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Activity Based Protein Profiling Leads to the Identification of Novel Protein Targets for the Nerve Agent VX Daniel Carmany, Andrew J. Walz, Fu-Lian Hsu, Bernard J. Benton, David Burnett, Jennifer A. Gibbons, Daan Noort, Trevor G Glaros, and Jennifer W Sekowski Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00438 • Publication Date (Web): 07 Mar 2017 Downloaded from http://pubs.acs.org on March 9, 2017

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Chemical Research in Toxicology

Activity Based Protein Profiling Leads to the Identification of Novel Protein Targets for the Nerve Agent VX Dan Carmany†, Andrew J. Walz§, Fu-Lian Hsu§, Bernard Benton§, David Burnett§, Jennifer Gibbons§, Daan Noort‡, Trevor Glaros§*, Jennifer W. Sekowski§* †

Excet, Inc. 6225 Brandon Ave, Suite 360, Springfield, VA 22150, USA

‡

TNO Defense, Security and Safety, P.O. Box 45, 2280 AA Rijswijk, The Netherlands.

§

Research and Technology Division, US Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, MD 21010, USA

*Corresponding Authors [email protected], Dr. Jennifer Sekowski, 5183 Blackhawk Road, RDCB-DRBD, Aberdeen Proving Ground- Edgewood Area, MD 21010; 410-436-5546 [email protected], Dr. Trevor Glaros, 5183 Blackhawk Road, RDCB-DRB-D, Aberdeen Proving Ground- Edgewood Area, MD 21010; 410-436-3616

Keywords: Organophosphate, OP, VX, CWA, ABPP, Protein Adducts, Nerve Agent, Poisoning

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Abstract. Organophosphorus (OP) nerve agents continue to be a threat at home and abroad during the war against terrorism. Human exposure to nerve agents such as VX results in a cascade of toxic effects relative to the exposure level, including ocular miosis, excessive secretions, convulsions, seizures, and death. The primary mechanism behind these overt symptoms is the disruption of cholinergic pathways. While much is known about the primary toxicity mechanisms of nerve agents, there remains a paucity of information regarding impacts on other pathways and systemic effects. These are important for establishing a comprehensive understanding of the toxic mechanisms of OP nerve agents. In order to identify novel proteins that interact with VX, and which may give insight into these other mechanisms, we used activitybased protein profiling (ABPP) employing a novel VX-probe on lysates from rat heart, liver, kidney, diaphragm and brain tissue. Making use of a biotin linked VX-probe, proteins covalently bound by the probe were isolated and enriched using streptavidin beads. The proteins were then digested, labeled with isobarically distinct tandem mass tag (TMT) labels, and analyzed by liquid chromatography mass spectrometry (LC-MS/MS). Quantitative analysis identified 132 bound proteins, with many proteins found in multiple tissues. As with previously published ABPP OP work, monoacylglycerol lipase associated proteins and fatty acid amide hydrolase (FAAH) were shown to be targets of VX. In addition to these two and other predicted neurotransmitter-related proteins, a number of proteins involved with energy metabolism were identified. Four of these enzymes, mitochondrial isocitrate dehydrogenase 2 (IDH2), isocitrate dehydrogenase 3 (IDH3), malate dehydrogenase (MDH), and succinyl CoA (SCS) ligase, were assayed for VX inhibition. Only IDH2 NADP+ activity was shown to be inhibited directly. This result is consistent with other work reporting animals exposed to OP compounds exhibit reduced IDH activity. Though clearly a secondary mechanism for toxicity, this is the first time VX has been shown to directly interfere with energy metabolism. Taken together, the ABPP work described here suggests the discovery of novel protein-agent interactions which could be useful for the development of novel diagnostics or potential adjuvant therapeutics.

Introduction. Understanding the spectrum of toxicity pathways for OP nerve agent exposure is essential for providing the highest quality of care for exposed patients. Current countermeasure research uses a traditional reductionist approach focusing on single mechanisms of action, which limits the solution space for development of new modes of diagnosis and treatment of exposure. This type of approach has identified the primary mechanism for toxicity, which is caused by OPs binding covalently to and inhibiting acetylcholinesterase. This is accepted as the primary binding event related to a broad range of dose dependent cholinergic symptoms including salivation, lacrimation, urination, defecation, gastric cramps, and emesis (SLUDGE).1 However, several studies over the past decade have suggested that secondary mechanisms of action may also play a role in OP nerve agent toxicity.



