Abacavir Forms Novel Cross-Linking Abacavir Protein Adducts in

Article pubs.acs.org/crt

Abacavir Forms Novel Cross-Linking Abacavir Protein Adducts in Patients Xiaoli Meng,† Alexandre S. Lawrenson,‡ Neil G. Berry,‡ James L. Maggs,† Neil S. French,† David J. Back,† Saye H. Khoo,† Dean J. Naisbitt,† and B. Kevin Park*,† †

MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, University of Liverpool, Sherrington Building, Ashton Street, Liverpool L69 3GE, United Kingdom ‡ Department of Chemistry, University of Liverpool, Crown Street, Liverpool L69 7ZD, United Kingdom S Supporting Information *

ABSTRACT: Abacavir (ABC), a nucleoside-analogue reverse transcriptase inhibitor, is associated with severe hypersensitivity reactions that are thought to involve the activation of CD8+ T cells in a HLA-B*57:01-restricted manner. Recent studies have claimed that noncovalent interactions of ABC with HLAB*57:01 are responsible for the immunological reactions associated with ABC. However, the formation of hemoglobin-ABC aldehyde (ABCA) adducts in patients exposed to ABC suggests that protein conjugation might represent a pathway for antigen formation. To further characterize protein conjugation reactions, we used mass spectrometric methods to define ABCA modifications in patients receiving ABC therapy. ABCA formed a novel intramolecular crosslinking adduct on human serum albumin (HSA) in patients and in vitro via Michael addition, followed by nucleophilic adduction of the aldehyde with a neighboring protein nucleophile. Adducts were detected on Lys159, Lys190, His146, and Cys34 residues in the subdomain IB of HSA. Only a cysteine adduct and a putative cross-linking adduct were detected on glutathione S-transferase Pi (GSTP). These findings reveal that ABC forms novel types of antigens in all patients taking the drug. It is therefore vital that the immunological consequences of such pathways of haptenation are explored in the in vitro models that have been used by various groups to define new mechanisms of drug hypersensitivity exemplified by ABC.

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INTRODUCTION Abacavir (ABC; Scheme 1), a nucleoside-analogue reverse transcriptase inhibitor, is associated with a high incidence of hypersensitivity reactions. The reactions involve the HLA-B*57:01restricted activation of drug-specific IFN-γ-producing cytotoxic CD8+ T cells.1 Multiple hypotheses have been proposed to explain the biochemical or physiological basis of ABC’s hypersensitivity. Most recently, several groups have shown that ABC can interact directly with exquisite selectivity to endogenous and cell surface HLA-B*57:01.2−6 Modification of the tertiary structure of endogenous HLA-B*57:01 by ABC alters the loading of self-peptides into the antigen binding groove, and it has been proposed that the T-cell response in hypersensitive patients might be directed against peptide antigens only displayed on the cell surface in the presence of the drug. Nevertheless, these findings do not exclude a parallel hapten mechanism of T-cell activation or the involvement of ABC metabolites in costimulatory signaling. ABC undergoes extensive oxidative metabolism in humans,7 yielding a number of isomeric carboxylic acids, which is inactivated by glucuronidation.8 We have also demonstrated that three carboxylic acids are formed in the cytosol of human antigen presenting cells.8−10 Formation of the carboxylate has been shown to proceed through a two-step oxidation process, via a © 2014 American Chemical Society

reactive aldehyde intermediate. A metabolic pathway involving double bond migration and epimerization of the aldehyde has been proposed.8 Oxidation by alcohol dehydrogenase8 and bond migration yields two putative intermediates: an unconjugated aldehyde and a more electrophilic conjugated aldehyde (Scheme 1), which rationalizes the observation of isomeric carboxylic acids. Synthetic β,γ-unsaturated ABC aldehyde (ABCA) readily rearranges to the α,β-unsaturated form.10 The α,β-unsaturated intermediate could react spontaneously with protein by Michael addition and/or Schiff base adduction, generating a conjugate with the potential to activate T-cells.10 Charneira et al. have shown that the conjugated aldehyde reacts with the N-terminal valine residue of hemoglobin in vitro10 and in rats and patients.11,12 The aims of this study, therefore, were to define, in detail, the chemical mechanisms of protein conjugate formation by ABCA in vitro, using human serum albumin (HSA) and glutathione S-transferase pi (GSTP) as model proteins, and to search rigorously for similar HSA conjugates formed in patients receiving ABC therapy that have the potential to trigger the immunological signals responsible for ABC hypersensitivity in patients. Received: November 4, 2013 Published: February 17, 2014 524

dx.doi.org/10.1021/tx400406p | Chem. Res. Toxicol. 2014, 27, 524−535

Chemical Research in Toxicology

Article

Scheme 1. Possible Metabolic Pathways of Abacavir

Table 1. ABCA-Modified Cys34 Peptide of HSA Detected in Patientsa time courseb (h) patient

gender

age

ABC daily dose (mg)

1 2

M M

36 51

600 600

3 4

F F

44 38

600 600

medication Kivexa, Nevirapine, calcium, ergocalciferol Kivexa, Nevirapine, Citalopram, Acyclovir, Qvar Kivexa, Efavirenz Kivexa, Kaletra

period of treatment (years)

adr to abc

0

2

4

6

8

2.5 3.7

no no

+ +

+ +

+ +

+ +

+ +

3.8 4.4

no no

− +

− +

− +

− +

+ +

a A tryptic digest of HSA isolated by immunoaffinity chromatography was analyzed by LC-MS/MS. ALVLIAFAQYLQQC*PFEDHVK was modified by the addition of ABCA to Cys34. bBlood samples were taken at predosing (0 h), then 2, 4, 6, and 8 h postdosing.

