Comparison of the Reactivity of Trapping Reagents toward

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Comparison of the Reactivity of Trapping Reagents toward Electrophiles: Cysteine Derivatives Can Be Bifunctional Trapping Reagents Kazuko Inoue,* Katsuyuki Fukuda, Tsutomu Yoshimura, and Kazutomi Kusano Drug Metabolism and Pharmacokinetics Japan, Eisai Product Creation Systems, Eisai Co., Ltd., Tsukuba, Japan S Supporting Information *

ABSTRACT: Trapping reagents are powerful tools to detect unstable reactive metabolites. There are a variety of trapping reagents based on chemical reactivity to electrophiles, and we investigated the reactivity of thiol and amine trapping reagents to metabolically generated electrophiles and commercially available electrophilic compounds. Glutathione (GSH) and N-acetylcysteine (Nac) trapped soft electrophiles, and amine derivatives such as semicarbazide (SC) and methoxyamine (MeA) reacted as hard nucleophiles to trap aldehydes as imine derivatives. Cysteine (Cys) and homocysteine (HCys) captured both soft electrophiles and hard electrophilic aldehydes. There were no qualitative differences in trapping soft electrophiles among Cys, HCys, GSH, and Nac, although quantitative reactivity to trap soft electrophiles varied likely depending on the pKa values of their thiol group. In the reactivity with aldehydes, Cys and HCys showed relatively lower reactivity as compared with SC and MeA. Nonetheless, they can trap aldehydes, and the resulting conjugates were stable and detected easily because their amino group formed imines after reaction with aldehydes, which are successively attacked by the intramolecular thiol group to form stable ring structures. This report demonstrated that Cys and HCys are advantageous to evaluate the formations of both soft electrophiles and aldehyde-type derivatives from a lot of drug candidates at early drug discovery by their unique structural characteristics.

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INTRODUCTION Trapping methodology has been developed to capture reactive species mainly generated from CYP-mediated oxidation in vitro. Nucleophilic small molecular trapping reagents enable us to visualize unstable reactive metabolites as liquid chromatography/ mass spectrometry (LC/MS) detectable stable conjugates and give us awareness for optimization of drug candidates which are devoid of metabolic activation. Glutathione (GSH) is one of the most common nucleophiles employed as a trapping reagent for reactive metabolites. For screening drug candidates in the potentials for metabolic activation, other trapping reagents have also been developed. According to the hard and soft acids and bases principle, soft and hard electrophiles feasibly react with soft and hard nucleophiles, respectively.1 Metabolically generated soft electrophiles representing epoxides, quinones, and α,β-unsaturated aldehydes are trapped by soft nucleophilic thiol derivatives such as GSH, N-acetylcysteine (Nac), and cysteine (Cys). GSH analogues have also been reported as trapping reagents for assessing the potential risk of drug candidates © 2015 American Chemical Society

not only qualitatively but also quantitatively. Dansylated GSH has a luminescent moiety to quantify trapped reactive metabolites, and structural analysis of the dansylated GSH adduct can be conducted by LC/MS at the same time.2 Commercially available 35S-GSH and 35S-Cys have also been reported as semiquantitative trapping reagents, and the obtained radioactive adducts with electrophiles can be quantitatively detected by a radio-detector paralleled with LC/MS for structural analysis.3,4 However, iminium intermediates and aldehydes are classified as hard electrophiles and mainly trapped by hard nucleophilic cyanide and amine derivatives, respectively. Cyanide has been applied to trap iminium intermediates generated from the oxidation of aliphatic amine derivatives, and 14C-cyanide has been reported as a quantitative probe for the evaluation of the potential risk of covalent binding to protein by iminium.5,6 For trapping aldehyde derivatives, Received: March 26, 2015 Published: July 14, 2015 1546

DOI: 10.1021/acs.chemrestox.5b00129 Chem. Res. Toxicol. 2015, 28, 1546−1555

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Chemical Research in Toxicology amine trapping reagents, semicarbazide (SC) and methoxyamine (MeA), have been reported to capture them as imine derivatives.7,8 Although there are lots of options to evaluate reactive metabolites by various trapping reagents, the reactivity of trapping reagents toward electrophiles is not fully assessed. Thus, we addressed here the quantitative and qualitative evaluations for the reactivity of common nucleophiles we usually use for the trapping assay, by using well-investigated toxic compounds, alpidem, clozapine, troglitazone, and abacavir, and commercially available soft electrophilic monochlorobimane (MCB) and electrophilic aldehydes, 4-hydroxynonenal (4HN), 2-bromo-5(hydroxyl)benzaldehyde (BHBA), and N-benzyl piperidine-4carboxaldehyde (NBPC). Regarding Cys and HCys, they would work as bifunctional trapping reagents to trap soft electrophiles and hard electrophilic aldehydes based on their thiol and amine groups in their structures. Thus, we also compared the reaction mechanism of Cys and HCys with those of other common trapping reagents toward soft and hard electrophiles to clarify their characteristics.