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In recent years, several studies have been published using various genomic, metabolomic, and chemoproteomic techniques to identify these secondary targets. Many results suggest effects in energy metabolism. Work performed at the United States Army Medical Institute for Chemical Defense (USAMRICD) demonstrated that something as seemingly inconsequential as diet can greatly enhance or diminish the observed effects of the OP soman exposure.2-3 Metabolomic studies on the livers of salmon exposed to the OP insecticide, chlorpyrifos (CP), found decreased level of downstream glycogen metabolites, including available glucose. From the same animals, TCA cycle intermediates were increased, suggesting possible interruption of key metabolic enzymes.4 Similarly, the livers of rats exposed to the OP insecticide chlorfenvinphos have been shown to contain diminished isocitrate dehydrogenase (IDH) activity, an enzyme critical in the mitochondrial TCA pathway.5 Furthermore, work by Nomura and Casida, using the activity-based protein profiling (ABPP) strategy applied to brains of mice exposed to OP and thiocarbamate (TC) pesticides, revealed a wide variety of enzyme inhibition.6 In particular, inhibition of monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) enzymes were observed. Those enzymes would normally terminate endogenous cannabinoid ligands, thus, their inhibition would lead to a wide range of downstream endocannabinoid effects including changes in memory, neurogenesis, feeding, energy homeostasis and metabolism. Overall, clues from the OP literature suggest that changes in energy metabolism, including glycolysis, gluconeogenesis, and glycogenesis, may be an important secondary effect of OP compound toxicity. Although OPs are typically associated with rapid and acute toxicity, delayed effects of up to 2-3 weeks have been observed including delayed neuropathy or polyneuropathy (OPIDN or OPIDP).7 Some of those effects follow an intense cholinergic response while others have long term effects seen even after a minimal depression of cholinergic enzymes.8 Physiological consequences of exposure can be observed systemically such as paralysis,9 while other effects are organ-specific.10 Taken together, these observations suggest that OP compounds likely bind to and possibly alter the function of many secondary enzymes or proteins collectively involved with other cellular functions and pathways which have not been recognized or correlated. Identifying these targets may help to facilitate development of novel strategies to provide a better quality of care to those with OP poisoning. In this work, we describe the use of the ABPP strategy to elucidate novel targets and pathways of the OP nerve agent, VX. The basis for the method was developed largely by the Cravat laboratory 6, 11-12 and employed in many other laboratories including investigations into targets of OPs such as sarin.13-16 Generally, ABPP utilizes a chemically synthesized probe molecule consisting of an active group, termed the “warhead”, and a detectable moiety on the other end, termed the “tail”.17 In this work, the warhead was the nerve agent VX, which is expected to covalently bind to target proteins. The VX warhead is tethered by an 11-carbon chain to an amide-linked biotin tail. By virtue of the biotin moiety, the probe-protein complexes are captured using streptavidin-coated beads. However, unlike the method used for the sarin or thiocarbamate work,13-14 which relied on visual identification and selection of specific bands in an SDS-PAGE gel prior to digestion and liquid chromatography tandem mass spectrometry (LCMS/MS), we have developed a new, more sensitive and quantitative method. A similar approach 4 ACS Paragon Plus Environment

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using an ABPP probe for adenosine triphosphate (ATP) was published to characterize various kinase inhibiting compounds using Thermo Fisher’s ActivX kinase enrichment kit.18 Using our new technique, all digested peptides from the ABPP assay were specifically labeled with isotopically distinct tandem mass tag (TMT) reagents, pooled, and then analyzed by LC-MS/MS. The integration of the TMT reagents made a more sensitive and quantitative assay, resulting in the identification of a number of novel VX protein targets, including metabolic enzymes shown to be critical in glycolysis and the citric acid (TCA) cycle. This improved ABPP method provided a unique opportunity to discover other OP binding proteins. The data resulting from this work are strongly suggestive that lesser known OP binding proteins may play secondary, synergistic and potentially important roles in the overall toxicity profile of OP nerve agents. These targets may also provide new opportunities for diagnostics as well as improved adjuvant therapeutic targets.