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9 volumes of ice-cold methanol to remove noncovalently bound ABCA, resuspended in 50 μL of phosphate buffer, and then reduced with 10 mM dithiothreitol (15 min) and alkylated with 55 mM iodoacetamide (15 min) at room temperature. The protein was precipitated with methanol once more and finally dissolved in 100 μL of 50 mM ammonium hydrogencarbonate, and 165 μg (1.25 nmol) of protein was digested with 16 μg of trypsin overnight at 37 °C. The samples were processed for liquid chromatography−tandem mass spectrometry (LC-MS/MS) analysis as described previously.14 Time-Dependent Modification of Serum Albumin by ABCA in Vitro. ABCA freshly dissolved in methanol/0.067 M potassium phosphate buffer (1:1, v/v) was incubated with HSA (0.6 mM, 300 μL) in phosphate buffer at 37 °C (final ABCA concentration, 6 mM). The final concentration of methanol was 5%. Aliquots of 50 μL were removed after 30 min, 1, 2, 3, 16, and 24 h and processed for LC-MS/MS analysis as described above. Concentration-Dependent Modification of His-GSTP by ABCA in Vitro. His-GSTP was expressed in E.coli as described previously.15 Purified His-GSTP captured on nickel beads was incubated with a range of molar ratios of ABCA to protein (1:1−100:1) in phosphate buffer at pH 7.4 for 16 h. The beads were then washed 5 times with 1000 μL of phosphate buffer. The protein was subjected to on-bead tryptic digestion as described previously.16 In brief, a suspension of beads in 30 μL of 50 mM ammonium bicarbonate buffer was incubated with 20 ng of trypsin for 16 h at 37 °C, and the digest was analyzed by LC-MS/MS. Time-Dependent Modification of His-GSTP by ABCA in Vitro. Purified His-GSTP captured on nickel beads was incubated with ABCA at an aldehyde-to-protein molar ratio of 10:1 in phosphate buffer at pH 7.4 and 37 °C. The incubations were stopped at the following time points: 12, 30, 60, and 90 min. The recovered beads were then washed 5 times with1000 μL of phosphate buffer. The modified protein was subjected to on-bead tryptic digestion followed by LC-MS/MS analysis. Isolation of Serum Albumin from Plasma by Affinity Chromatography. HSA was isolated by affinity chromatography as described previously.14,17 A POROS anti-HSA affinity cartridge

METHODS AND MATERIALS

Chemicals. HSA (97−99% pure) was purchased from SigmaAldrich (St.Louis, MO), trypsin from Promega (Madison, WI), LC-MS grade solvents from Fisher Scientific UK Ltd. (Loughborough, Leicestershire), and all other standard reagents from Sigma-Aldrich. Synthetic α,β-unsaturated ABCA was provided by Dr. Alexandra Antunes, Universidade Técnica de Lisboa. The composition of this material was confirmed by reversed-phase LC-electrospray MS (Rt 11.9 min; Agilent Eclipse column, 5-μm C18, 150 mm × 4.6 mm; acetonitrile/0.1% formic acid−10 mM ammonium formate, pH 4.1, 5% to 20%, 40 min, 1 mL/min), parent ion ([M+H]+ at m/z 285; aminocyclopropylaminopurine fragment at m/z 191) and by derivatization with methoxylamine.8 The isomeric aldoxime derivatives (minor, Rt 24.5 min; major, Rt 26 min; ratio of peak heights in the m/z 314 chromatogram, 5.8) yielded [M+H]+ at m/z 314 and indistinguishable electrospray product-ion spectra containing abundant peaks at m/z 282 (loss of CH3OH) and m/z 191 (base peak). Patients. Plasma samples were obtained from the Liverpool HIV Therapeutic Drug Monitoring Registry, which has received ethics approval from The Northwest Multicenter Research Ethics Committee. The samples were from four patients (Table 1) who had received ABC at 600 mg once daily for at least two years and were considered fully adherent (self-report and with durable virological suppression (plasma VL < 50 copies/mL)). On the morning of the sampling visit, a predose blood sample was drawn at baseline 0 h. Further sampling was performed at 0, 2, 4, 8, and 12 h postdosing (Table 1). Blood (5 mL) was collected in lithium heparin-containing tubes and was centrifuged at 700g and 4 °C within 6 min to obtain plasma. Plasma was heatinactivated (58 °C, 40 min) and stored at −20 °C.13 Concentration-Dependent Modification of Serum Albumin by ABCA in Vitro. ABCA freshly dissolved in methanol/0.067 M potassium phosphate buffer (1:1, v/v) was incubated with HSA (0.6 mM, 100 μL) in phosphate buffer at 37 °C for 16 h. The molar ratios of ABCA to protein were 1:1, 10:1, 50:1, and 100:1. The final concentration of methanol was 5%. Protein was precipitated twice with 525



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Figure 1. Analysis of human GSTP modified by ABCA in vitro. (A−C) MS/MS spectra of unmodified tryptic peptide 45ASCLYGQLPK54 containing the highly reactive Cys47 residue (A); ABCA-modified peptide 45ASC*LYGQLPK54 with a mass addition of 284 amu (ABCA) at Cys47 (B); in-source generated fragment of ABCA-modified 45ASC*LYGQLPK54 with a mass addition of 76 amu at Cys47 (assigned to a residue of ABCA remaining on the peptide after cleavage of a cross-linking adduct between the cysteine and serine residues) (C); and semiquantitative analysis of ABCA-modified GSTP showing the modifications were concentration dependent (red line, addition of ABCA; blue line, cross-linking adducts) (D). Mass Spectrometric Characterization of ABCA-Modified Proteins. In order to acquire accurate MS/MS characterization of modified peptides, samples were analyzed using a Q-TOF instrument.18 Samples were delivered into a Triple TOF 5600 mass spectrometer (AB Sciex) by automated in-line reversed phase liquid chromatography, using an Eksigent NanoUltra cHiPLC System (AB Sciex) mounted with

(Applied Biosystems, Foster City, CA) attached to a Vision Workstation (Applied Biosystems) was used to affinity-capture HSA from 50 μL of freshly thawed plasma. The HSA was eluted with 12 mM HCl and precipitated with 9 volumes of ice-cold methanol. The eluted protein was processed for LC-MS/MS analysis as described above. 526