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Figure 1. Structures of test compounds. Niles, IL) and a refrigerated vapor trap (RVT400, Thermo Fisher Scientific Inc.), and the residue was reconstituted with 50 μL of acetonitrile/water (3:7, v/v). The resulting suspension was centrifuged (4 °C, 9000g, 10 min, Kubota3520). The supernatant was subject to a Synapt G2 high definition mass spectrometer (HDMS)/ ultraperformance liquid chromatography (UPLC) system (Waters Corp., Milford, MA) to analyze the structures of adducts formed from test compounds and nucleophiles. Reaction Conditions for Quantitative Analysis. Quantitative analysis for the reactivity of nucleophilic trapping reagents to MCB, 4HN, BHBA, and NBPC was conducted by calculating the half-life (t1/2) values of disappearance of MCB, 4HN, BHBA, and NBPC in the presence of individual nucleophiles in 100 mM potassium phosphate buffer (pH7.4). MCB (10 μM) was incubated with 1 mM individual nucleophiles (GSH, Nac, Cys, HCys, SMCys, Lys, SC, and MeA) at 37 °C for 0−60 min in duplicate. The reactivity of nucleophiles to BHBA was investigated by incubating with BHBA (50 μM) and 5 mM individual nucleophiles (GSH, Nac, Cys, HCys, Lys, SMCys, MeA, and SC) in 100 mM potassium phosphate buffer (pH7.4) for 0−100 min (GSH, Nac, Lys, and SMCys), 0−80 min (Cys and HCys), and 0−20 min (SC and MeA), respectively, at 37 °C in duplicate. NBPC (50 μM) was incubated with 5 mM individual nucleophiles (GSH, Nac, Cys, HCys, Lys, SMCys, MeA, and SC) in 100 mM potassium phosphate buffer (pH7.4) for 0−120 min at 37 °C in duplicate. The reactions of MCB, BHBA, and NBPC were monitored by LC/MS after termination of the reactions by the addition of equal volumes of acetonitrile/methanol (2:1, v/v) with 5% formic acid (by volume) and 1 μM niflumic acid as internal standard. The reactivity of individual nucleophiles (each of 1 mM, GSH, Nac, Cys, HCys, SMCys, and Lys) to 4HN (20 μM) was investigated in triplicate at room temperature by monitoring UV at λ 225 nm (V-550, Rev. 1.00, JASCO International Co. Ltd., Tokyo, Japan) with 5-s intervals as previously reported.9 Reactions were kept for 0−10 min (Cys and HCys), 0−20 min (GSH and Nac), and 0−60 min (SMCys and Lys). As reference, individual nucleophiles without 4HN were prepared for each analysis. As negative control for the 4HN assessment, samples of 4HN without nucleophiles were used, and the reactions were monitored by UV at λ 225 nm for 0−60 min. Analytical Conditions by LC/MS. For qualitative analysis, structures of adducts formed from alpidem, clozapine, troglitazone, ABC, MCB, 4HN, BHBA, and NBPC reacting with each nucleophile were analyzed with an LC/MS system consisting of Synapt G2 HDMS and Acquity UPLC H-class operated by Masslynx software (version 4.1, Waters Corp.). Mass spectral analysis was conducted in positive (alpidem, clozapine, ABC, MCB, 4HN, and NBPC) and negative (troglitazone and BHBA) ion modes. Typical analytical settings of Synapt G2 HDMS were as follows: analyzer mode, resolution; capillary, 0.3−2.0 kV; sampling cone, 15−40 V; source temperature,

MATERIALS AND METHODS

Materials. Alpidem, niflumic acid, N-acetylcysteine (Nac), S-methylcysteine (SMCys), clozapine, monochlorobimane (MCB), and N-benzylpipelidine-4-carboxaldehyde (NBPC) were purchased from Sigma-Aldrich (St. Louise, MA). Abacavir (ABC) was purchased from Toronto Research Chemicals Inc. (Toronto, Canada). Reduced glutathione (GSH), dimethyl sulfoxide (DMSO), L-cysteine (Cys), L-lysine (Lys), acetonitrile, distilled water, troglitazone, leucineenkephalin, β-diphosphopyridine nucleotide from yeast (β-NAD), and formic acid were obtained from Wako Pure Chemical Industries (Osaka, Japan), and 2-bromo-5-(hydroxy)benzaldehyde (BHBA) was obtained from Matrix Scientific (Columbia, SC). β-Nicotine amideadenine dinucleotide phosphate, reduced form (β-NADPH), was purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). Semicarbazide hydrochloride (SC) and methoxyamine hydrochloride (MeA) were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Homocysteine (HCys) was obtained from Nakarai Tesque (Kyoto, Japan), and 4-hydroxynonenal (4HN) was obtained from Calbiochem (San Diego, CA). Human liver microsomes (mixed gender, pool of 50 individuals) and human liver cytosol (mixed gender, pool of 200 individuals) were purchased from Xenotech LLC (Lenexa, KS). Other reagents used were of analytical grade. Incubation Conditions for Qualitative Analysis. Structures of test compounds are shown in Figure 1. Alpidem, clozapine, troglitazone, ABC, MCB, NBPC, and BHBA were individually dissolved in DMSO, and 4HN was dissolved in acetonitrile to achieve 10 mM at final concentration. Each solution for GSH, Nac, Cys, HCys, SC, and MeA was prepared with 100 mM potassium phosphate buffer (pH7.4) at a final concentration of 50 mM. The incubation mixture consisted of 50 μM individual test compound (alpidem, clozapine, and troglitazone), 5 mM individual nucleophiles (GSH, Nac, and Cys), 1 mg/mL human liver microsomes, and 5 mM β-NADPH at final concentrations in 100 mM potassium phosphate buffer (pH7.4). Incubation was conducted for 120 min at 37 °C, and the reaction was terminated by the addition of equal volumes of acetonitrile/methanol (2:1, v/v). The resulting mixture was centrifuged at 4 °C and 9000g for 10 min (Kubota 3520, Kubota Corp., Tokyo, Japan). ABC (50 μM) was incubated with individual nucleophiles (each of 5 mM, GSH, Nac, Cys, HCys, Lys, SC, MeA, and SMCys) in 10 mM β-NADsupplemented human liver cytosol (1 mg/mL) at 37 °C for 24 h to achieve a total volume of 500 μL. The reaction was terminated by the addition of equal volumes of acetonitrile/methanol (500 μL, 2:1, v/v), and the resulting mixture was centrifuged at 4 °C and 9000g for 10 min (Kubota3520). The supernatant was evaporated by a Speedvac concentrator (SPD121P, Thermo Fisher Scientific Inc., Waltham, MA) coupled with a Chemstar vacuum pump (1402N, Welch Ilmvac, 1547

