The sulfation of benzylic alcohols, allylic alcohols, and N-hydroxy arylamines is often the critical step in their biotransformation into chemically reactive metabolites that can form covalent bonds with cellular macromolecules, the initial step leading to various toxicological responses including cellular necrosis, mutagenesis, and carcinogenesis. The long-term goal of this research is to more fully understand and predict the roles that aryl and alcohol sulfotransferases play in these toxic responses. The research proposed in this application addresses fundamental aspects of the molecular recognition of substrates and inhibitors as well as intrahepatic expression of aryl sulfotranferase IV (AST IV) and alcohol (hydroxysteroid) sulfotransferase (STa).
Specific Aims 1 and 2 of the proposal involve the development and refinement of three-dimensional models of structure-activity relationships for AST IV and STa.
Aim 1 is based on the hypothesis that specific amino acid residues lining the sulfuryl acceptor sites of these enzymes are major determinants of the molecular recognition and stereoselectivity of these enzymes for substrates and inhibitors. This hypothesis will be tested using a multi-faceted approach wherein kinetic analyses of stereochemically defined substrates are coupled with site-directed mutagenesis, protein homology modeling, and conformer modeling analysis based on structure-alignment. Investigations on stereochemical aspects of the sulfation of alpha-hydroxytamoxifen (a potentially critical step involved in the carcinogenic effects seen in a small percentage of women treated with this drug) and several related model allylic alcohols will also be continued.
Aim 2 is centered on refinement of the homology models from the C-terminal regions of AST IV and STa. In the third specific aim, homology models and three-dimensional structure-activity relationships will be utilized to design, synthesize, and evaluate isoform-specific inhibitors of rat AST IV and STa. Results from these studies on isoform-specific inhibitors will then be extended to the related human isoforms of aryl and alcohol sulfotransferases. Finally, Specific Aim 4 of the proposal is to explore the expression and activity of AST IV and STa within both cholangiocytes (bile duct epithelial cells) and Kupffer cells, two types of nonparenchymal cells that play critical roles in the pathophysiology of the liver. The results to be forthcoming from the proposed continuation of this grant will, therefore, provide significant new insight into factors, such as molecular recognition of substrates and inhibitors and intrahepatic localizations, that regulate aryl and alcohol sulfotransferase-mediated xenobiotic and endobiotic metabolism and their roles in liver pathophysiology.
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