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a microfluidic trap and analytical column (15 cm × 75 μm) packed with ChromXP C18−CL at 3 μm. A NanoSpray III source was fitted with a 10 μm inner diameter PicoTip emitter (New Objective, MS). Samples were loaded in 0.1% formic acid onto the trap, which was then washed with 2% ACN/0.1% FA (v/v) for 10 min at 2 μL/min before switching in-line with the analytical column. A gradient of 2−50% (v/v) ACN/ 0.1% (v/v) FA over 90 min was applied to the column at a flow rate of 300 nL/min. Spectra were acquired automatically in positive ion mode using information-dependent acquisition powered by Analyst TF 1.5.1 software, using mass ranges of 400−1600 amu in MS and 100− 1400 amu in MS/MS. Up to 25 MS/MS spectra were acquired per cycle (approximately 10 Hz) using a threshold of 100 counts per s, with dynamic exclusion for 12 s and rolling collision energy. Modified peptides were identified by filtering for specific fragment ions in PeakView 1.2.0.3 (AB Sciex) and manual inspection of the spectra. For multiple reaction monitoring (MRM) detection of modified peptides, samples were reconstituted in 2% ACN/0.1% formic acid (v/v), and aliquots of 2.4−5 pmol of tryptic digests were delivered into a QTRAP 5500 (AB Sciex, Framingham, MA, USA) fitted with a NanoSpray II source by automated in-line liquid chromatography (U3000 HPLC System, 5 mm C18 nanoprecolumn and 75 μm × 15 cm C18 PepMap column [Dionex, Sunnyvale, CA]) via a 10 μm inner diameter PicoTip (New Objective). A gradient from 2% ACN/0.1% FA (v/v) to 50% ACN/0.1% FA (v/v) in 60 min was applied at a flow rate of 300 nL/min. The ion spray potential was set to 2,200−3,500 V, the nebulizer gas to 19, and the interface heater to 150 °C. MRM transitions specific for ABCA-modified peptides were selected as follows: the mass/ charge ratio (m/z) values were calculated for all possible peptides containing a histidine, lysine, or cysteine residue together with a characteristic b or y fragment ion of each of the selected peptides. MRM transitions were acquired at unit resolution in both the Q1 and Q3 quadrupoles to maximize specificity. They were optimized for collision energy and collision cell exit potential, and the dwell time was 50 ms. MRM survey scans were used to trigger enhanced product ion scans of ABCA-modified peptides, with Q1 set to unit resolution, dynamic fill selected, and dynamic exclusion for 20 s. Total ion counts were determined from a second aliquot of each sample analyzed by conventional LC-MS and were used to normalize sample loading on the column. Relative quantification of modified peptides was performed by comparing the relative normalized intensity of MRM peaks for each of the modified residues against total ion counts across samples. Data were analyzed using Analyst software (AB Sciex). Computer Modeling of the Docking of ABC and ABCA to HSA and HLA-B*57:01. ABC and its aldehyde tautomers underwent energy minimization in preparation for docking. This was performed in Spartan ’08 software (Wave function Inc., Irvine, CA) using Equilibrium Geometry, Molecular Mechanics−MMFF. The protein structures into which these compounds were docked were those of HSA protein (PDB accession code 2bxm) and HLA-B*57:01 protein (PDB accession code 3upr). Prior to the docking study, suitable protocols were developed that could reproduce the binding pose of the native ligands in these crystal structures. Details of the modeling method are provided in Supporting Information.

The modification of Cys47 by ABCA resulted in two types of adducts corresponding to doubly charged ions at m/z 682.36 and 578.26, respectively. The adduct corresponding to m/z 682.36, which eluted at 36.2 min, represented a mass increase of 284 compared to that of the unmodified peptide and corresponded to a Michael adduct formed by the reaction of ABCA and Cys47. The sequence of the ABCA-modified peptide 45ASCLYGQLPK54 was confirmed by the MS/MS spectrum (Figure 1B). An abundant ion at m/z 285.15 corresponding to protonated ABCA provided the first-line evidence of modification. A series of b ions such as b4* (m/z 659.3) and b7*(m/z 1007.5), and a ions such as a5* (m/z at 794.4), a5*+2 (m/z 397.7), and a7*+2 (m/z 490.2), all with adduction of 284 amu, confirmed the addition of ABCA to Cys47. This was the only adduct detected as a complete structure in the GSTP samples. However, because of the presence of a reactive aldehyde functional group, we anticipated that the initial Michael adduct would further react with an adjacent nucleophilic residue to form a cross-linking adduct (Scheme 2). Indeed, a peptide corresponding to a doubly charged ion of m/z 578.26 was detected. We deduced that the ion at m/z 578.26 could correspond to a hemiacetal formed by reaction of the aldehyde of the ABCA-Cys47 Michael adduct with the adjacent serine residue in the sequence 45ASCLYGQLPK54. Further loss of a water molecule and in-source cleavage of the purine moiety would give a mass shift of 76 amu (Scheme 2). Although the initial crosslinking adduct would be expected to exhibit the same mass increment as the Michael adduct, it is worth noting that it was eluted at a different retention time compared to that of the Michael adduct (38.6 versus 36.2 min), indicating that these two types of adducts were present independently prior to chromatographic separation. The deduction was supported by the MS/MS spectrum shown in Figure 1C. The peptide sequence was confirmed by a series of y product ions, and the site of the modification was evidenced by the presence of b3*(m/z 338.1), y8* (m/z 997.51), and y9* (m/z 1084.53), all with a mass increment of 76 amu. Interestingly, an abundant ion of m/z 580.26 corresponding to a mass addition of 80 amu to peptide 45ASCLYGQLPK54 was also detected (Figure S2A,B, Supporting Information). Comparison of extracted chromatographic peaks corresponding to 76 and 80 amu adducted peptides revealed the same retention time, indicating that the 80 amu adduct was a MS-induced product from the cross-linking adduct. As such, both the 76 and 80 amu adducts will be referred to as cross-linking adducts in this study. Although GSTP contains four cysteine residues, only Cys47 was found to be modified by ABCA. The modification of Cys47 was detectable at the lowest molar ratio of ABCA to protein (1:1), while the extent of modification increased when the molar ratio of aldehyde to protein was increased to 10:1 and above (Figure 1D). Characterization of HSA Modified by ABCA in Vitro. To probe the chemical basis of the putative Michael and Schiff base adducts of ABCA and HSA, HSA was incubated with a range of molar ratios of ABCA (1:1 to 50:1). LC-MS/MS analysis of the tryptic digests revealed six ABCA-modified peptides containing cysteine, histidine, or lysine residues after incubation at the highest concentration of ABCA and three modified peptides when the aldehyde to protein ratio was 1:1 (Table 2). Similar to the modifications observed in GSTP, modification of Cys34 in HSA resulted in two types of adducts: Michael and cross-linking adducts. A triply charged ion of m/z 906.4 was detected, corresponding to the Michael adduct derived from the peptide 21ALVLIAFAQYLQQCPFEDHVK41 with an additional

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RESULTS Characterization of ABCA-Modified GSTP in Vitro. To characterize the Michael addition adducts of synthetic α,βunsaturated ABCA and protein, human GSTP which contains several reactive cysteine residues was chosen as a model.19 HisGSTP captured on nickel beads was exposed to a range of molar ratios of ABCA to protein (1:1−1:50). Mass spectrometric analysis of tryptic digests in the absence of ABCA identified the unmodified Cys47-containing peptide 45ASCLYGQLPK54. The peptide corresponding to a doubly charged ion of m/z 540.3 that eluted at 37.2 min was detected, and the peptide sequence was confirmed by a series of y product ions (m/z 244.2, 357.3, 542.3, 705.4, 818.5, and 921.4) shown on the MS/MS spectrum (Figure 1A). 527



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Scheme 2. Proposed Structure of ABCA Protein Conjugates

Table 2. ABCA-Modified HSA and GSTP Tryptic Peptides Detected in Vitroa molar ratio (ABCA/protein)b protein HSA

GSTP a

time course (min)

mass (obs.)