DOI: 10.1021/acs.chemrestox.5b00129 Chem. Res. Toxicol. 2015, 28, 1546−1555

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Chemical Research in Toxicology 150 °C; desolvation temperature, 600 °C; cone gas flow, 50 L/h; and desolvation gas flow, 600−1000 L/h. The mass spectrometer was calibrated with sodium formate (10 mM solution dissolved in water/ isopropanol = 1/9 by volume) in the mass range of 100−1500 Da. The mass spectral data were acquired by a centroid and continuously corrected by an external standard, leucine-enkephalin (2 ng/mL, at m/z 556.2771 in positive and at m/z 554.2615 in negative ion modes) infused by 10-s intervals at a flow rate of 50 μL/min. Full scan mass spectra were acquired from the mass range of m/z 100 to 1000 Da. Samples prepared from alpidem, clozapine, troglitazone, MCB, 4HN, BHBA, and NBPC were injected onto an Acquity BEH 2.1 × 50 mm column (Waters Corp.) with 5−10 μL at a flow rate of 0.7 mL/min. For alpidem, clozapine, and troglitazone, mobile phases consisted of 0.1% formic acid in water (mobile phase A) and of 0.1% formic acid in acetonitrile (mobile phase B). The initial mobile phase was 5% B and was kept for 0.5 min. Then, it was linearly increased to 90% B over 2.5 min and was kept 90% B in the next 0.5 min. For equilibration, the mobile phase was returned to the initial condition, and it was maintained over 1.5 min. For MCB, 4HN, BHBA, and NBPC, the initial mobile phase was 5% B and was kept for 0.5 min. Then, it was linearly increased to 50% B over 2.3 min and to 90% B in the next 0.2 min, and it was kept for 0.5 min. For equilibration, the mobile phase was returned to the initial condition, and it was maintained over 1.5 min. Samples prepared from ABC incubation were injected onto an Acquity BEH 2.1 × 50 mm column (Waters Corp.) with 5 μL at a flow rate of 0.7 mL/min. Mobile phases consisted of 100 mM ammonium acetate/water (1:9, v/v, mobile phase C) and of 100 mM ammonium acetate/acetonitrile (1:9, v/v, mobile phase D). The initial mobile phase was 5% D and was kept for 0.5 min. Then, it was linearly increased to 35% D over 2.3 min and to 90% D in the next 0.2 min, and was kept at 90% D for 0.5 min. For equilibration, the mobile phase was returned to the initial condition, and it was maintained over 1.5 min. Autosampler and column oven were kept at 10 and 40 °C, respectively, throughout the analysis. For quantitative analysis by LC/MS, a TSQ Quantum mass spectrometer coupled with an Accela Ultra HPLC system operated by Xcalibur software (version 2.0 SR, Thermo Fisher Scientific) was used. Typical mass analytical settings were as follows: capillary temperature, 300 °C; capillary voltage, 3500 V; vaporized temperature, 300 °C; sheath gas pressure, 60; and auxillary gas pressure, 30. Samples were injected onto an Acquity UPLC BEH 2.1 × 50 mm column (Waters Corp.) with 5 μL at a flow rate of 0.5 mL/min. Mobile phases consisted of 0.1% formic acid in water (mobile phase A) and of 0.1% formic acid in acetonitrile (mobile phase B). Gradient conditions and product ions monitored for each test compound were as follows: MCB, 0−0.5 min, 0% B; 0.5−2.8 min, 90% B; 2.8−3.5, 90% B; and 3.6−5.0 min, 0% B monitoring product ions at m/z 149, 164, and 191 of m/z 227 for MCB, and at m/z 265 of m/z 283 for niflumic acid (IS) in positive ion mode. NBPC: 0−1.0 min, 5% B; 1.0−3.0 min, 90% B; 3.0−4.9, 90% B; and 5.0−7.0 min, 5% B monitoring product ions at m/z 65 and 91 of m/z 204 for NBPC, and at m/z 265 of m/z 283 for IS in positive ion mode. Chromatographic separation conditions for BHBA; 0−0.5 min, 5% B; 0.5−3.0 min, 90% B; 3.0−4.9 min, 90% B; 5.0−7.0 min, 5% B monitoring product ions at m/z 79 of m/z 199 for BHBA, and at m/z 237 of m/z 281 for IS in negative ion mode. Data Analysis. Reactivity of nucleophiles to electrophiles tested (MCB, BHBA, and NBPC) was expressed as t1/2 (min) of disappearance of each electrophile under incubation with individual nucleophiles in this study using the following equation:

Graph Pad Prism 6 (GraphPad Software Inc., San Diego, CA) according to the following equation:

y = (y0 − plateau) × exp−kx + plateau t1/2 (min) = ln 2/k where k represents the reaction constant when electrophiles decrease by the reaction with nucleophiles. Plateau and y0 are y values at infinite time and y value at time zero, respectively. The disappearance curves of electrophiles in the incubation mixture were depicted in Supporting Information (MCB, Figure S1; BHBA, Figure S2; NBPC, Figure S3; and 4HN, Figure S4). Predicted pKa values were calculated using ACD software (version 10, Advanced Chemistry Development Inc., Ontario, Canada).

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RESULTS Qualitative Comparisons of Thiol Reagents as Nucleophiles to Trap Soft and Hard Electrophiles. Trapping properties for commonly used trapping reagents, GSH, Nac, Cys, HCys, SMCys, Lys, SC, and MeA, and their reactivity toward soft electrophiles and hard electrophilic aldehydes were quantitatively evaluated, and the mass spectral information on the adducts detected by an accurate mass spectrometer are summarized in Table 1. The mass spectra of each parent ion detected in this study are shown in Supporting Information (Figures S5−S43). To compare the trapping ability of thiol reagents (GSH, Nac, and Cys) to soft electrophiles, alpidem, clozapine, and troglitazone which have been reported to generate reactive metabolites10−13 were incubated with each of thiol reagent in NADPH-supplemented human liver microsomes. Alpidem formed conjugates with GSH, Nac, and Cys after incubation with NADPH-supplemented human liver microsomes, showing protonated molecular ions at m/z 709.1974 for the mono-GSH adduct (+305 Da, C31H39Cl2N6O7S+, +0.1 ppm, Figure S5, Supporting Information), m/z 565.1439 for the mono-Nac adduct (+161 Da, C26H31Cl2N4O4S+, +0.2 ppm, Figure S6, Supporting Information), and m/z 523.1328 for the mono-Cys adduct (+119 Da, C24H29Cl2N4O3S+, −0.8 ppm, Figure S7, Supporting Information) (Table 1). As shown in Figure 2A, the product ion spectra of each adduct showed at m/z 436 (parent +32 Da) corresponding to thiolated alpidem resulting from the neutral loss of the glutamylalanylglycine moiety from GSH, the N-acetylalanine moiety from Nac, and the alanine moiety from Cys added to alpidem. Alpidem was reported to be bioactivated at the chlorophenyl moieties, and the MS/MS spectra observed as the thiolated alpidem (m/z 436) suggested that nucleophiles tested would bind to the aromatic moieties of alpidem. The structures predicted from the product ions indicated that the nucleophiles would bind around the chlorophenyl rings of alpidem that could be identical to the previous report.10 Clozapine is known to be bioactivated to the reactive nitrenium intermediate leading to the formations of 6- and 9-glutathionyl clozapine11,13 with GSH, and mono-GSH, monoNac, and mono-Cys adducts of clozapine with protonated molecular ions at m/z 632.2053 (+305 Da, C28H35ClN7O6S+, 0 ppm, Figure S8, Supporting Information), 488.1525 (+161 Da, C23H27ClN5O3S+, +1.4 ppm, Figure S11, Supporting Information), and 446.1408 (+119 Da, C21H25ClN5O2S+, −0.9 ppm, Figure S15, Supporting Information), respectively, were detected (Table 1). The representative product ion at m/z 302 as shown in Figure 2B was thought to be the thiolated clozapine with a loss of 57 Da from the piperazine moiety of clozapine, suggesting that nucleophiles could trap the reactive

