Chemoproteomic Profiling Reveals Ethacrynic Acid Targets Adenine

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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 highblood 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

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INTRODUCTION Ethacrynic acid (EA) is a diuretic drug developed over 50 years ago.1 Because it can produce a prompt and profound diuresis, the drug has been widely employed in the treatment of highblood pressure and edematous states caused by congestive heart failure, liver failure, or kidney failure.2 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 ascending limb of Henle’s loop to block the re-absorption of NaCl.3 Chemically, the conjugation with free cysteines occurs 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.4 In addition, the EA-GSH adduct was demonstrated to impose a strong inhibition on the activity of members of the glutathione S-transferase (GST) family.5 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 healthy level of reactive oxygen species (ROS) for tumors to survive.6 Based on these properties, EA is currently being investigated as © XXXX American Chemical Society

a chemotherapy enhancing agent and has been combined with anticancer drugs to reduce drug resistance during cancer treatment.7,8 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 inducing cell apoptosis.9 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 residue.10 It was able to react with the p65 subunit of NF-kB at Cys38 and inhibit its binding with DNA.11 EA could also directly bind to the LEF-1 protein and destabilize its complex formation with β-catenin, which in turn suppresses the recruitment of LEF1 to promoter regions on DNA.12 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. Chemical proteomic strategies, such as activity-based protein profiling (ABPP), have become an enabling tool to discover Received: March 9, 2018 Revised: May 2, 2018 Accepted: May 4, 2018

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DOI: 10.1021/acs.molpharmaceut.8b00250 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

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-H2. (B) EA but not EA-H2 imposes dose-dependent cytotoxicity to A375 cells 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.

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EXPERIMENTAL METHODS Gel-Based ABPP. The A375 cells were lysed using a probe 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(2carboxyethyl)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%

and interrogate functional proteins directly in complex proteomes. 13 In addition to identifying residues with heightened intrinsic reactivity for functional annotation,14,15 ABPP-based methods have also been developed to map the sites of modification by endogenous metabolites,16 natural products,17 or anticancer drugs18,19 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 EAinduced cytotoxicity, including the deformation of mitochondrial 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. B

DOI: 10.1021/acs.molpharmaceut.8b00250 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics SDS-PAGE gel. The gels were scanned by fluorescense and stained by Coomassie brilliant blue to demonstrate equal loading. 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−chloroform 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 washed with H2O 3 times. The enriched proteins were digested by trypsin on-bead in 100 mM TEAB buffer and subjected to reductive dimethylation labeling.16 Finally, heavy and light labeled peptide samples were mixed, concentrated, separated by the Fast-seq protocol,20 and analyzed on a Q Exactive Plus mass spectrometer (Thermo Fisher). For identifying EA-hyperreactive 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. Mapping EA-Reactive Cysteines by Competitive isoTOP-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 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 IAprobe at room temperature in the dark for 1 h. Click chemistry was performed by the addition of either 100 μM of the heavyTEV-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 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.

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 Nterminal of a peptide and lysines. For competitive isoTOPABPP data, the isotopic modifications (464.28596 and 470.29977 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 done by an in-house software CIMAGE, accordingly.16,21 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.

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RESULTS EA Induces Noncanonical Cell Death with Mitochondrial 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 57.26 ± 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-H2, which lacks the double bond at 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 LC3-II as distinct markers, respectively.22,23 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 (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 plasma membrane rupture.24 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 mitochondria 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 C

DOI: 10.1021/acs.molpharmaceut.8b00250 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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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 EAprobe (3) with the alkyne functional group. Reagents and conditions: (a) Ethyl bromoacetate, K2CO3, acetone, 50 °C, 3 h, and 98%; (b) 5bromovaleryl chloride, AlCl3, CH2Cl2, 0 °C, 2 h, r.t., 48 h, and 60% brsm; (c) Ag2O, acetone/H2O (3/1), 60 °C, 48 h, and 60%; (d) DMP, CH2Cl2, 1 h, and 80%; (e) dimethyl (1-diazo-2-oxopropyl)phosphonate, K2CO3, MeOH, 2 h, and 29%; and (f) 37 wt % HCHO, K2CO3, EtOH/H2O (1/1), 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 NaBH4 and completed by EA.