ΔM (amu)

Rt (min)

peptide sequence

modified sites

1:1

5:1

10:1

50:1

12

30

60

90

533.6 712.4 660.4 546.5 608.3 676.4 838.4 906.4 682.4 578.3

266 266 266 284 266 284 266 284 284 266

31.1 76.8 83.3 69.5 91.2 85.1 72.1 75.9 36.2 38.5

LDELRDEGK*ASSAK RHPYFYAPELLFFAK*R* R*H*PYFYAPELLFFAK RH*PYFYAPELLFFAK H*PYFYAPELLFFAK H*PYFYAPELLFFAK ALVLIAFAQYLQQ*C*PFEDHVK ALVLIAFAQYLQQC*PFEDHVK ASC*LYGQLPK AS*C*LYGQLPK

Lys190/? Lys159/Arg160 His146/Arg145 His146 His146/N-terminus-NH2 His146 Cys34/Gln33 Cys34 Cys47 Cys47/Ser46

− − + − + − + − + +

− − + − + − + − + +

+ + + + + − + + + +

+ + + + + + + + + +

− − − − − − + + + +

− − + + − + + + + +

− − + + − + + + + +

− − + + + + + + + +

ABCA and protein were incubated in 0.067 M potassium phosphate buffer at pH 7.4, at 37 °C. bIncubated for 16 h.

cross-linking adduct as it was eluted at the same chromatographic time as the 94 amu adduct (Figure 2B and Figure S3b, Supporting Information). Two of the ABCA-modified peptides contained a histidine residue (His146). Figure 2C shows a representative MS/MS spectrum for a triply charged ion at m/z 660.35, corresponding to the tryptic peptide 145RHPYFYAPELLFFAK160 with an additional mass of 80 amu. The peptide sequence was confirmed by MS/MS fragmentations that generated partial singly charged y and b series ions. The modification site was determined to be a cross-linking between His146 and Arg145 because all fragment ions including b2* (m/z 374.21), a4*(m/z 607.34), b4* (m/z 634.32), b5* (m/z 781.4), b6* (m/z 944.47), b7* (m/z 1015.52), and b9* (m/z 1241.63) are 80 amu higher than those expected. (Figure 2C). The cross-linking adduct could be formed from the reaction of the Arg145 side chain with the initial ABCA-His146 Michael adduct in the context of the intact protein (Scheme 2). Alternatively, this adduct could be formed by reaction of the N-terminal amino group of Arg145 with the

mass of 284 amu (Figure S3a, Supporting Information). This ABCA-modified Michael adduct eluting at 75.9 min was only present at extremely low levels as its chemically labile aldehyde group could further react with other nucleophiles. In fact, a peptide with a mass addition of 94 amu, corresponding to a triply charged ion of m/z 843.11, was eluted at a different chromatographic time (72 vs 75.9 min), indicating that these two types of adducts were present independently prior to chromatographic separation. The addition of 94 amu is attributed to cross-linking between the Michael adduct of Cys34 and the adjacent Gln33, and in-source cleavage of the purine moiety would give a mass shift of 94 amu (Scheme 2). The sequence of the peptide was confirmed by the MS/MS spectrum shown in Figure 2A: a series of y ions including y16* +2 (m/z 1009.98), y17*+2 (m/z 1066.53), and y18* (m/z 1123.07), all with a mass addition of 94 amu, providing substantive evidence of cross-linking between Cys34 and Gln33. In addition, an adduct corresponding to a mass addition of 80 amu to the Cys34 containing peptide (+3, m/z 838.48) was also detected, presumably derived from the 528



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Figure 2. continued

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Figure 2. Identification of HSA residues modified by ABCA in vitro. MS/MS spectrum showing the ABCA-modified tryptic peptide 21ALVLIAFAQYLQQC*PFEDHVK41 with a cross-linking adduct formed between Gln33 and Cys34 with a mass addition of 94 amu (A) and 80 amu (B); MS/MS spectrum of 145RH*PYFYAPELLFFAK159 modified by cross-linking ABCA at His146 and Arg145 with a mass addition of 80 amu (C); semiquantitative analysis of ABCA-modified HSA peptides showing that ABCA modification is concentration-dependent (D) and time-dependent (E); and tertiary structure of HSA showing the ABCA binding sites detected in vitro. Protein was rendered as ribbons, the modified histidine residue rendered as a blue sphere, cysteine residue as a red sphere, and lysine residues as green spheres (F).

Figure 3. ABCA-modified HSA was isolated from abacavir-treated patients. MS/MS spectrum of the tryptic peptide 21ALVLIAFAQYLQQC*PFEDHVK41 with ABCA conjugated at Cys34 by Michael addition (A); MS/MS spectrum of a cross-linking adduct formed between Gln33 and Cys34 (B). 530



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Figure 4. Time courses of formation of ABCA−protein conjugates. Kinetics of the ABCA-modified Cys47 peptide of GSTP formed in vitro (A); kinetics of the ABCA-modified Cys34 peptide of HSA formed in vitro (B); and kinetics of the modified Cys34 and His146 peptides of HSA detected in plasma isolated from three of the four patients (C, D, and E).

confirmed by the presence of a characteristic purinyl fragment ion (m/z 191.2). The observation of several doubly charged ions, e.g., y9*+2 (m/z 694.1) and y12*-H2O+2 b4*(m/z 887.6), provided further evidence that ABCA was attached to Cys34. The Michael adducts only presented at low abundance; the crosslinking adducts appeared to be predominant. The formation of crosslinking adducts was evidenced by a triply charged ion at m/z 838.26, corresponding to the peptide 21ALVLIAFAQYLQQCPFEDHVK41 with an additional mass of 80 amu (Figure 3B). The presence of y13*+2 (m/z 857.39), y15*+2 (m/z 966.45), y16*+2 (m/z 1001.97), y*17+2 (m/z 1058.51), y18*+2 (m/z 1115.06), and y19*+2 (m/z 1164.59), all with a mass increment of 80 amu, provided further evidence for the formation of cross-linking adducts. In addition to modified cysteine, ABCA-modified His146 containing peptide HPYFYAPELLFFAK was also detected in the tryptic digest of one patient’s HSA (Figure S3e, Supporting Information). Time Course and Reversibility of Protein Modification by ABCA. A semiquantitative analysis was carried out to measure the ABCA modification over time. The reaction of ABCA with Cys47 in GSTP appeared to be extremely fast, with modification detected at 10 min. The level of modification peaked after 1 h of incubation, followed by a decline (Figure 4A). The same trend was observed in in vitro incubations with HSA (Figure 4B). Molecular Modeling of Interactions of ABCA and ABC with HSA and HLA-B*57:01. Docking of ABC and ABCA with HSA revealed several potential binding areas including a site in subdomain IB (Figure S4 a, Supporting Information). High docking scores were observed when either ABC or ABCA was docked into the IB site, indicating strong binding affinities at this site (Figure S4 b, Supporting Information). ABCA were then covalently linked to Cys34, His146, Lys159, and Lys190 to perform covalent docking. In all cases, covalent docking formed similar or stronger affinities than were observed in noncovalent binding. In the cases of His146, Lys159, and Lys190, binding was considerably stronger (Figure S4 c, Supporting Information). In order to investigate how the cross-linking adducts might be formed on HSA, docking of ABCA cross-linked to Cys34 and

