Protein and Nonprotein Cysteinyl Thiol Modification by N-Acetyl-p-benzoquinone Imine via a Novel Ipso Adduct

Abstract

N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminophen (APAP), can arylate and oxidize protein and nonprotein thiols in the pathogenesis of APAP-induced hepatotoxicity. We report the first direct evidence for the formation of a labile ipso adduct between glutathione (GSH) and NAPQI using a combination of techniques including liquid chromatography/tandem mass spectrometry and liquid chromatography/NMR spectroscopy. Decomposition kinetics of the GSH-NAPQI ipso adduct and product ratios suggested that the ipso adduct was readily reversible back to NAPQI under neutral and basic conditions. The significance of the ipso adduct is that it may migrate from its site of formation to other cell compartments where it can either oxidize protein thiols or covalently modify them. Ipso adduct formation with protein thiols was demonstrated with a cysteine protease, papain, whose catalytic activity relies on the presence of an active site cysteinyl thiol. The formation and reactions of cysteinyl thiol ipso adducts of NAPQI provides significant new insights into possible reactions of quinone imines with cellular peptides and proteins.

Inhibition of macrophage migration inhibitory factor (MIF) tautomerase and biological activities by acetaminophen metabolites

“…NAPQI (4) was significantly more potent with an IC 50 value (40 M) that was 250-fold lower than that of acetaminophen. The most potent compound in this series was the hydroxyquinone (HQ, 6), also a known acetaminophen metabolite (43,44), which had an IC 50 of 0.7 M, corresponding to Ͼ715-fold more inhibition than dihydroxyacetaminophen (5). Of Ϸ60 related quinones tested, the most active compounds were observed to contain an ortho-or para-quinoid structure similar to that found in dopachrome 1 ( Table 1).…”

“…1). NAPQI (4) for instance has the iminoquinone functionality of L-dopachrome methyl ester and is known to be an electrophile and alkylating agent (39,40,43,44).…”

“…NAPQI and HQ are known to react with nucleophiles, particularly thiols (43,44). It is noteworthy that no inhibition of MIF enzymatic activity was obtained with the thiol-reactive agents iodoacetamide (17) and N-ethylmaleimide (18), suggesting that NAPQI and HQ are reacting at sites other than free cysteines present in MIF.…”

“…Under these reaction conditions, the inhibitory activity of these compounds is exceedingly efficient and consistent with irreversible, covalent modification of MIF. We considered that dopachrome-related quinones may comprise a general class of irreversible MIF enzyme inhibitors and focused our attention on NAPQI, which is the major metabolite of acetaminophen (43,44). Fig.…”

“…Unless otherwise indicated, all chemicals including compounds 3, 12, 13, 15, 17, and 18 were from Aldrich or Sigma and were of the highest grade commercially available. Compounds 4, 5, 8, 9, and 10 were prepared according to previously reported methods and had 1 H NMR characteristics that were identical to the known compounds (39)(40)(41)(42)(43)(44)(45). Compound 4, which is known to be unstable (39), was characterized by GC͞MS and stored as a solid at Ϫ70°C.…”