Protein Adducts As Prospective Biomarkers of Nevirapine Toxicity

Abstract

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor used against the 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.

Evidence for nevirapine bioactivation in man: Searching for the first step in the mechanism of nevirapine toxicity

“…An N-alkyl Edman procedure (Antunes et al, 2010b) was used for the specific detachment of NVP adducts with the N-terminal valine of Hb (Fig. 2).…”

“…After Hb isolation from the red blood cells each sample was subjected to N-alkyl Edman degradation with phenyl isothiocyanate (Antunes et al, 2010b). Briefly, each 50 mg sample was dissolved in N,N-dimethylformamide (1.5 mL), followed by the addition of 1 M NaOH (65 L) and phenyl isothiocyanate (10 L).…”

Biomonitoring Human Albumin Adducts: The Past, the Present, and the Future

“…79 The cooked meat carcinogen PhIP and the non-nucleoside reverse transcriptase inhibitor Nevaripine also form adducts at the Trp214 of human Alb in vitro. 195−197 Since rat and human Alb contain only a single tryptophan residue at Trp214 situated in a hydrophobic drug-binding site, its high selectivity for carcinogen binding suggests a unique role for Alb in the detoxification and/or transport of ultimate carcinogenic/toxic metabolites. 80 …”

Tandem Mass Spectrometry of Peptides

“…Another approach for this type of tandem MS application is described by Switzar and colleagues (2011), where the protein digestion protocol conditions were optimized (pH, temperature and time) in order to increase protein coverage and signal intensity of the modified peptides. This tandem mass spectrometry application has also been employed in the study of the toxicity effect of anti-viral drugs, more precisely nevirapine (NVP) employed against human immunodeficiency virus type-1 (HIV-1) (Antunes et al, 2010). MALDI-TOF-TOF-MS of tryptic digests was used to identify which human serum albumin and human hemoglobin amino acid residues were bound to NVP upon incubation with the synthetic model electrophile 12-mesyloxy-NVP, used as a surrogate for the Phase II metabolite 12-sulfoxy-NVP.…”

“…Due to this particularity, peptide tandem MS can be used in several applications involving protein characterization and identification, such as the study of proteomes (Franco et al, 2011a,b) and differential proteomics (Puerto et al, 2011) of different organs, tissues, cells or biological fluids. In our laboratory we have also been using this approach for the characterization of proteins with specific function in adhesives (Santos et al, 2009), the identification of protein adducts as potential biomarkers of toxicity (Antunes et al, 2010), and protein glycation (Gomes et al, 2008), it has also been useful in the identification of peptides with immunomodulation properties (Koči et al, 2010). Two types of information obtained by mass spectrometry experiments are used for the study of proteins and peptides.…”