ABCA-His146 Michael adduct during tryptic digestion. Initial modification of His146, by Michael addition, was confirmed by the observation of ABCA-modified peptide 145RHPYFYAPELLFFAK160 with a mass addition of 284 amu (Figure S3c, Supporting Information). In addition to histidine modification, ABCA was found to form lysine adducts on HSA. Two ABCA-modified peptides containing a lysine residue, namely, Lys159 and Lys190, were detected when HSA was incubated with an aldehyde to protein ratio above 10:1. The cross-linking adduct in 145RHPYFYAPELLFFAKR160 could be formed between Lys159 and Arg160. However, the cross-linking sites in 182LDELRDEGKASSAK195 cannot be determined by a MS/MS spectrum. A typical MS/MS spectrum representing 145RHPYFYAPELLFFAKR160 modified by ABCA at Lys159 is shown in Figure S3d (Supporting Information). The modification of HSA by ABCA was found to be concentrationand time-dependent. A semiquantitative analysis of modification at each site was performed by determining the peak area for the extracted masses of the modified peptides, followed by normalization of the ion intensity using the total ion count for the sample. A concentration- and time-dependent increase in normalized ion counts was observed for each modified peptide (Figure 2D and E). In addition, a significant depletion of unmodified peptides from the native HSA over time was also observed (Figure 2D). A summary of ABCA-modified HSA is presented in Figure 2E. Modification of HSA by ABCA in Patients. ABCAmodified HSA was isolated from HIV patients receiving ABC 600 mg once daily for at least two years. The ABCA-modified Cys34 containing peptide 21ALVLIAFAQYLQQCPFEDHVK41 was detected in all four patients. Similarly, as observed in vitro, modification of Cys34 resulted in two types of adducts: a Michael adduct and a putative cross-linking adduct between Gln33 and Cys34 (Figure 3A and B). A triply charged ion of m/z 906.4 was observed, corresponding to the peptide 21 ALVLIAFAQYLQQCPFEDHVK41 with an additional mass of 284 amu. The peptide sequence was confirmed by the MS/MS spectrum as shown in Figure 3A. Modification by ABCA was also 531



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Figure 5. Molecular modeling presentations showing the interactions of ABC and ABCA with HSA and HLA B*57:01. The cross-linking adduct formed between ABCA and peptide 21ALVLIAFAQYLQQCPFEDHVK41 (Cys34 and Gln33) of HSA is stabilized by several potential H-bond and van der Waals interactions (A); the interactions of ABCA with PepV (HSITYLLPV) were greatly improved when ABCA is covalently bound to Ser116 of HLA B*57:01 (B); replacement of Ser116 with Tyr116 led to the reduction of binding affinity between ABC and PepV, the steric hindrance being observed at the hemiacetal covalent bond (orange circle) (C); and the comparison of docking scores observed when either ABC or ABCA was docked into HLA B*57:01 (D). Protein was rendered as cyan ribbons and other molecules rendered as sticks (carbon, gray; nitrogen, blue; and oxygen, red).

Gln33 of 21ALVLIAFAQYLQQCPFEDHVK41 was performed. As shown in Figure 5A, the cross-linking adduct is stabilized by a few of the surrounding amino acids: the potential for a good Hbond between Thr83 and a purine H-atom, and an H-bond between the NH2 and Asp107 was observed; there are also good van der Waals interactions at the cyclopropane group. We next explored the potential interaction of ABCA with HLA B*57:01, a leukocyte antigen variant which is intimately associated with the hypersensitivity of ABC. Ser116 is a crucial HLA B*57:01 residue with regards to the cellular process initiating the hypersensitivity reaction, and the exchange of Ser116 for Tyr116 is enough to abrogate recognition by ABCspecific CD8+ T cells in vitro.2 Covalent docking was therefore performed between ABCA and Ser116. As shown in Figure 5B, ABCA binds very well in the pocket, and strong H-bond interactions are observed between PepV (HSITYLLPV) and the ligand at the amine of the purine ring, and the amine linker between the purine and the cyclopropane group. The docking score was greatly improved, suggesting that ABCA has a strong binding interaction when bound covalently to Ser116, certainly much stronger than the noncovalently bound (free) molecule (Figure 5D). Because of the critical functional consequences of replacing Ser116 with Tyr116, further docking experiments were performed to try and rationalize this observation with regard to the result above. When ABC was docked noncovalently with Tyr116 form of HLA B*5701, the docking score fell (Figure 5D). This effect may be due to the steric bulk of the Tyr116 residue which distorts the orientation of ABC in this site, as well as the loss of

the interaction with the original Ser116 residue and Ile124 (Figure S4 d, Supporting Information) Additionally, ABC no longer interacts well with PepV, suggesting that the Tyr116 exchange might indeed result in the loss of key interactions and hindered binding. This was consistent with the previous observations.3 Importantly, when ABCA was covalently docked into the Tyr116 form of HLA B*57:01, the score dropped further, suggesting further loss of binding interactions (Figure 5D). In addition, there are no close contacts between ABCA and PepV, and the steric hindrance observed at the hemiacetal covalent bond (Figure 5C, orange circle) would suggest that this binding mode is largely unfavorable.