y = exp−kx t1/2 (min) = ln 2/k where k represents the reaction constant when electrophiles decrease by the reaction with nucleophiles. Because the reactivity of GSH, Nac, Cys, and HCys to 4HN was high, t1/2 values (min) of 4HN in the presence of these nucleophiles were calculated by fitting with a one phase exponential model using 1548

DOI: 10.1021/acs.chemrestox.5b00129 Chem. Res. Toxicol. 2015, 28, 1546−1555

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Chemical Research in Toxicology

Table 1. Summary of Mass Spectral Data of Adducts with Metabolically Generated or Commercially Available Electrophiles and Nucleophilesa adduct [M + H]+/[M − H]− (m/z) test compod (m/z)

+/−b

alpidem (404)

+

clozapine (327)

+

troglitazone (440) ABC (287)

−

MCB (227)

+

4HN (157)

+

BHBA (199) NBPC (204)

GSH 709.1974 (+GSH) 632.2053 (+GSH) 648.2011 (+GSH+O) 598.2449 (-Cl+GSH)

Nac

Cys

HCys

MeA

523.1328 (+Cys)

-d

-

-

488.1525 (+Nac)

446.1408 (+Cys)

-

-

-

504.1455 (+Nac+O) 506.1617 (+Nac+H2O) 474.1370 (-Me+Nac) 601.1672 (+Nac)

462.1293 (+Cys+O)

559.1575 (+Cys)

-

-

-

342.1794 (+SC-H2O) -

314.1726 (+MeA-H2O) -

236.1368 (+SC-O+Na) 255.9713 (+SC-O) 261.1721 (+SC-O)

168.1388 (MeA-O-H2O) 227.9642 (+MeA-O) 233.1649 (+MeA-O)

432.1262 (-Me+Cys) 412.1792 (-Cl+Cys)

745.2214 (+GSH) NDc

ND

388.1562 (+Cys-H2O)

402.1700 (+HCys-H2O)

376.0939 (+Nac-Cl+Na) 342.1351 (+2H +Nac+Na ND

312.1012 (+Cys-Cl)

326.1173 (+HCys-Cl)

381.1510 (+2H+2Cys-O)

409.1827 (+2H+2HCys-O)

−

498.1660 (+GSH-Cl) 464.2073 (+2H+GSH) ND

301.9491 (+Cys-O)

315.9636 (+HCys-O)

+

ND

ND

307.1484 (+Cys-O)

321.1639 (+HCys-O)

+

SC

565.1439 (+Nac)

a c

Mass spectra of the parent ions of the adducts above were shown in Supporting Information (Figures S5−S43). bPolarity in mass measurement. Evaluated but not detected. dNot evaluated.

Figure 2. Product ion spectra of alpidem, clozapine, and troglitazone incubated with GSH or Nac or Cys in NADPH-fortified human liver microsomes.

clozapine (m/z 598, Figure S10, Supporting Information) was formed possibly via glutathione S-transferases (GSTs) as reported previously.13 The same as GSH, Cys also generated a des-chlorinated Cys adduct with clozapine.14 Since Cys is not a cofactor of GSTs, a des-chlorinated Cys adduct with clozapine (m/z 412, Figure S18, Supporting Information) would be formed due to a spontaneous chemical reaction. Further

metabolite generated around the phenyl moieties of clozapine possibly via nitrenium intermediate formation. Since clozapine was metabolized to hydroxylated and des-methylated metabolites, which would be also subjected to bioactivation possibly generating the nitrenium intermediates, the variety of adducts with nucleophiles were similarly detected among GSH, Nac, and Cys (Table 1). The des-chlorinated GSH adduct with 1549

DOI: 10.1021/acs.chemrestox.5b00129 Chem. Res. Toxicol. 2015, 28, 1546−1555

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Chemical Research in Toxicology

Figure 3. Product ion spectra of ABC adducted with Cys (A), HCys (B), SC (C), and MeA (D) generated in β-NAD-fortified human liver cytosol.

