The overall goal of the research described in this renewal is to investigate the hypothesis that acyl-CoA thioester metabolites of carboxylic acid-containing drugs are reactive acylating species that, in addition to, but possibly more important than, reactive acyl glucuronides, contribute to the covalent binding of acidic drugs to proteins and which may be responsible for, among other toxicities, untoward allergic side-effects. This hypothesis will be focused on by a range of in vitro and in vivo studies, as directed by the following specific aims: 1) To evaluate the chemical reactivity of acyl-CoA thioester derivatives with biological nucleophiles and compare such reactivity with that of their respective acyl glucuronides; 2) To evaluate the effect of enzyme inducers and inhibitors, with respect to acyl glucuronidation and acyl-CoA formation, on the covalent binding of selected carboxylic acids to protein; 3) To quantitatively determine and compare the levels of drug-protein acylation, acyl glucuronidation and acyl-CoA formation of selected carboxylic acid drugs in vivo in selected tissues; 4) To develop improved methodology for the analysis of acyl-CoA thioester derivatives from biological samples; 5) To determine the effect of alpha-fluoro-substitution of carboxylic acid drugs on xenobiotic acyl-CoA formation and protein acylation; 6) To assess the enantioselectivity of covalent binding of chiral NSAIDs to protein and their metabolism to reactive acylating derivatives in vivo and in vitro, 7) To identify the hepatic protein targets for reactive metabolites of diclofenac, resulting from either glucuronide or CoA-thioester intermediates, using 2D-PAGE and mass spectrometry; 8) To investigate the role of the enzymes of fatty acylation on the selective acylation of cellular proteins by acidic drugs through their acyl-CoA thioester derivatives; 9) To determine if drug-protein conjugates formed by the reaction of xenobiotic-acyl-CoA thioester derivatives with protein are antigenic. This application proposes studies designed to: (a) characterize the chemical reactivity of acyl-CoA thioester metabolites of a number of acidic drugs, (b) compare this reactivity with corresponding acyl glucuronide metabolites, (c) elucidate the mechanisms by which protein acylation may occur for carboxylic acid-containing drugs through the CoA thioester pathway, as well as define the chemical structure of these products and (d) evaluate the immunotoxic potential of these thioester metabolites. In addition, since activation of endogenous fatty acids to their corresponding high-energy acyl-CoA thioester derivatives is an important enzymatic step required prior to the utilization of fatty acids for many cellular reactions, the findings of this work should provide important insights beyond immunotoxicity questions.
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