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DISCUSSION Diverse experimental approaches have led to multiple mechanisms to explain ABC hypersensitivity.3−6,20 Consistent with the hapten hypothesis,21 reactive metabolites of ABC, specifically the aldehyde intermediates, form adducts with self-proteins or peptides, generating neo-antigens that could induce an immune response. However, it has not been possible to explore this mechanism in patients because neither the proteins that ABC modifies in drug-exposed patients nor the epitopes formed on proteins which may function as antigens have been characterized. In this study, we have found that ABCA forms an intramolecular cross-linking adduct on HSA in patients and in vitro via Michael addition followed by nucleophilic adduction of the aldehyde with a neighboring nucleophile on the protein. As ABC is not protein reactive per se, certain bioactivation pathways must 532



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the formation of both the Michael adducts and cross-linking adducts is reversible. Therefore, the reversible binding with HSA may serve as a “transport system” to deliver aldehyde metabolites to extrahepatic tissues, including immune cells. Such a mechanism has been invoked for the intracellular transport of the reactive metabolite of paracetamol, NAPQI, from the endoplasmic reticulum to the mitochondria. A 1,2 addition product of GSH serving as a latent form of NAPQI has been proposed to distribute the metabolite to other target organelles.30 ABCA was found to react selectively with nucleophilic side chains in the subdomain IB of HSA. Many acidic, neutral, and basic small molecules are known to bind non-covalently to subdomain IB,31,32 which is now recognized, together with the more familiar Sudlow sites in subdomains IIA and IIIA,32,33 as a major drug binding region of the protein. Whereas subdomain IIA contains residues such as Lys195 and Lys199 that are well known to react with a wide variety of low molecular weight electrophiles, including drugs and drug metabolites,14,18,23,24 none of those residues was modified detectably by ABCA. This selectivity can be explained by the presence of adjacent aromatic and basic side chains, which favor, respectively, noncovalent interactions that facilitate covalent binding and the neutral, i.e., nucleophilic, forms of the adducted residues. The docking analyses confirmed that ABCA has strong binding interactions at this site, positioning ABCA in a pose conducive to the Michael adduct. In addition, much stronger interactions were observed in the case of covalent binding than noncovalent binding of ABCA to HSA. More importantly, with respect to biological effects, docking of ABCA with HLA B*57:01 confirmed that ABCA has strong interactions when bound covalently to Ser116, a key residue with regards to recognition by ABC-specific CD8+ T cells. Given that ABCA could form adducts with Ser46 in GSTP in vitro, a possible role of covalent binding of ABCA to HLA B*5701 in ABC hypersensitivity could not be discounted. The role of ABCA-protein adducts in the adverse reactions to ABC in patients remains to be determined. Given that the median time to onset of symptoms is 11 days,34 a long-lasting interaction between ABC and the immune system would be expected. It is therefore plausible that ABC-haptenated proteins or peptides may be involved in immune-mediated reactions. Nevertheless, ABCA could attach covalently to MHC molecules and subsequently alter the presentation of self-peptides. In this case, a strong and stable immune complex among ABCA, peptides, and MHC will be formed as demonstrated in the modeling studies. Taken together, these observations indicated that the involvement of reactive metabolites of ABC in the activation of the immune system, most-likely after the formation of protein or peptide adducts, could not be totally excluded at present. Further studies should explore in detail whether ABCA-protein or ABCA-peptide conjugates are able to be immunogenic and therefore play a role in ABC hypersensitivity, through various types of interactions with HLA B*57:01. In summary, we have shown that ABCA can form novel crosslinking adducts and Michael adducts with proteins in vitro. Furthermore, the same types of adduct were detected in HSA isolated from ABC-treated patients. Although further studies will be needed to determine whether such protein adducts have any involvement in the mechanisms of ABC’s hypersensitivity reactions, our discoveries provide a unique framework from which to identify naturally processed HLA peptides and explore the biological function of ABCA-haptenated proteins.

have been involved in protein conjugation. A mass balance study has shown that following a 600 mg oral dose of 14C-ABC, the majority of radioactivity detected in plasma (92%) was attributed to ABC and its two major metabolites, a carboxylic acid and a glucuronide.7 ABC undergoes extensive oxidative metabolism in humans,7 forming a number of isomeric carboxylic acids and glucuronide metabolites (Scheme 1).8−10 Consistent with the previous findings, a number of isomeric carboxylic acids were also detected in the plasma of all patients recruited in this study (Figure S1a, Supporting Information). Formation of the carboxylate has been shown to proceed through a two-step oxidation process, via a reactive aldehyde intermediate. However, these intermediates have only been identified in vitro as stabilized derivatives. The N-terminal hemoglobin (Hb) adducts found previously11 thus provided substantive evidence for the formation of reactive aldehyde metabolites in patients that bind covalently to endogenous proteins. To probe the structures of ABCA adducts formed on HSA in patients, in vitro studies were performed using HSA and human GSTP as model proteins because both proteins are targets for numerous electrophilic drugs and drug metabolites.14,18,19,22−24 We showed that ABCA covalently binds to cysteine, histidine, and lysine residues on HSA and to the highly reactive Cys47 in GSTP in vitro. ABCA, as an α,β-unsaturated aldehyde, has the potential to form adducts with protein nucleophiles through two distinct mechanisms: Schiff base formation, in which the carbonyl carbon reacts with the amino group of lysine or N-terminal residues to form an unstable imine via dehydration of a carbinolamine, and Michael (1,4-) addition to the activated β-carbon of ABCA. Moreover, the aldehyde group of the Michael addition adducts could, in theory, react further with other nucleophilic groups, producing intermolecular and intramolecular cross-linking adducts. Interestingly, although several types of adducts from Michael addition and subsequent cross-linking were assigned in the present study (Scheme 2), no simple Schiff base adducts, such as the familiar glycation derivatives of ε-amino groups,24 were detected even when high molar ratios of aldehyde to protein were employed. The cross-linking adducts could be formed within proteins, e.g., the adduct between Arg145 and His146, and the one between Gln33 and Cys34. Alternatively, cross-links could be formed by reaction of the N-terminal amino group with the initial Michael adducts during and after trypsin digestion, e.g., the cross-linking adducts at His146 (HPYFYAPELLFFAK). ABCA appeared to favor Michael addition, which is consistent with the protein reactivity of other α,β-unsaturated aldehydes25−28 and the diminished adduction of HSA by [14C] dihydroabacavir.8 This preference can be explained by electronwithdrawing carbonyl groups, enhancing the α-carbon’s electrophilicity and the electron donating double bond decreasing the carbonyl carbon’s electrophilicity. The relative rates of ABCA adduct formation at cysteine and histidine residues of HSA, estimated from LC-MS/MS analyses of modified tryptic peptides in time-course experiments, are apparently much faster than the rates at lysine residues. At low aldehyde concentrations and at early time-points, Cys34 and His146 appeared to be the kinetically preferential targets for ABCA. On the basis of the time course of ABCA adduct formation, the reactivity of residues decreases in the following order: Cys34 > His146 > Lys159 ≈ Ly190. This ranking confirms generally the observation that alkyl α,β-unsaturated aldehydes combine with side chains of a multi-nucleophile peptide in the rank order Cys ≫ His > Lys > Arg.29 Interestingly, a decline of ABCA-HSA adducts over time was also observed, indicating that 533