investigation will be needed to clarify the mechanism for the formation of the des-chlorinated Cys adduct with clozapine. Troglitazone also yielded adducts with GSH, Nac, and Cys with molecular ions of the mono-GSH adduct at m/z 745.2214 (+305 Da, C34H41N4O11S2−, −0.7 ppm, Figure S19, Supporting Information), mono-Nac adduct at m/z 601.1672 (+161 Da, C29H33N2O8S2−, −2.0 ppm, Figure S20, Supporting Information), and the mono-Cys adduct at m/z 559.1575 (+119 Da, C27H31N2O7S2−, −0.5 ppm, Figure S21, Supporting Information) after incubation in NADPH-fortified human liver microsomes (Table 1). The adducts of troglitazone with nucleophiles afforded the same patterns of the product ion spectra shown at m/z 438, which correspond to dehydrogenated troglitazone with a neutral loss of GSH (−307 Da), Nac (−163 Da), and cysteine (−121 Da) molecule from each adduct. Troglitazone was known to be bioactivated around chromane and thiazolidinedione moieties,12 and the neutral loss of nucleophile in the product ion scan in this study also suggested that the reactive metabolite would be formed around thiazolidinedione or chromane moieties (Figure 2 C). Additionally, MCB, a substrate of GSTs,15 chemically formed mono-GSH (MCB-Cl+GSH, m/z 498.1660, C20H28N5O8S+, +1.4 ppm, Figure S26, Supporting Information), mono-Nac (MCB-Cl+Nac+Na, m/z 376.0939, C15H19N3NaO5S+, +0.3 ppm, Figure S27, Supporting Information), and mono-Cys (MCB-Cl +Cys, m/z 312.1012, C13H18N3O4S+, −0.3 ppm, Figure S28, Supporting Information) adducts with GSH, Nac, and Cys in phosphate buffer (pH7.4), respectively, resulting from the displacement of chlorine of MCB with nucleophiles (Table 1). The typical chlorine isotope pattern of MCB disappeared in the mass spectrum of each adduct indicating that the displacement of chlorine with nucleophile should occur to form each deschlorinated MCB conjugated with nucleophile (Figures S26−S28, Supporting Information). On the basis of these results, thiol groups of GSH, Nac, and Cys are qualitatively identical in the reactivity to these soft electrophiles. Qualitative reactivity of nucleophiles toward hard electrophilic aldehydes was investigated using ABC that was known to form a reactive aldehyde metabolite and commercially available

aldehydes such as 4HN (α,β-unsaturated aldehyde), BHBA (benzaldehyde), and NBPC (aliphatic aldehyde). Since ABC was known to form a reactive aldehyde metabolite catalyzed by alcohol dehydrogenase localized in the liver cytosolic fraction, ABC was incubated with each nucleophile in β-NAD-fortified human liver cytosol for 24 h. Although GSH, Nac, Lys, and SMCys did not form any adducts with ABC, Cys, HCys, SC, and MeA formed adducts with ABC showing protonated molecular ions at m/z 388.1562 (ABC-H2O+Cys, C17H22N7O2S+, +3.1 ppm, Figure S22, Supporting Information), m/z 402.1700 (ABC-H2O+HCys, C18H24N7O2S+, −1.7 ppm, Figure S23, Supporting Information), m/z 342.1794 (ABC-H2O+SC, C15H20N9O+, +0.9 ppm, Figure S24, Supporting Information), and m/z 314.1726 (ABC-H2O +MeA, C15H20N7O+, −1.0 ppm, Figure S25, Supporting Information), respectively (Table 1). The estimated formula based on accurate mass detections (ABC-H2O+nucleophiles) indicated that ABC was metabolized to the aldehyde metabolite (−2H), and the aldehyde reacted with nucleophiles accompanied by the loss of oxygen (−O) to generate imine derivatives as adducts (Table 1). The product ion spectra of each ABC-adduct with nucleophile exhibited almost the same pattern in each other as shown in Figure 3, and the product ions at m/z 134, 174, and 191 suggested that SC and MeA would bind to the cyclopenten moiety as imine as shown in Figure 3C and D, respectively. Since SMCys did not react with ABC, Cys and HCys were estimated to bind to the cyclopenten moiety of ABC as cyclized forms as shown in Figure 3A and B, respectively. As a result, Cys, HCys, SC, and MeA trapped the metabolically formed reactive aldehyde generated from ABC. Meanwhile, 4HN, an α,β-unsaturated aldehyde,16,17 reacted with both of the thiol and amine reagents, such as GSH, Nac, Cys, HCys, SC, and MeA. GSH and Nac formed adducts with 4HN showing the protonated molecular ions at m/z 464.2073 (4HN+2H+GSH C19H34N3O8S+, +2.6 ppm, Figure S30, Supporting Information) as a hydrogenated mono-GSH adduct and at m/z 342.1351 (4HN+2H+Na+Nac, C14H25NNaO5S+, +1.5 ppm, Figure S31, Supporting Information) as a hydrogenated mono-Nac adduct (Table 1). Amine derivatives, SC and MeA, 1550

DOI: 10.1021/acs.chemrestox.5b00129 Chem. Res. Toxicol. 2015, 28, 1546−1555

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Chemical Research in Toxicology

Figure 4. Adducts of 4HN with soft and hard nucleophiles.

NBPC, an aliphatic aldehyde, also formed adducts with Cys, HCys, SC, and MeA corresponding to imine derivatives which showed protonated molecular ions at m/z 307.1484 (NBPC +Cys-O, C16H23N2O2S+, +2.9 ppm, Figure S40, Supporting Information), at m/z 321.1639 (NBPC +Cys-O, C17H25N2O2S+, +2.5 ppm, Figure S41, Supporting Information), at m/z 261.1721 (NBPC +SC-O, C14H21N4O+, +4.2 ppm, Figure S42, Supporting Information), and at m/z 233.1649 (NBPC+MeA-O, C14H21N2O+, +0.4 ppm, Figure S43, Supporting Information) with elimination of an oxygen from NBPC (Table 1). GSH, Nac, Lys, and SMCys did not generate any adducts with BHBA and NBPC in this study. The results of the reactivity of nucleophiles toward aldehydes indicated that Cys, HCys, SC, and MeA could trap any types of aldehydes qualitatively; however, the reaction mechanisms in Cys and HCys are different from those in SC and MeA. Quantitative Analysis for the Reactivity of Nucleophilic Trapping Reagents. Quantitative analyses for the reactivity of nucleophilic trapping reagents to soft and hard electrophiles were investigated using commercially available electrophiles, MCB (soft electrophile), 4HN (soft and hard electrophilic aldehyde), BHBA (aromatic aldehyde), and NBPC (aliphatic aldehyde). Reaction was conducted in physiological conditions (100 mM potassium phosphate buffer, pH 7.4) at room temperature (4HN) or 37 °C (MCB, BHBA, and NBPC). Reactivity was expressed as t1/2 for the disappearance of electrophiles tested, and the results are summarized in Table 2. MCB reacted with soft nucleophiles, GSH, Nac, Cys, and HCys, resulting in t1/2 values of 18, 53, 17, and 39 min, respectively, although it did not react with hard nucleophiles, SMCys, Lys, SC, and MeA. The extent of reactivity to MCB was as follows: GSH = Cys > HCys > Nac. On the basis of the predicted pKa values of thiol groups of GSH (9.25), Cys (8.54), Nac (10.13), and HCys (9.27) shown in Table2, the extent of the reactivity in physiological conditions (pH7.4), which are usually employed in the trapping assay system, would relate to

