title: Protein adducts as prospective biomarkers of nevirapine toxicity.
abstract: Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor used against human immunodeficiency virus type-1 (HIV-1), mostly to prevent mother-to-child HIV-1 transmission in developing countries. Despite its clinical efficacy, NVP administration is associated with a variety of toxic responses that include hepatotoxicity and skin rash. Although the reasons for the adverse effects of NVP administration are still unclear, increasing evidence supports the involvement of metabolic activation to reactive electrophiles. In particular, Phase II activation of the NVP metabolite 12-hydroxy-NVP is thought to mediate NVP binding to bionucleophiles, which may be at the onset of toxicity. In the present study, we investigated the nature and specific locations of the covalent adducts produced in human serum albumin and human hemoglobin by reaction in vitro with the synthetic model electrophile 12-mesyloxy-NVP, used as a surrogate for the Phase II metabolite 12-sulfoxy-NVP. Multiple sites of modification were identified by two different mass spectrometry-based methodologies, liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and matrix-assisted laser desorption ionization tandem mass spectrometry (MALDI-TOF-TOF-MS). These two distinct methodologies, which in some instances afforded complementary information, allowed the identification of multiple adducts involving cysteine, lysine, tryptophan, histidine, serine, and the N-terminal valine of hemoglobin. Tryptophan, which is not a common site of covalent protein modification, was the NVP-modified amino acid residue detected in the two proteins and consistently identified by both LC-ESI-MS/MS and MALDI-TOF-TOF-MS. The propensity of tryptophan to react with the NVP-derived electrophile is further emphasized by the fact that human serum albumin possesses a single tryptophan residue, which suggests a remarkable selectivity that may be useful for biomonitoring purposes. Likewise, the NVP adduct with the terminal valine of hemoglobin, detected by LC-ESI-MS/MS after N-alkyl Edman degradation, appears as an easily assessed marker of NVP binding to proteins. Our results demonstrate the merits and complementarity of the two MS-based methodologies for the characterization of protein binding by NVP and suggest a series of plausible biomarkers of NVP toxicity that should be useful in the monitoring of toxicity effects in patients administered NVP.
authors.affiliation: Centro de Química Estrutural, Instituto Superior Técnico, Universidade Técnica de Lisboa, Lisboa, Portugal. alexandra.antunes@ist.utl.pt
authors.forename: Alexandra M M
authors.initials: AM
authors.lastname: Antunes
authors.forename: Ana L A
authors.initials: AL
authors.lastname: Godinho
authors.forename: Inês L
authors.initials: IL
authors.lastname: Martins
authors.forename: M Conceição
authors.initials: MC
authors.lastname: Oliveira
authors.forename: Ricardo A
authors.initials: RA
authors.lastname: Gomes
authors.forename: Ana V
authors.initials: AV
authors.lastname: Coelho
authors.forename: Frederick A
authors.initials: FA
authors.lastname: Beland
authors.forename: M Matilde
authors.initials: MM
authors.lastname: Marques
mesh_terms: D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000378:metabolism* / Q000627:therapeutic use / Q000633:toxicity; D015415:Biomarkers / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D015658:HIV Infections / Q000188:drug therapy; D006454:Hemoglobins / Q000737:chemistry; D006801:Humans; D008698:Mesylates / Q000633:toxicity; D008969:Molecular Sequence Data; D019829:Nevirapine / Q000031:analogs & derivatives / Q000378:metabolism* / Q000627:therapeutic use / Q000633:toxicity; D018894:Reverse Transcriptase Inhibitors / Q000378:metabolism* / Q000627:therapeutic use / Q000633:toxicity; D012709:Serum Albumin / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014364:Tryptophan / Q000737:chemistry
chemical_list: C551532:12-mesyloxynevirapine; D019380:Anti-HIV Agents; D015415:Biomarkers; D006454:Hemoglobins; D008698:Mesylates; D018894:Reverse Transcriptase Inhibitors; D012709:Serum Albumin; D014364:Tryptophan; D019829:Nevirapine
