Structural basis of inactivation of thiol protease by N-acetyl-p-benzoquinone imine (NAPQI). A knowledge-based molecular modeling of the adduct of NAPQI with thiol protease of the papain family

Inhibition of hepatic cysteine proteases by non-steroidal anti-inflammatory drug (NSAID) metabolites is implicated in several pathological conditions. It has been reported in the literature that N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminophen (APAP) can quickly arylate and oxidize thiol (cysteine) protease of the papain family to form an adduc t in the pathogenesis of acetaminophen-induced hepatotoxicity. It was also clarified by earlier NMR studies that the 3-position of the aromatic ring ( C-3) is the only site of conjugation with cysteinyl thioethers for protein arylation. In a recent study, the adduct of NAPQI has been identified and c haracterized by LC/MS/MS, LC/NMR and UV spectroscopy, and two possible cova lent binding modes corresponding to the 2-position (model-1) and the 3-posi tion (model-2) of the aromatic ring of NAPQI have been proposed. The work p resented here has been initiated to check the structural viability of inhib ition for the two proposed adducts at the atomic level. Results of our inve stigation by computer-assisted molecular modeling structurally demonstrate why model-2 would be more applicable to the static x-ray structure of the c omplex at physiological pH. This coordinated computational and molecular bi ology experiment can be used for metabolic screening of NSAIDs. A combinato rial approach of this kind alleviates the doubts in interpreting the result s of metabolic function and enhances our insights obtained from either comp utational or experimental studies alone.