formed adducts with 4HN accompanied by a loss of an oxygen to generate an imine substructure with 4HN, and protonated molecular ions were observed at m/z 236.1368 (4HN-O+SC, C10H19N3NaO2+, −0.4 ppm, Figure S34, Supporting Information) as sodium adduct for SC and at m/z 168.1388 as the dehydrated form (4HN-O+SC-H2O, C10H18NO+, +3.0 ppm, Figure S35), which would be due to ion source fragmentation with the loss of water, for MeA (Table 1). Different from other reagents, two molecules of Cys or HCys reacted with one molecule of 4HN to form di-Cys and di-HCys adducts of 4HN with loss of oxygen and addition of hydrogens, respectively, and the protonated molecular ions were detected at m/z 381.1510 (4HN+2H−O+2Cys, C15H29N2O5S2+, −0.5 ppm, Figure S32, Supporting Information) as the di-Cys adduct and m/z 409.1827 (4HN+2H−O2HCys, C17H33N2O5S2+, +0.5 ppm, Figure S33, Supporting Information) as the di-HCys adduct (Table 1). The structures of di-Cys and di-HCys adducts of 4HN were proposed in Figure 4. One molecule of Cys or HCys would bind to 4HN with the addition of hydrogens (+2H) the same as thiol derivatives, GSH and Nac, and another molecule would bind to the aldehyde moiety to form imine structure the same as amine derivatives, SC and MeA. On the basis of these results, the adduct formation mechanism of Cys and HCys toward electrophilic aldehydes was thought to be different from GSH and Nac as well as SC and MeA. BHBA, a benzaldehyde, formed imine derivatives with Cys, HCys, SC, and MeA showing protonated molecular ions at m/z 301.9491 (BHBA+Cys-O, C10H9BrNO3S−, −0.3 ppm, Figure S36, Supporting Information), at m/z 315.9636 (BHBA+HCys-O, C11H11BrNO3S−, −4.1 ppm, Figure S37, Supporting Information), at m/z 255.9713 (BHBA+SC-O, C8H7BrN3O2−, −5.5 ppm, Figure S38, Supporting Information), and at m/z 227.9642 (BHBA+MeA-O, C8H7BrNO2−, −10.5 ppm, Figure S39, Supporting Information) with a loss of an oxygen from BHBA (Table 1). Since BHBA contains bromine in the structure, bromine specific signals enable the easy detection of adducts with nucleophiles (Figures S36−S39, Supporting Information). 1551

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Chemical Research in Toxicology Table 2. Predicted pKa and t1/2 of Electrophiles to Nucleophiles Tested pKaa nucleophiles

-SH

GSH Nac Cys HCys SMCys Lys MeA SC

9.25 10.13 8.54 9.27

trapping reagents GSH and Nac as soft nucleophiles and SC and MeA as hard nucleophiles. Cys and HCys had a similar reactivity with GSH toward soft electrophiles generated from well-known toxicants such as alpidem, clozapine, and troglitazone, bioactivated mainly by CYP.10−12 As summarized in Table 1, accurate mass spectra of adducts of test compounds with GSH, Nac, and Cys indicated that these nucleophiles formed the same types of adducts with each toxicant. As shown in Figure 2, representative adducts of GSH, Nac, and Cys with metabolically formed reactive metabolites of alpidem (A), clozapine (B), and troglitazone (C) gave the same patterns of product ions with a loss of substructure of GSH (−273 Da or −307 Da), Nac (−129 Da or −163 Da), and Cys (−87 Da or −121 Da). It suggested that Nac and Cys could trap reactive metabolites in the same manner with GSH. Qualitative and quantitative assessments of reactivity of nucleophiles as thiol trapping reagents were investigated by using MCB15 that is known to form a GSH adduct accompanied by the displacement of chlorine of MCB with GSH. MCB also formed adducts with Nac and Cys in the same manner with GSH to generate des-chlorinated MCB adducts with nucleophiles (Table 1), indicating that thiol groups of Cys and Nac have the same ability with that of GSH in the adduct formation with soft electrophiles, qualitatively. Since Nac is a cysteine derivative which has an amino residue modified by an N-acetyl group, the amino groups in Cys would not be involved in trapping soft electrophiles. However, quantitative analysis of reactivity for thiol reagents in this study demonstrated the different reactivities with MCB among GSH, Nac, Cys, and HCys. As shown in Table 2, the reactivity of thiol groups in nucleophiles was expressed as the half-life of MCB in the presence of GSH, Nac, Cys, and HCys, and Nac exhibited the weakest reactivity to MCB with a t1/2 value of 53 min compared with those of 18 and 17 min by GSH and Cys, respectively. To consider this difference in chemical reactivity among these thiol reagents, the predicted pKa values of each nucleophile as shown in Table 2 may explain that since Nac was estimated to show the highest pKa value of the thiol group among them, the reactivity of Nac to MCB in phosphate buffer (pH7.4) would be the lowest of all. In addition, based on the expected pKa values, Cys and HCys would show the same quantitative reactivity with GSH toward soft electrophiles. These results suggested that Cys and HCys could work as thiol trapping reagents for the soft electrophiles comparable to GSH. The reactivity of the cysteinyl amino group toward aldehydes was also investigated to demonstrate that Cys and HCys have the potential to trap hard electrophilic aldehydes the same as amine derivatives for trapping reagents. As the model compounds, commercially available BHBA and NBPC, aromatic and aliphatic aldehydes, respectively, were used for the quantitative and qualitative evaluations. Aldehydes were known to be captured by amine derivatives such as SC and MeA, and aldehydes tested in this study successfully formed adducts with SC and MeA as shown in Table 2. Cys and HCys also showed reactivity toward BHBA and NBPC. Although NBPC itself gradually hydrolyzed to a corresponding carboxylic acid in phosphate buffer (pH 7.4), MeA, SC, Cys, and HCys reacted with NBPC to form adducts (Table 2). The same as NBPC, MeA, SC, Cys, and HCys also reacted with BHBA (Table 2). However, GSH, Nac, SMCys, and Lys did not give any adducts with both BHBA and NBPC, indicating that their thiol and amino groups were inactive to trap aldehyde derivatives. As proposed in Figure 6, amine derivatives (Cys, HCys, MeA,

t1/2b (min)

