydrogen peroxides
38
(
H O ) and maintain the balance in the redox signaling.
2 2
Consistent with their roles in reducing H O and associated
2
2
cytosolic ROS, we observed that EA treatment could induce
increased cytosolic ROS, as monitored by the oxidation of the
DCFH-DA probe. So, it is likely that EA-induced oxidative
stress stems from its modification and impairment of multiple
antioxidant enzymes, including, but not limited to, GSTs and
PRDXs. In this regard, caution should be taken in the future to
use EA to inhibit GSTs in a cellular setting.
In summary, we have performed a comprehensive chemo-
proteomic profiling of protein targets and sites of modification
of EA. Our findings suggest that EA-induced cytotoxicity is
multifaceted in scope with interference on various functional
pathways, including energy transportation and antioxidant
defense. Our results will provide rich information to guide
improvement on the clinical safety profile of EA. The novel
mode of actions discovered in this study will also offer new
As reduction of the α,β-double bond in EA completely
abolished its cytotoxicity, it confirms that the cysteine-reactive
property is critical for the biological activity of this drug. In gel-
based ABPP, we observed a concentration-dependent EA-probe
labeling in proteomes that can be enhanced with reduction by
NaBH (Figure 2C), and it suggests that EA-cysteine Michael
4
adducts on proteins require further stabilization. However,
conditions remain to be optimized in order to directly identify
H
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Molecular Pharmaceutics
Article
opportunities to repurpose this widely used drug for treating
cancers.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.molpharma-
ceut.8b00250.
■
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AUTHOR INFORMATION
Corresponding Authors
E-mail: xglei@pku.edu.cn
E-mail: chuwang@pku.edu.cn
■
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*
ORCID
2
(
Xiaoguang Lei: 0000-0002-0380-8035
Chu Wang: 0000-0002-6925-1268
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⊥
Z.Y. and X.Z. contributed equally to this work. X.L and C.W.
conceived the project. Z.Y. and T. S. performed the
chemoproteomic and biological experiments. X.Z. synthesized
the EA compounds and probes. Y.Z. and X.C. performed the
TEM experiments. Z.Y., X.Z., X.L., and C.W. analyzed data and
wrote the article.
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
in the human proteome. Nat. Chem. 2017, 9 (12), 1181−1190.
We thank Prof. Li Yu from Tsinghua University for collecting
preliminary EM data and helpful discussions. We thank Dr.
Yuan Liu and Mr. Jinjun Gao for help with proteomic and
structural data analysis. We thank the Computing Platform of
the Center for Life Science for supporting the proteomic data
analysis. This work was supported by the Ministry of Science
and Technology of China (2016YFA0501500 to C.W.;
017YFA0505200 to X.L.), National Science Foundation of
China (21472008, 21521003, and 81490740 to C.W.;
1472010, 21561142002, and 21625201 to X.L.), and a
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derived electrophiles by bioorthogonal aminooxy probe. Redox Biol.
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