Experimental Procedures. Caution. Due to the acute hazards associated with VX, all experiments involving VX or the synthesis of the VX- probe were performed in certified chemical fume cabinets equipped with an advanced filtration system that protects the user and the environment at the US Army’s Edgewood Chemical Biological Center (Edgewood, MD) Probe synthesis. The overall strategy to introduce the VX-functionality onto an ABPP probe was based on previous work on VX analogs containing (N,N-diethylammonium) ethanthiol19 and is outlined in Figure 1. Compound 2: Briefly, methylphosphonothioic dichloride (0.259 g, 1.74 mmol) was dissolved in 10 mL chloroform (CHCl3) and cooled in an ice-water bath under nitrogen. A solution of BOC-aminoundecan-1-ol (0.500 g, 1.74 mmol), triethylamine (TEA) (0.185 g, 1.83 mmol), and 4-dimethylaminopyridine (DMAP) (0.213 g, 1.74 mmol) in 10 mL CHCl3 was added dropwise. The ice-water bath was removed and the reaction was stirred at room temperature for 3h. A solution of 2-(diisopropylamino) ethanol (2-DIPEA) (0.248 g, 1.74 mmol) and TEA (0.185 g, 1.83 mmol) in 8 mL CHCl3 was added dropwise. The reaction was stirred at room temperature for 16h. The solvents were evaporated and the residue was taken up in a mixture of CHCl3 and a saturated solution of sodium bicarbonate. The organic layer was separated and the aqueous solution was extracted two times with CHCl3. The combined organic extracts were dried over sodium sulfate, filtered, and volatiles were evaporated. The residue was purified by column chromatography using a 4:1 to 2:1 hexanes: ethyl acetate gradient which provided 0.720 g of compound 2 as an oil in an 82% yield. HRMS (ESI-TOF) m/z: [M+H]+ Calculated for C25H54N2O4PS 509.3541, Found 509.3530; 1H NMR (CDCl3) δ 4.48 (s, 1H), 4.10-3.92 (m, 4H), 3.87-3.78 (m, 2H), 3.11-2.92 (m, 4H), 2.65 (t, 2H, J = 7.56 Hz), 1.78 (d, 3H, J = 15.11 Hz), 1.661.59 (m, 2H), 1.51-1.22 (m, 23H), 0.99 (d, 12 H, J = 6.41 Hz); 13C NMR (CDCl3) δ 156.05, 79.09, 66.80 (d, J = 7.62 Hz), 66.56 (d, J = 6.67 Hz), 49.56, 45.24 (d, J = 6.67 Hz), 40.71, 30.44, 30.37, 30.15, 29.60, 29.57, 29.37, 29.28, 28.52, 26.89, 25.67, 22.11, 20.95; 31P NMR (CDCl3) δ 95.97. 5 ACS Paragon Plus Environment