-NH2

MCB

4HN

BHBA

NBPC

11.05 10.44 8.97 10.64(ε) 9.48 (α) 4.66 3.5

18 53 17 39 ND ND ND ND

5 11 0.6 2 >60 >60 ND ND

>100 >100 79 74 >100 >100 3 1

>60 >60 49 51 >60 52 35 59

a Predicted pKa of the sulfhydryl group (ACD software). bt1/2, calculated half-life; ND, evaluated but not detectable. MCB was incubated at 10 μM with nucleophiles at 1 mM. Incubation of 4HN was conducted at 20 μM and room temperature with nucleophiles at 1 mM. BHBA and NBPC were incubated at 50 μM with nucleophiles at 5 mM.

acidity of their thiol groups. These results also suggested that Cys and HCys would have the same reactivity with GSH toward soft electrophiles and that Nac would show the weakest reactivity among them. The reactivity of nucleophiles to 4HN was investigated by monitoring the decline of 4HN in the presence of nucleophiles by UV at λ 225 nm. Without nucleophiles, 4HN was stable in the 60 min incubation period. In the presence of GSH, Nac, Cys, and HCys, 4HN showed t1/2 values of 5, 11, 0.6, and 2 min, respectively (Table 2). SC and MeA were highly reactive toward 4HN. However, since the resulting adducts of 4HN with SC or MeA also showed the same absorbance with unchanged 4HN, the reactivity of SC and MeA to 4HN could not be evaluated as t1/2 values. The adduct formation with 4HN and SC or MeA ended in a few seconds based on the LC/MS measurement by consuming all of the 4HN added resulting in showing almost zero percentage of remaining 4HN. From these results and observation, the reactivity order of nucleophiles to 4HN was shown to be as follows: SC = MeA > Cys > HCys > GSH > Nac, and the reaction of 4HN with SMCys and Lys to 4HN was not observed. BHBA did not react with GSH, Nac, Lys, and SMCys, and it reacted with Cys, HCys, SC, and MeA with t1/2 values of 79, 74, 3, and 1 min, respectively (Table 2). The reactivity order of nucleophiles to BHBA was as follows: MeA = SC > Cys = HCys. NBPC was gradually decomposed to the corresponding carboxylic acid (N-benzylpiperidine-4-carboxylic acid) in the potassium phosphate buffer (pH7.4), and it showed a certain extent of t1/2 value (>60 min), even though there was no nucleophile added in the mixture. As shown in Table 2, t1/2 values of NBPC were shorter under the presence of Cys, HCys, Lys, and MeA than that without nucleophiles. Formation of imine derivatives of NBPC with Cys, HCys, SC, and MeA were observed based on structural analysis as described above (Table 1). There were no adducts of NBPC with GSH, Nac, Lys, and SMCys. On the basis of these results, NBPC reacted with Cys, HCys, SC, and MeA to form imine derivatives but not with GSH, Nac, Lys, and SMCys.

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DISCUSSION To show Cys and HCys as useful bifunctional trapping reagents, we investigated the reactivity of Cys and HCys with soft and hard electrophiles by comparing with common 1552

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mass detections (Table 1), and the product ions of these adducts showed almost the same pattern as that shown in Figure 3, suggesting that Cys and HCys also trap the reactive aldehyde of ABC as reported previously.17 GSH, Nac, Lys, and SMCys did not form any adducts with ABC implied thiol groups of GSH and Nac, and amino groups of SMCys and Lys could not trap the aldehyde metabolite of ABC as the stable adduct. However, since aldehyde can be trapped by Cys but not by SMCys, the thiol groups of Cys and HCys would also play an important role to trap reactive aldehyde as the stable conjugate by cyclization. Additionally, 4HN, a reactive α,β-unsaturated aldehyde, was used as a substrate to assess the reactivity of Cys and HCys. Both of the soft nucleophilic thiol reagents, GSH, Nac, Cys, and HCys, and hard electrophilic amine derivatives, SC and MeA, reacted with 4HN in potassium phosphate buffer (pH 7.4). As shown in Table 1, GSH and Nac generated adducts with 4HN accompanied by the addition of two hydrogens (4HN + 2H + nucleophile), possibly via Michael addition to 4HN (Figure 4). In the meantime, SC and MeA reacted with the aldehyde moiety of 4HN with elimination of oxygen resulting in the formation of imines as proposed in Figure 4. Interestingly, two molecules of Cys or HCys formed adducts with one molecule of 4HN with the addition of hydrogens (+2H) accompanied by loss of oxygen (−O). As proposed in Figure 4, the estimated structure of these adducts indicated one molecule of Cys or HCys would bind to 4HN via Michael addition the same as GSH and Nac, and another molecule of Cys or HCys reacted with aldehyde moiety to form imine-like structure as observed in SC and MeA. Cys is reported to react with 4HN by two molecules of it.16,17 On the basis of these reports, Cys can react with the aldehyde moiety of 4HN to form a thiazolidine structure as product, and therefore, the structures of adducts of 4HN with Cys and HCys observed in this study would also be cyclized five- and six-membered ring structures as shown in Figure 4. To investigate importance of the intramolecular thiol group of Cys to trap 4HN, SMCys that has an amino group to trap aldehyde but does not have a free thiol group in the structure was exposed to 4HN in potassium phosphate buffer. SMCys did not give any adduct with 4HN, indicating that the intramolecular thiol group of Cys and HCys should play a

Figure 5. Proposed reaction schemes of Cys derivatives with aldehyde.

