Chemical Profile pubs.acs.org/crt
A Chemoproteomic Platform To Assess Bioactivation Potential of Drugs Rui Sun,†,‡,# Fuguo Shi,§,# Keke Liu,† Ling Fu,† Caiping Tian,† Yong Yang,‡ Keri A. Tallman,∥ Ned A. Porter,∥ and Jing Yang*,† †
State Key Laboratory of Proteomics, National Center for Protein Sciences, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China ‡ State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, Center for New Drug Safety Evaluation and Research, China Pharmaceutical University, Nanjing 211198, China § Department of Pharmacology, Key Laboratory of Basic Pharmacology of Ministry of Education and Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Zunyi 563003, China ∥ Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37232, United States S Supporting Information *
ABSTRACT: Reactive metabolites (RM) formed from bioactivation of drugs can covalently modify liver proteins and cause mechanism-based inactivation of major cytochrome P450 (CYP450) enzymes. Risk of bioactivation of a test compound is routinely examined as part of lead optimization efforts in drug discovery. Here we described a chemoproteomic platform to assess in vitro and in vivo bioactivation potential of drugs. This platform enabled us to determine reactivity of thousands of proteomic cysteines toward RMs of diclofenac formed in human liver microsomes and living animals. We pinpointed numerous reactive cysteines as the targets of RMs of diclofenac, including the active (heme-binding) sites on several key CYP450 isoforms (1A2, 2E1 and 3A4 for human, 2C39 and 3A11 for mouse). This general platform should be applied to other drugs, drug candidates, and xenobiotics with potential hepatoxicity, including environmental organic substances, bioactive natural products, and traditional Chinese medicine.
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INTRODUCTION Reactive metabolites (RMs) generated from bioactivation of drugs can covalently modify liver proteins and cause mechanism-based inactivation of key drug metabolizing enzymes such as cytochrome P450 (CYP450) family.1,2 These unwanted side-effects of drugs may result in potential drug−drug interactions (DDIs) and idiosyncratic drug toxicity in both experimental animals and humans.3 In the pharmaceutical industry, two complementary strategies are mainly applied to determine potential bioactivation effect of drug candidates on hepatic CYP450 enzymes.4 First, small molecule nucleophiles, such as glutathione, N-acetylcysteine, and cyanide, are used to directly trap and detect RMs of a test compound formed in liver microsomes. Second, in vitro CYP450 inhibition assays are used to evaluate whether RMs of a test compound lead to mechanism-based inactivation of its metabolizing enzymes. Although these traditional strategies are commonly used in the pharmaceutical industry, they have many shortcomings. First, these strategies are laborious and timeconsuming. Second, in vitro CYP450 inhibition assays rely highly on the specificity of probe substrate, which may result in potential false discovery. Last but not the least, these strategies are only focused on a few CYP450 isoforms, including 1A2, © 2017 American Chemical Society
2C8, 2C9, 2C19, 2D6, and 3A4. Nonetheless, whether bioactivation of a test compound affects other isoforms or non-CYP450 proteins remains to be assessed. In the last two years, various innovative proteomic methodologies have emerged to directly identify protein targets of RMs in vitro and in vivo. Leeming et al. reported a nontargeted mass spectrometry-based approach and a software called Xenophile for identification of acetaminophen-derived RMs protein adducts in liver microsome.5 Yamazaki et al. used 2D-gel combined with accelerator mass spectrometry to identify CYP3A as a major target of RMs of 14C-labeled 5hydroxoythalidomide in humanized mice.6 Pierce et al. developed a clickable probe for NSAIDs to investigate target profiles of its RMs in cells using mass spectrometry-based shogun proteomics.7 More recently, Cravatt and his colleagues described a similar chemoproteomic strategy and conducted a comprehensive survey of the in vivo targets of RMs from several hepatoxic drugs.8 Despite of these impressive methodological advances, they may not be feasible in early drug discovery processes, because they required either biorthogonal modReceived: July 4, 2017 Published: September 29, 2017 1797
DOI: 10.1021/acs.chemrestox.7b00183 Chem. Res. Toxicol. 2017, 30, 1797−1803
Chemical Profile
Chemical Research in Toxicology