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Compound 3: Compound 2 (0.200 g, 0.393 mmol) was dissolved in 4.5 mL of dichloromethane at room temperature under nitrogen. Trifluoroacetic acid (TFA) (4.5 mL) was added and the reaction was stirred for 1h. The volatiles were evaporated and 2 equivalents of TEA were added. The residue was taken up in a mixture of CHCl3 and a saturated solution of sodium bicarbonate. The organic layer was separated and the aqueous solution was extracted two times with CHCl3. The combined organic extracts were dried over sodium sulfate, filtered, and volatiles were evaporated. The residue was dissolved in 4.5 mL of N,N-dimethyl formamide (DMF) and 0.011 g of DMAP was added followed by NHS-Biotin (0.134 g, 0.393 mmol). The reaction was stirred at room temperature for 24h under nitrogen. The solvent was evaporated and the residue purified by column chromatography using 15% methanol:85% chloroform as the eluent which provided 0.185 g of Compound 3 as an amorphous white solid in an 74% yield. HRMS (ESI-TOF) m/z: [M+H]+ calculated for C30H60N4O4PS2 635.3793, found 635.3796; 1H NMR (D6-DMSO) δ 7.68 (t, 1H, J = 5.50 Hz), 6.39 (s, 1H), 6.32 (s, 1H), 4.28-4.07 (m, 2H), 3.97-3.71 (m, 4H), 3.07-2.88 (m, 5H), 2.78 (dd, 1H, J = 12.37 Hz, 5.04 Hz), 2.59-2.52 (m, 3H), 2.00 (t, 2H, J = 7.56 Hz), 1.75 (d, 3H, J = 15.11 Hz), 1.58-1.15 (m, 24H), 0.91 (d, 12H, J = 6.87 Hz); 13C NMR (D6-DMSO) δ 172.27, 163.21, 66.64 (d, J = 7.62 Hz), 66.30 (d, J = 6.67 Hz), 61.56, 59.70, 55.99, 49.32, 44.95 (d, J = 6.68 Hz), 38.88, 35.75, 30.19 (d, J = 7.63 Hz), 29.72, 29.54, 29.48, 29.31, 29.09, 28.75, 28.58, 26.97, 25.74 (d, J = 28.75 Hz), 21.83, 21.26, 20.70; 31P NMR (D6-DMSO) δ 96.66. VX-probe: Compound 3 (0.292 g, 0.460 mmol) was dissolved in 26 mL DMF and heated in a 95°C oil bath for 1h and 45min. The solvent was evaporated and the residue was purified by column chromatography using the following method. 18.7 g of silica gel was washed with 400 mL of fresh eluent (2.0% TEA:2.5% MeOH: 95.5% CHCl3). The same fresh eluent was used to load and elute the target compound to provide 0.205 g of VX-probe as an amorphous white solid in a 70% yield. HRMS (ESI-TOF) m/z: [M+H]+ Calculated for C30H60N4O4PS2 635.3793, Found 635.3797; 1H NMR (CD3OD) 4.47 (dd, 1H, J = 7.79 Hz, 5.04 Hz), 4.28 (dd, 1H, J = 7.96 Hz, 4.30 Hz), 4.12-3.98 (m, 2H), 3.21-2.67 (m, 11H), 2.17 (t, 2H, J = 7.73 Hz), 1.81 (d, 3H, J = 15.57 Hz), 1.75-1.27 (m, 24H), 1.03 (d, 12H, J = 6.87 Hz); 13C NMR (CD3OD) δ 174.60, 164.77, 65.48 (d, J = 6.67 Hz), 62.04, 60.29, 55.70, 48.90, 46.30 (d, J = 4.77 Hz), 39.73, 39.04, 35.49, 30.78, 30.02 (d, J = 6.67 Hz), 29.36, 29.31, 29.11, 28.94, 28.46, 28.19, 26.69, 25.51 (d, J = 25.88 Hz), 19.71, 18.55, 17.46; 31P NMR (CD3OD) δ 58.69. For routine use, the rearrangement of compound 3 to the VX-probe was conducted in DMSO. A 1 mg/mL stock solution of the probe in DMSO was created and stored at -20°C. Prior to each assay run, LC-MS analysis of the probe solution was performed for quality control purposes. Probe Activity Assay. The functional cholinesterase activity of the probe was tested by using a variation of the Ellman’s Assay.20-21 Briefly, whole human blood was exposed to VX, the VX-probe, or EtOH for 20min while mixing at 37°C. These exposed lysates were combined with a 2X master mix of buffer, substrate, and developer. All available (non-bound by VX or VX-probe) cholinesterases in the blood hydrolyzed the substrate generating thiocholine. The thiocholine reacted with the developer to generate 5-thio-2-nitrobenzoate anion. The anion was 6 ACS Paragon Plus Environment