and SC in this work) can trap aldehydes as imine derivatives, and in the case of Cys and HCys, further reaction between the resulting imine and intramolecular thiol group would occur, resulting in cyclization to five- or six-membered ring structures, respectively. On the basis of these structural characters of Cys and HCys, they can qualitatively work as hard nucleophiles like amine derivatives such as SC and MeA to trap hard electrophilic aldehydes. Since Cys and HCys successfully trap both soft electrophiles and hard electrophilic aldehydes, the bifunctionality of Cys and HCys toward soft electrophiles and hard electrophilic aldehydes in the trapping assay system was investigated. ABC is known to form a reactive α,β-unsaturated aldehyde metabolite mediated by alcohol dehydrogenase, and the aldehyde of ABC was thought to covalently bind to protein leading to toxic incidence in humans. ABC was reported to form adducts with MeA as the imine derivative17 and with Cys as a conjugate with the α,β-unsaturated aldehyde moiety of ABC aldehyde via Michael addition.18 In the present study, Cys, HCys, SC, and MeA formed adducts with the aldehyde metabolite of ABC with a loss of water (ABC-H2O+Nucleophiles) based on accurate

Figure 6. Proposed reaction schemes of amine derivatives with aldehydes. 1553

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possibly resulting in toxic incidence, and the use of appropriate trapping reagents to detect reactive species should be helpful to optimize the prototype structures for a safe drug without metabolic activation. In conclusion, among the nucleophiles tested in this study, Cys (and HCys) was the most powerful and versatile trapping reagent for reactive metabolites at an early stage to optimize the compounds against potential toxicity caused by metabolic activation.

significant role to trap 4HN as shown in Figure 5. These results clearly indicated that both Cys and HCys could work as bifunctional trapping reagents to trap both soft electrophiles and hard electrophilic aldehyde. Aldehydes sometimes generate toxicological implications likely to form imine with amine groups of protein as the covalent products,18−20 and therefore, they should also be paid attention to as one of the reactive metabolites. In this study, Lys that possesses an ε-amino group as the nucleophile responsible for covalent binding with aldehydes did not react with aldehydes in potassium phosphate buffer (pH7.4). Lys was reported to form an imine with n-octanal in the presence of H2O2, and reactive aldehydes such as α,β-unsaturated aldehydes are known to form covalent adducts with the Lys residue in protein.21,22 The amino group of Lys was thought to react with aldehyde derivatives to give corresponding imine derivatives; however, the imine derivatives would be easily hydrolyzed in the common conditions of the trapping experiment, whereas Cys and HCys were able to finally form stable ring structures with aldehydes. Thus, Cys or HCys seemed to work as one of the suitable probes to evaluate the potential risk of aldehyde formation in drug candidates. The unique reactivity of Cys derivatives to form a stable adduct with aldehyde has been published. Barve et al. reported that Cys and HCys formed five- and six-membered ring structures with aldehyde probes as Cys derived thiazolidine-4carboxylic acid and HCys derived thiaziname-4-carboxylic acid, respectively, to monitor several diseases.23 Paroxetine, a known CYP2D6 inactivator, formed several GSH conjugates with an orthoquinone metabolite resulting from oxidation of methylene dioxide moiety of paroxetine, and a unique cyclized cysteinylglycine conjugate of paroxetine after hydrolysis of the glutamyl moiety of the GSH conjugate was reported.24 A homomorpholine derivative formed corresponding thiazolidines with Cys derivatives after the elimination of glutamic acid by γ-glutamyltranspeptidase from the GSH adduct.25 Furthermore, the GSH thioester of diclofenac was reported to be converted from the S-cysteinylglycine conjugate to the N-cysteinylglycine conjugate via intramolecular nucleophilic attack by the free amino group after hydrolysis of the GSH adduct mediated by γ-glutamyltranspeptidase.26 Taking into account these unique characteristics of Cys, Cys and HCys were expected to show unique reactivity to not only soft electrophiles but also hard electrophilic aldehyde derivatives based on the structural characteristic to form stable five- and six-membered ring structures. Since any type of reactive metabolite in many compounds is better to be assessed as much as possible at drug discovery in a simple way, the current study indicated that Cys (and HCys) can work as the most effective trapping reagent to evaluate the risk of bioactivation of drug candidates. In summary, we demonstrated the reactivity of nucleophiles toward reactive metabolites and commercially available electrophiles. The thiol group of Cys exhibited the same reactivity to soft electrophiles as that of GSH, qualitatively and quantitatively. Different from GSH and Nac, Cys and HCys can also trap a variety of aldehydes qualitatively similar to amine derivatives such as SC and MeA by forming stable five (Cys)or six (HCys)-membered ring derivatives. These results indicated that Cys and HCys could work as bifunctional trapping reagents to trap both soft and hard electrophiles. Trapping methodology is one of the effective ways to assess the potential risk of drug candidates to form covalent binding

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ASSOCIATED CONTENT

S Supporting Information *

Disappearance curves of MCB, BHBA, NBPC, and 4HN as results of reactions with or without nucleophiles (GSH, Nac, Cys, HCys, and SMCys for MCB, and GSH, Nac, Cys, HCys, SMCys, Lys, SC, and MeA for BHBA, NBPC, and 4HN) and mass spectra of parent ions of adducts discussed in this study and included in Table 1. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.5b00129.

■

AUTHOR INFORMATION

Corresponding Author

*5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, Japan. Tel: +81-29-847-5684. Fax: +81-29-847-5672. E-mail: k12-inoue@ hhc.eisai.co.jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. George Lai for scientific discussions in this work. We also thank Tomoki Nishioka for calculation of the expected pKa values of nucleophiles and Dr. Tomomi Ishida for editorial support of this work.

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ABBREVIATIONS ABC, abacavir; BHBA, 2-bromo-5-(hydroxy)benzaldehyde; Cys, cysteine; HCys, homocysteine; GSTs, glutathione S-transferases; HDMS, high definition mass spectrometer; 4HN, 4-hydroxynonenal; LC/MS, liquid chromatography/mass spectrometry; Lys, L-lysine; MCB, monochlorobimane; MeA, methoxyamine; Nac, N-acetylcysteine; NBPC, N-benzylpiperidine 4-carboxaldehyde; SC, semicarbazide; SMCys, S-methyl cysteine; UPLC, ultraperformance liquid chromatography

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REFERENCES

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