Figure 3. Chemproteomic profiling of EA-reactive proteins by RD-ABPP. (A) Scheme of profiling EA modified proteins by RD-ABPP with EAcompetitive probe labeling. (B) Ratio distribution of a representative RD-ABPP profiling experiment in which proteins with 0.9 ≤ Rprobe vs probe ≤ 1.1 and RDMSO vs EA ≥ 2.0 are considered as EA-reactive proteins. (C) A total number of 277 EA-reactive proteins are commonly identified from two 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.

ability than that of the aromatic ring part and carboxyl group.27 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 Oalkylation 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

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 (CuAAC).25,26 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

DOI: 10.1021/acs.molpharmaceut.8b00250 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

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 ≤ R10:40 ≈ R20:40 ≈ R40:40 ≤ 1.2) were defined as EA-hyper-reactive proteins. (D) GO analysis of the EA-hyperactive proteins by molecular functions. (E) Quantified ratios for members from the ANTs and VDACs family (n = 2).

quantitative profiling of EA-reactive proteins using the EAprobe by combining competitive ABPP with stable isotope reductive dimethylation (RD-ABPP).16 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. Probe-labeled proteins were reduced with NaBH4, conjugated 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 chromatography (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 EAreactive 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 voltagedependent anion channels (VDAC) families localized on the

chloride through Friedel−Crafts acylation in the presence of AlCl3. We next tried 5-bromovaleryl chloride and successfully transformed 5 to bromide 6. Treatment of 6 with Ag2O 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 K2CO3, followed by ester hydrolysis, successfully generated the EAprobe 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 NaBH4 for another 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 NaBH4 (Figure 2C). This observation is consistent with the premise that NaBH4 could 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 E

DOI: 10.1021/acs.molpharmaceut.8b00250 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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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 EAadducted 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.

a similar approach hoping to detect a subset of EA-hyperreactive 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) fragmentation. We instead applied this strategy at the protein level and performed three parallel quantitative RD-ABPP experiments 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-hyperreactive target. We applied the filter of 0.8 ≤ R10:40 ≈ R20:40 ≈ R40:40 ≤ 1.2 and identified 115 targets, which showed nearly 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

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.28 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− 4) and our quantitative profiling identified ANT1, ANT2, and ANT3,29 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.30 The genomes of humans, as well as other higher eukaryotes, encode three different VDACs (VDAC1−3),31 and we identified VDAC2 and VDAC3 as EA-reactive proteins (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 hyperreactive cysteines in native proteomes.32 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 EAhyper-reactive proteins. 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 modification by endogenous lipid-derived electrophiles21 and recently expanded to discover druggable cysteines by smallmolecule electrophilic fragments in proteomes.33 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, respectively, 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)ABPP protocol,34 from which the IA-probe-labeled cysteines are identified, and their extent of labeling quantified. EAsensitive 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 10, 20, and 40 μM of EA and collectively quantified more than 120 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).

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 (Figure S4 and Table S4). We immunoprecipitated the EAprobe-modified ANT3 variants from these cells by streptavidin for Western blot analysis. The results showed that the EAprobe 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. 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 abnormalities.29 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 G

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Molecular Pharmaceutics 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). 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 EAinduced 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.

EA-adducted peptides by LC-MS/MS, especially that alternative fragmentation methods (e.g., electron-transfer dissociation) 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 (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). 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 side of ANT,37 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 (H2O2) and maintain the balance in the redox signaling.38 Consistent with their roles in reducing H2O2 and associated 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 chemoproteomic 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

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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 include hearing loss and jaundice.35,36 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. 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 gelbased ABPP, we observed a concentration-dependent EA-probe labeling in proteomes that can be enhanced with reduction by NaBH4 (Figure 2C), and it suggests that EA-cysteine Michael adducts on proteins require further stabilization. However, conditions remain to be optimized in order to directly identify H

DOI: 10.1021/acs.molpharmaceut.8b00250 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

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opportunities to repurpose this widely used drug for treating cancers.

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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/acs.molpharmaceut.8b00250. Visualization of A375 and HeLa cell death induced with EA by TMS; quantitated ratios for all six members of the PRDX family; methods of cell culture, MTT assay, immunoblotting of cell death markers, TEM, flow cytometry, GO analysis, stable expression of recombinant ANTs in HeLa cells, quantitative proteomics for determination of the relative abundances of endogenous ANT3 and overexpressed ANT-WT or ANT-C257A, validation of ANT3 as an EA-modified protein, and validation of C257 of ANT3 as an EA-reactive site; and synthetic procedures of 2, 3, 4, 5, 6, and 7 (PDF) Tables of quantitative proteomic data (XLSX)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] *E-mail: [email protected] ORCID

Xiaoguang Lei: 0000-0002-0380-8035 Chu Wang: 0000-0002-6925-1268 Author Contributions ⊥

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. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS 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.; 2017YFA0505200 to X.L.), National Science Foundation of China (21472008, 21521003, and 81490740 to C.W.; 21472010, 21561142002, and 21625201 to X.L.), and a “1000 Talents Plan” Young Investigator Award (C.W.).

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DOI: 10.1021/acs.molpharmaceut.8b00250 Mol. Pharmaceutics XXXX, XXX, XXX−XXX