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measured at an absorbance of 405 nM. The colorimetric change was plotted over time to gauge the enzymatic inhibitory activity of the probe versus VX on the blood cholinesterases. Tissue collection. Male Sprague-Dawley rats (275-350 g) were obtained from Charles River Laboratories (Wilmington, MA). They were housed in a temperature-controlled room with a 12h light/12h dark cycle and given food and water ad libitum. All animal work was conducted at ECBC, which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International. All animal procedures were approved by the Institute Animal Care and Use Committee and conducted in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996) and the Animal Welfare Act of 1966, as amended. Animals were euthanized by decapitation. The brain, heart, kidney, liver, and diaphragm tissue were immediately snap-frozen in liquid nitrogen and stored at -80°C until use. Preparation of rat tissue lysates. Frozen rat tissues (brain, heart, liver, kidney, diaphragm) were thawed on ice and homogenized with a Potter homogenizer (Glas-Co) at 8001000 RPM in 10 volumes (w/v) of ice cold 0.1% Triton X-100 (Sigma-Aldrich). The suspension was centrifuged at 1,000 x g for 10min at 4˚C and the supernatant was centrifuged at 17,000 x g for 55min at 4˚C. The resulting supernatant was then centrifuged at 100,000 x g for 60min at 4˚C. The final supernatant was incubated with 100µL High Capacity Streptavidin Beads in a 50% (w/v) aqueous slurry (Thermo Scientific) overnight at 4˚C with gentle continuous mixing. The suspension was centrifuged at 3,200 x g for 10min and the supernatant was collected. The supernatant protein (lysate) concentration was determined with a BCA protein assay (Thermo Scientific) as per manufacturer’s instructions. Exposure of rat tissue lysate to VX and VX-probe. Equal protein amounts of each tissue lysate (brain, heart, liver, kidney or diaphragm) from 3 animals were pooled. Each lysate was tested in a positive (probe only), negative (lysate only) and competition (pre-incubated with 10x VX, 100uM, prior to adding VX-probe) ABPP assay. To account for the solvent of the VX, equal molar ethanol was added into negative and positive assays. These were incubated at RT for 60min with gentle mixing. After this pre-incubation, the VX-probe was added to a final concentration of 10 µM to the competition and positive samples while the negative sample received an identical volume of DMSO. All samples were incubated at room temperature for 60min with gentle mixing. Each sample was then passed through a PD-10 size exclusion chromatography column (GE Healthcare) which had been equilibrated 3X with 5mL of Phosphate Buffered Saline with 0.1% Triton X-100 (PBST). The columns were eluted with PBST buffer. The eluate was treated with a final concentration of 0.5% SDS, heated to 85˚C for 5min, cooled to RT and then diluted 2.5-fold with PBST. The mixtures were incubated with 75 µl streptavidin-agarose beads at 4˚C overnight on a rotator. The beads were then pelleted and transferred to filter spin column (Thermo Scientific). The beads were washed 5X with 0.5 mL 1.0% SDS (w/v), 5X with 0.5mL 6M urea, and finally 10X with 0.5 mL PBS. Protein digestion and TMT labeling. The washed streptavidin-agarose beads were resuspended in 500 μL of PBS with 6M urea. DTT was added to yield a final concentration of 10 mM. The samples were heated to 65 °C for 15min while mixing. After mixing, the samples 7 ACS Paragon Plus Environment