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Cells from HLA-B*57:01+ human subjects are activated with abacavir through two independent pathways and induce cell death by multiple mechanisms. Chem. Res. Toxicol. 26, 759−766. (7) McDowell, J. A., Chittick, G. E., Ravitch, J. R., Polk, R. E., Kerkering, T. M., and Stein, D. S. (1999) Pharmacokinetics of [14C]abacavir, a human immunodeficiency virus type 1 (HIV-1) reverse transcriptase inhibitor, administered in a single oral dose to HIV-1infected adults: a mass balance study. Antimicrob. Agents Chemother. 43, 2855−2861. (8) Walsh, J. S., Reese, M. J., and Thurmond, L. M. (2002) The metabolic activation of abacavir by human liver cytosol and expressed human alcohol dehydrogenase isozymes. Chem.-Biol. Interact. 142, 135− 154. (9) Bell, C. C., Santoyo Castelazo, A., Yang, E. L., Maggs, J. L., Jenkins, R. E., Tugwood, J., O’Neill, P. M., Naisbitt, D. J., and Park, B. K. (2013) Oxidative bioactivation of abacavir in subcellular fractions of human antigen presenting cells. Chem. Res. Toxicol. 26, 1064−1072. (10) Charneira, C., Godinho, A. L., Oliveira, M. C., Pereira, S. A., Monteiro, E. C., Marques, M. M., and Antunes, A. M. (2011) Reactive aldehyde metabolites from the anti-HIV drug abacavir: amino acid adducts as possible factors in abacavir toxicity. Chem. Res. Toxicol. 24, 2129−2141. (11) Charneira, C., Grilo, N. M., Pereira, S. A., Godinho, A. L., Monteiro, E. C., Marques, M. M., and Antunes, A. M. (2012) N-terminal valine adduct from the anti-HIV drug abacavir in rat haemoglobin as evidence for abacavir metabolism to a reactive aldehyde in vivo. Br. J. Pharmacol. 167, 1353−1361. (12) Grilo, N. M., Antunes, A. M., Caixas, U., Marinho, A. T., Charneira, C., Conceicao Oliveira, M., Monteiro, E. C., Matilde Marques, M., and Pereira, S. A. (2013) Monitoring abacavir bioactivation in humans: Screening for an aldehyde metabolite. Toxicol. Lett. 219, 59−64. (13) Almond, L. M., Edirisinghe, D., Dalton, M., Bonington, A., Back, D. J., and Khoo, S. H. (2005) Intracellular and plasma pharmacokinetics of nevirapine in human immunodeficiency virus-infected individuals. Clin. Pharmacol. Ther. 78, 132−142. (14) Jenkins, R. E., Meng, X., Elliott, V. L., Kitteringham, N. R., Pirmohamed, M., and Park, B. K. (2009) Characterisation of flucloxacillin and 5-hydroxymethyl flucloxacillin haptenated HSA in vitro and in vivo. Proteomics Clin. Appl. 3, 720−729. (15) Chang, M., Bolton, J. L., and Blond, S. Y. (1999) Expression and purification of hexahistidine-tagged human glutathione S-transferase P1−1 in Escherichia coli. Protein Expression Purif. 17, 443−448. (16) Jenkinson, C., Jenkins, R. E., Maggs, J. L., Kitteringham, N. R., Aleksic, M., Park, B. K., and Naisbitt, D. J. (2009) A mechanistic investigation into the irreversible protein binding and antigenicity of pphenylenediamine. Chem. Res. Toxicol. 22, 1172−1180. (17) Greenough, C., Jenkins, R. E., Kitteringham, N. R., Pirmohamed, M., Park, B. K., and Pennington, S. R. (2004) A method for the rapid depletion of albumin and immunoglobulin from human plasma. Proteomics 4, 3107−3111. (18) Jenkins, R. E., Yaseen, F. S., Monshi, M. M., Whitaker, P., Meng, X., Farrell, J., Hamlett, J., Sanderson, J. P., El-Ghaiesh, S., Peckham, D., Pirmohamed, M., Park, B. K., and Naisbitt, D. J. (2013) beta-Lactam antibiotics form distinct haptenic structures on albumin and activate drug-specific T-lymphocyte responses in multiallergic patients with cystic fibrosis. Chem. Res. Toxicol. 26, 963−975. (19) Jenkins, R. E., Kitteringham, N. R., Goldring, C. E., Dowdall, S. M., Hamlett, J., Lane, C. S., Boerma, J. S., Vermeulen, N. P., and Park, B. K. (2008) Glutathione-S-transferase pi as a model protein for the characterisation of chemically reactive metabolites. Proteomics 8, 301− 315. (20) Norcross, M. A., Luo, S., Lu, L., Boyne, M. T., Gomarteli, M., Rennels, A. D., Woodcock, J., Margulies, D. H., McMurtrey, C., Vernon, S., Hildebrand, W. H., and Buchli, R. (2012) Abacavir induces loading of novel self-peptides into HLA-B*57: 01: an autoimmune model for HLA-associated drug hypersensitivity. AIDS 26, F21−29.

ASSOCIATED CONTENT

S Supporting Information *

Additional MS/MS spectra of ABCA-modified peptides and computer modeling of the protein docking of ABC and ABCA. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +44 151 7945559. Fax: +44 151 7945540. E-mail: bkpark@ liv.ac.uk. Funding

This work was supported by the Medical Research Council Centre for Drug Safety Science (Grant Number G0700654). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are greatly indebted to Dr. Alexandra M. M. Antunes, Centro ́ de Quimica Estrutural, Instituto Superior Técnico, Universidade Técnica de Lisboa, Portugal, for supplying abacavir aldehyde. We are grateful to Professor Paul O’Neill, Department of Chemistry, University of Liverpool, for obtaining the aldehyde. We thank Dr. L. J. Else, Sara Gibbons, and Helen Reynolds for providing plasma samples and Dr. Rosalind Jenkins and Dr. Anahi Santoyo Castelazo, Department of Molecular and Clinical Pharmacology, University of Liverpool, for the assistance with sample analysis by mass spectrometry.