Protein Sciences (Beijing). Male CD-1(ICR) mice (6−8 weeks old) were purchased from Charles River Laboratories (Beijing, China). Mice weighing 33−35 g were allowed free access to commercial mouse chow and water. All mice were housed under standard conditions (a specific pathogen-free, temperature-controlled microenvironment with a 12 h day/night cycle). The four mice were separated into two groups according random allocation. A 0.9% w/v NaCl solution (pH 7.0) containing 10 mg/mL diclofenac or vehicle was then administered at 200 mg/kg by i.p. injection. After 4 h of treatment, the mice were then anesthetized with phenobarbital sodium and euthanized by cervical dislocation. Fresh liver tissue samples were collected and washed with cold PBS three times, followed by homogenization in cold lysis buffer with tissue grinding pestles (DVS, China) and sonicated in the presence of 200 unit/mL catalase. Samples were centrifuged at 20,000 g for 10 min at 4 °C. The liver tissue homogenate from two mice were mixed and measured by BCA assay (Pierce Thermo Fisher). The protein concentrations were adjusted to 2 mg/mL for each sample in a volume of 500 μL for further preparation. Sample Preparation for Proteomic Analysis. Protein samples from HLMs or mouse liver tissues were labeled with 100 μM IPM probe in the presence of 200 unit/mL catalase for 1 h at room temperature. The labeled proteome was further incubated with 8 mM DTT (Research Products International) at 75 °C for 15 min to reduce the reversibly oxidized cysteines. Reduced cysteines then were alkylated with 32 mM IAM for 30 min in the dark at room temperature. Proteins were then precipitated with a methanolchloroform system (aqueous phase/methanol/chloroform, 4:4:1, v/ v/v) as previously described.11 The precipitated protein pellets were resuspended with 50 mM ammonium bicarbonate containing 0.2 M urea. Resuspended protein concentrations were determined with the BCA assay (Pierce Thermo Fisher) and adjusted to a concentration of 1 mg/mL. Resuspended proteins were first digested with sequencing grade trypsin (Promega) at a 1:50 (enzyme/substrate) ratio overnight at 37 °C. A secondary digestion was performed by adding additional trypsin to a 1:100 (enzyme/substrate) ratio, followed by incubation at 37 °C for additional 4 h. The tryptic digests were desalted with HLB extraction cartridges (Waters). The desalted samples were then evaporated to dryness under vacuum. Click Chemistry, Capture, and Enrichment. Desalted tryptic digests were reconstituted in a solution containing 30% ACN at pH ∼ 6. Click chemistry was performed by the addition of 1 mM either light or heavy Azido-UV-biotin (1 μL of a 40 mM stock), 10 mM sodium ascorbate (4 μL of a 100 mM stock), 1 mM TBTA (1 μL of a 50 mM stock), and 10 mM CuSO4 (4 μL of a 100 mM stock). Samples were allowed to react at room temperature for 2 h with rotation and light protection. The light and heavy isotopic tagged samples were then mixed immediately following click chemistry. The samples were cleaned by strong cation exchange (SCX) chromatography as previously described and then allowed to interact with pre-washed streptavidin sepharose for 2 h at room temperature.11 Streptavidin sepharose then was washed with 50 mM NaOAc (sodium acetate, pH4.5), 50 mM NaOAc containing 2 M NaCl (pH4.5), and water twice each with vortexing and/or rotation to remove non-specific binding substances, and resuspended in 25 mM ammonium bicarbonate. The suspension of streptavidin sepharose was transferred to several glass tubes (VWR) and irradiated with 365 nm UV light (Entela, Upland, CA) for 2 h at room temperature with stirring. The supernatant was collected, evaporated to dryness under vacuum, and stored at −20 °C until analysis. LC-MS/MS Analysis. LC-MS/MS analyses were performed on a Q Exactive plus (Thermo Fisher Scientific) coupled with an EasynLC1000 system (Thermo Fisher Scientific). Samples were reconstituted in 0.1% formic acid and pressure-loaded onto a 2 cm microcapillary precolumn packed with C18 (3 μm, 120 Å, SunChrom, USA). The precolumn was connected to a 12 cm 150 μm-inner diameter microcapillary analytical column packed with C18 (1.9 μm, 120 Å, Dr. Maisch GebH, Germany) and equipped with a homemade electrospray emitter tip. The spray voltage was set to 2.0 kV and the heated capillary temperature to 320 °C. LC gradient consisted of 0 min, 7% B; 14 min, 10% B; 51 min, 20% B; 68 min, 30% B; 69−75
ification or expensive stable isotopic labeling of test compounds. Moreover, none of these methods can determine the influence of drug bioactivation on active sites of most major CYP450 isoforms. Development of a general strategy to assess in vitro and in vivo bioactivation potential of drugs while determining global protein adduction by RMs in parallel remains a daunting challenge. Here we described a chemoproteomic platform to assess the influence of potential bioactivation of drugs on liver proteins in vitro and in vivo (Figure 1). Using this platform, we
Figure 1. Scheme of chemoproteomic workflow for assessment of in vitro (A) and in vivo (B) bioactivation potential of drugs.
simultaneously determined reactivity of thousands of proteomic cysteines toward RMs of diclofenac formed in human liver microsomes (HLMs) and mouse liver tissues. Among the targets of RMs of this drug were the active (heme-binding) sites of several important CYP450 isoforms (1A2, 2E1 and 3A4 for human, 2C39 and 3A11 for mouse) and a number of proteins involved in cellular redox regulation.