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were cooled to RT and iodoacetamide added to yield a final concentration of 25 mM. The samples were then incubated at RT for 30min protected from light. The samples were centrifuged at 1400 x g for 2min and the supernatant removed. The pelleted beads were washed 5X with 1 ml PBS to remove the urea and then resuspended in 200 μL of PBS. To this suspension, 2 μL of 100 mM CaCl2 was added followed by 4 μL of 0.5 mg/mL trypsin (2 µg total) and incubated at 37°C overnight with shaking. The following day, the samples were centrifuged at 1400 × g for 2min and the supernatant collected. The beads were resuspended with 100 μL of PBS, briefly mixed, centrifuged, and the supernatant was again collected. Both supernatants were pooled together for a final volume of 300 μL of digested proteins. For quantitative comparison, TMT tags were used per the manufacturer’s instructions (Thermo Fisher Scientific) to label the N-terminus and/or each assay sample: negative, positive, and competitor. Briefly, 20 uL of each peptide digest was diluted with 80 μL of triethylammonium bicarbonate (TEAB) and added to the label (resuspended in 41uL of anhydrous acetonitrile) and incubated at RT for 1h while mixing. The reaction was quenched with 8 μl of 5% (w/v) hydroxylamine in TEAB. The negative, positive, and competition samples were then pooled together and dried completely in a speedvac. Samples were stored at -80C prior to liquid chromatography mass spectrometry analysis (LC-MS/MS). LC-MS/MS Analysis for VX and VX- probe QC. Prior to performing ABPP assay, VX and VX-probe samples were diluted with IPA (Sigma) as necessary, spiked with d5VX internal standard (prepared in-house), and analyzed using LC-MS/MS. Quantitative analysis of the probe and agent was conducted using high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS, Agilent 1260 LC triple-quadrupole mass spectrometer with MassHunter® data acquisition and analysis software, Wilmington, DE). The LC was fitted with an Agilent Eclipse® XDB-C18 column (5 µm, 4.6 x 150 mm). Sample injections were 1 µL. A 13min separation method was used; the composition of mobile phase A was 0.1% formic acid (v/v) in H2O and mobile phase B was 0.1% formic acid (v/v) in methanol (MeOH). The column eluent was delivered to an electrospray ionization source maintained in positive ion mode. MS/MS discrimination was performed via the multiple reaction monitoring (MRM) technique using collision induced dissociation (CID) with a fixed collision energy (CE) of 20V. Quantitation was performed by incorporating d5VX as the isotopically labeled internal standard (ISTD). The transitions used were: d5VX (Da 273128), VX quantitation (Da 268128), and VX confirmation (Da 26886). LC-MS/MS Analysis of TMT labeled peptides. Peptides were analyzed (in technical quintuplicate) on a Velos Orbitrap ELITE mass spectrometer coupled with the Easy-nLC II liquid chromatography pump system. Dried peptides were reconstituted in 3% acetonitrile/0.1% formic acid (v/v) and resolved on virgin Picofrit 15 cm X 75 µm ID HPLC column packed with 5µm BioBasic C18 particles 300Å (New Objective) using a 190 minute multistep gradient [05min: 5-10% (v/v) B, 6-160 min: 10-35% (v/v) B, and 161-190 min: 35-95% (v/v) B ]. For the gradient, the A buffer was 3% acetonitrile/ 0.1% formic acid (v/v) and the B buffer was 95% acetonitrile/0.1% formic acid (v/v). Orbitrap MS1 scans were performed at a resolution of 120,000 at 400 m/z, with a scan range of 350-1700 m/z. The top 15 precursors were selected for MS2 data-dependent fragmentation. MS2 spectra were acquired using the orbitrap at a resolution 8 ACS Paragon Plus Environment

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of 15,000 at 400 m/z (Top 15 Method). The minimum signal required to trigger a data-dependent scan was 2000. Higher-energy collisional dissociation (HCD) was used to generate MS2 spectra with the following settings: normalized collision energy 37%, default charge state 2, isolation width 1.5 m/z, and activation time 0.1ms. AGC target was set to 1 x 106 for MS and 5 x104 for MS/MS with a maximum accumulation time of 200 ms. Dynamic exclusion was set for 60 sec for up to 500 targets with a 5 ppm mass window. A lock mass of 445.120025 was used for internal calibration to improve mass accuracy. Mass spectrometry data and bioinformatic analysis. Spectra data was processed using Proteome Discoverer 1.4 with the SEQUEST HT search algorithm against the Rattus norvegicus RefSeq database (Tax ID: 10116). Dynamic modifications were set for carbamidomethylation of cysteine [+57.02 Da], oxidation of methionine [+15.99 Da], N-terminal TMT labeling [+229.16 Da], and TMT labeling of lysine [+229.16 Da]. MS/MS spectra were searched with a precursor mass tolerance of 10 ppm and a fragment mass tolerance of 0.6 Da. Trypsin was specified as the protease with a maximum number of missed cleavages set to 2. A false discovery rate was calculated using PERCOLATOR and was set at

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