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ABBREVIATIONS ABC, Abacavir; ABCA, abacavir aldehyde; HSA, human serum albumin; GSTP, glutathione S-transferase Pi; Hb, hemoglobin; MS, mass spectrometry; LC-MS/MS, liquid chromatography− tandem mass spectrometry; MRM, multiple reaction monitoring; ADR, adverse drug reaction; Rt, retention time

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REFERENCES

(1) Almeida, C. A., Martin, A. M., Nolan, D., Lucas, A., Cameron, P. U., James, I., Phillips, E., and Mallal, S. (2008) Cytokine profiling in abacavir hypersensitivity patients. Antiviral Ther. 13, 281−288. (2) Chessman, D., Kostenko, L., Lethborg, T., Purcell, A. W., Williamson, N. A., Chen, Z., Kjer-Nielsen, L., Mifsud, N. A., Tait, B. D., Holdsworth, R., Almeida, C. A., Nolan, D., Macdonald, W. A., Archbold, J. K., Kellerher, A. D., Marriott, D., Mallal, S., Bharadwaj, M., Rossjohn, J., and McCluskey, J. (2008) Human leukocyte antigen class Irestricted activation of CD8+ T cells provides the immunogenetic basis of a systemic drug hypersensitivity. Immunity 28, 822−832. (3) Ostrov, D. A., Grant, B. J., Pompeu, Y. A., Sidney, J., Harndahl, M., Southwood, S., Oseroff, C., Lu, S., Jakoncic, J., de Oliveira, C. A., Yang, L., Mei, H., Shi, L., Shabanowitz, J., English, A. M., Wriston, A., Lucas, A., Phillips, E., Mallal, S., Grey, H. M., Sette, A., Hunt, D. F., Buus, S., and Peters, B. (2012) Drug hypersensitivity caused by alteration of the MHC-presented self-peptide repertoire. Proc. Natl. Acad. Sci. U.S.A. 109, 9959−9964. (4) Adam, J., Eriksson, K. K., Schnyder, B., Fontana, S., Pichler, W. J., and Yerly, D. (2012) Avidity determines T-cell reactivity in abacavir hypersensitivity. Eur. J. Immunol. 42, 1706−1716. (5) Illing, P. T., Vivian, J. P., Dudek, N. L., Kostenko, L., Chen, Z., Bharadwaj, M., Miles, J. J., Kjer-Nielsen, L., Gras, S., Williamson, N. A., Burrows, S. R., Purcell, A. W., Rossjohn, J., and McCluskey, J. (2012) Immune self-reactivity triggered by drug-modified HLA-peptide repertoire. Nature 486, 554−558. (6) Bell, C. C., Faulkner, L., Martinsson, K., Farrell, J., Alfirevic, A., Tugwood, J., Pirmohamed, M., Naisbitt, D. J., and Park, B. K. (2013) T534



Article

(21) Lavergne, S. N., Park, B. K., and Naisbitt, D. J. (2008) The roles of drug metabolism in the pathogenesis of T-cell-mediated drug hypersensitivity. Curr. Opin. Allergy Clin. Immunol. 8, 299−307. (22) Rappaport, S. M., Li, H., Grigoryan, H., Funk, W. E., and Williams, E. R. (2012) Adductomics: characterizing exposures to reactive electrophiles. Toxicol. Lett. 213, 83−90. (23) Meng, X., Maggs, J. L., Pryde, D. C., Planken, S., Jenkins, R. E., Peakman, T. M., Beaumont, K., Kohl, C., Park, B. K., and Stachulski, A. V. (2007) Cyclization of the acyl glucuronide metabolite of a neutral endopeptidase inhibitor to an electrophilic glutarimide: synthesis, reactivity, and mechanistic analysis. J. Med. Chem. 50, 6165−6176. (24) Hammond, T. G., Regan, S. L., Meng, X. L., Maggs, J. L., Jenkins, R. E., Kenna, J. G., Sathish, J. G., Williams, D. P., and Park, B. K. (2011) In vitro assessment of the interactions between diclofenac and tolmetin acyl glucuronide drug metabolites and human serum albumin. Toxicology 290, 142−143. (25) Sayre, L. M., Lin, D., Yuan, Q., Zhu, X., and Tang, X. (2006) Protein adducts generated from products of lipid oxidation: Focus on HNE and ONE. Drug Metab. Rev. 38, 651−675. (26) Isom, A. L., Barnes, S., Wilson, L., Kirk, M., Coward, L., and Darley-Usmar, V. (2004) Modification of Cytochrome c by 4-hydroxy2-nonenal: evidence for histidine, lysine, and arginine-aldehyde adducts. J. Am. Soc. Mass. Spectrom. 15, 1136−1147. (27) Ishii, T., Kumazawa, S., Sakurai, T., Nakayama, T., and Uchida, K. (2006) Mass spectroscopic characterization of protein modification by malondialdehyde. Chem. Res. Toxicol. 19, 122−129. (28) Szapacs, M. E., Riggins, J. N., Zimmerman, L. J., and Liebler, D. C. (2006) Covalent adduction of human serum albumin by 4-hydroxy-2nonenal: kinetic analysis of competing alkylation reactions. Biochemistry 45, 10521−10528. (29) Doorn, J. A., and Petersen, D. R. (2003) Covalent adduction of nucleophilic amino acids by 4-hydroxynonenal and 4-oxononenal. Chem.-Biol. Interact. 143−144, 93−100. (30) Chen, W., Shockcor, J. P., Tonge, R., Hunter, A., Gartner, C., and Nelson, S. D. (1999) Protein and nonprotein cysteinyl thiol modification by N-acetyl-p-benzoquinone imine via a novel ipso adduct. Biochemistry 38, 8159−8166. (31) Zsila, F. (2013) Subdomain IB is the third major drug binding region of human serum albumin: toward the three-sites model. Mol. Pharmacol. 10, 1668−1682. (32) Ghuman, J., Zunszain, P. A., Petitpas, I., Bhattacharya, A. A., Otagiri, M., and Curry, S. (2005) Structural basis of the drug-binding specificity of human serum albumin. J. Mol. Biol. 353, 38−52. (33) He, X. M., and Carter, D. C. (1992) Atomic structure and chemistry of human serum albumin. Nature 358, 209−215. (34) Hetherington, S., McGuirk, S., Powell, G., Cutrell, A., Naderer, O., Spreen, B., Lafon, S., Pearce, G., and Steel, H. (2001) Hypersensitivity reactions during therapy with the nucleoside reverse transcriptase inhibitor abacavir. Clin. Ther. 23, 1603−1614.

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Abacavir Forms Novel Cross-Linking Abacavir Protein Adducts in

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