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EXPERIMENTAL SECTION
Chemicals and Reagents. IPM and 12C- and 13C-labeled azidoUV-biotin reagents were synthesized as described previously.9,10 HLMs including THP (X008061, pool of 10 Caucasian donors) and SUBK (LM-R-02M, pool of 10 male Mongolian donors) were purchased from RILD (China, Shanghai). NADPH was purchased from Roche (5650640001). Diclofenac (224122) and omeprazole (LS60R47) were purchased from J&K Chemicals. Dextromethorphan (P1057725) was purchased from Adamas Reagent, Ltd. (Shanghai, China). Phenacetin (WXBC0201 V), dextrorphan (UC205), and 5hydroxy omeprazole (43999) were purchased from Sigma-Aldrich. Taxol (Z-014-17046) was purchased from Chengdu Herbpurify Co., Ltd. 6α-OH taxol (10009027) was purchased from Cayman Chemicals. Acetaminophen (100018-200408) and letrozole (101045201101) were purchased from China National Institute for drug and biological products. HPLC-grade water, ACN, and MeOH were purchased from Thermo. Other chemicals and reagents were obtained from Sigma-Aldrich unless otherwise indicated. In Vitro Metabolism of Diclofenac. Twenty-five μL HLMs (20 mg/mL) were pre-suspended in 465 μL reaction buffer (0.1 M phosphate buffer containing 10 mM MgCl2, pH7.4) and pre-incubated with or without 100 μM diclofenac (0.5 μL of a 100 mM stock solution) at 37 °C for 5 min. Ten μL of a 50 mM NADPH in stock solution was added to initiate the reaction and then incubated at 37 °C for 1 h. In Vivo Metabolism of Diclofenac. This study received ethical and scientific approval in compliance with the animal care regulations of Institutional Animal Care and Use Committee, National Center for 1798
DOI: 10.1021/acs.chemrestox.7b00183 Chem. Res. Toxicol. 2017, 30, 1797−1803
Chemical Profile
Chemical Research in Toxicology
the linear regression lines of semilogarithmic plots (logarithm of remaining activity versus pre-incubation time). The reciprocal of kobs thus obtained was plotted against the reciprocal of the diclofenac concentration. Then, KI the inactivator concentration that produces half-maximal rate of inactivation, and kinact the inactivation rate constant were generated by the double reciprocal Lineweaver−Burk plot (1/kobs versus 1/[I]), which shows kinact estimate at reciprocal of the y-intercept and KI at negative reciprocal of the x-intercept.
min, 95% B (A = water, 0.1% formic acid; B = ACN, 0.1% formic acid) at a flow rate of 600 nL/min. MS1 spectra were measured with a resolution of 70,000, an AGC target of 3e6, a max injection time of 20 ms, and a mass range from m/z 300 to 1400. HCD MS/MS spectra were recorded in the data-dependent mode using a Top-20 method with a resolution of 17,500, an AGC target of 1e6, a max injection time of 60 ms, a 1.6 m/z isolation window and normalized collision energy of 30. Peptides m/z that triggered MS/MS scans were dynamically excluded from further MS/MS scans for 18 s. Peptide Identification and Quantification. Raw data files were searched against Homo sapiens Uniprot canonical database (December 2, 2016; 20,130 entries). Database search was performed with pFind studio (Version 3.0.11).12−14 Precursor ion mass and fragmentation tolerance was 10 and 20 ppm, respectively. The maximum number of modifications allowed per peptide was three, as was the maximum number of missed cleavages allowed. Different modifications of +15.9949 Da (methionine oxidation), +57.0214 Da (iodoacetamide alkylation), and +252.1222 (IPM-triazohexanoic acid) were searched as variable modifications. A differential modification of 6.0201 Da on IPM-derived modification was used for all analyses. The FDRs were estimated by the program from the number and quality of spectral matches to the decoy database. For all data sets, the FDRs at spectrum, peptide, and protein level were