references.citation: Chem Res Toxicol. 2010 May 17;23(5):888-99
references.pmid: 20392079
references.citation: Drug Metab Dispos. 1999 Aug;27(8):895-901
references.pmid: 10421616
references.citation: Chem Res Toxicol. 2003 Sep;16(9):1078-89
references.pmid: 12971795
references.citation: FEBS J. 2005 Jan;272(2):353-62
references.pmid: 15654874
references.citation: Methods Enzymol. 1994;231:650-7
references.pmid: 8041284
references.citation: Chem Res Toxicol. 2008 Jul;21(7):1443-56
references.pmid: 18597499
references.citation: Chem Res Toxicol. 2008 Jan;21(1):117-28
references.pmid: 18052106
references.citation: J Chromatogr A. 2007 Oct 12;1167(2):125-34
references.pmid: 17826786
references.citation: Drug Metab Dispos. 2010 Jan;38(1):122-32
references.pmid: 19797610
references.citation: N Engl J Med. 2004 Jul 15;351(3):217-28
references.pmid: 15247338
references.citation: Toxicol Sci. 2009 Apr;108(2):401-11
references.pmid: 19221146
references.citation: Int J Clin Pract. 2007 Jan;61(1):105-18
references.pmid: 17229185
references.citation: Curr Opin Allergy Clin Immunol. 2009 Aug;9(4):311-5
references.pmid: 19535973
references.citation: Contact Dermatitis. 2005 Oct;53(4):189-200
references.pmid: 16191014
references.citation: JAMA. 2008 Aug 6;300(5):555-70
references.pmid: 18677028
references.citation: Annu Rev Pharmacol Toxicol. 2007;47:513-39
references.pmid: 16879083
references.citation: Int J Hyg Environ Health. 2007 May;210(3-4):201-28
references.pmid: 17376741
references.citation: Drug Metab Dispos. 2008 Sep;36(9):1770-9
references.pmid: 18508880
references.citation: J Clin Oncol. 2009 Feb 20;27(6):884-90
references.pmid: 19114688
references.citation: Chem Res Toxicol. 2006 Jul;19(7):889-93
references.pmid: 16841955
references.citation: Chem Res Toxicol. 2008 Sep;21(9):1862-70
references.pmid: 18729332
references.citation: Clin Ther. 1998 Nov-Dec;20(6):1071-92
references.pmid: 9916603
references.citation: Chem Res Toxicol. 2005 Dec;18(12):1799-813
references.pmid: 16359170
references.citation: Annu Rev Pharmacol Toxicol. 2005;45:177-202
references.pmid: 15822174
references.citation: J Biol Chem. 1986 May 25;261(15):6747-57
references.pmid: 3009475
references.citation: Electrophoresis. 1988 Jun;9(6):255-62
references.pmid: 2466658
references.citation: Drug Metab Rev. 2005;37(1):41-213
references.pmid: 15747500
references.citation: Chem Res Toxicol. 2006 Sep;19(9):1205-14
references.pmid: 16978025
references.citation: Drug Metab Dispos. 2009 Jul;37(7):1557-62
references.pmid: 19364830
references.citation: Lancet. 2003 Sep 13;362(9387):859-68
references.pmid: 13678973
references.citation: HIV Med. 2008 Feb;9(2):72-81
references.pmid: 18257770
references.citation: Drug Metab Dispos. 1999 Dec;27(12):1488-95
references.pmid: 10570031
references.citation: N Engl J Med. 1972 Apr 6;286(14):748-57
references.pmid: 4554517
references.citation: J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Oct 5;778(1-2):279-308
references.pmid: 12376136
references.citation: Drug Metab Dispos. 1999 Dec;27(12):1434-47
references.pmid: 10570025
references.citation: Clin Pharmacokinet. 2000 Oct;39(4):281-93
references.pmid: 11069214
references.citation: Lancet. 1999 Sep 4;354(9181):803-9
references.pmid: 10485721
references.citation: Anal Biochem. 1999 Apr 10;269(1):143-8
references.pmid: 10094785
references.citation: Environ Health Perspect. 1993 Mar;99:51-5
references.pmid: 8319659
references.citation: Biochem J. 2008 Dec 15;416(3):317-26
references.pmid: 18651835
references.citation: J Chromatogr A. 2007 Jul 20;1157(1-2):10-6
references.pmid: 17467723
references.citation: N Engl J Med. 2007 Jan 11;356(2):135-47
references.pmid: 17215531
references.citation: Mass Spectrom Rev. 2009 Sep-Oct;28(5):725-84
references.pmid: 19127566
references.citation: Rapid Commun Mass Spectrom. 2007;21(16):2734-42
references.pmid: 17654464
references.citation: Biochem Pharmacol. 1993 Jan 26;45(2):271-9
references.pmid: 8435087
references.citation: Biomed Chromatogr. 2010 Jul;24(7):717-26
references.pmid: 19882749