MECHANISMS AND CHEMISTRY OF DEHYDROGENASE INACTIVATORS

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Hydroxysteroid dehydrogenases (HSD)s constitute an important class of steroid metabolizing enzymes. The reversible oxidoreductase reactions mediated by these enzymes have dramatic effects on the hormonal activities of corticosteroids, progestins, androgens, and estrogens. By using mechanism-based irreversible inhibitors of HSDs and studying their mechanisms of inactivation, information relevant to the pharmacologic manipulation of this class of enzymes can be obtained. The specific aims of this proposal are: 1) the synthesis of novel mechanism-based irreversible inhibitors of three different HSDs, 2) the enzymatic evaluation of these inhibitors, and 3) the elucidation of the structures of covalent HSD-inhibitor adducts. The proposed mechanism-based inhibitors are acetylenic alcohols whose enzymatic oxidation by a particular HSD will generate a conjugated acetylenic ketone that can be attacked by an enzyme nucleophile to form a covalent adduct and thus inactivate the enzyme. Three different series of acetylenic compounds, one series for each of the three hydroxysteroid dehydrogenases included in the study, will be prepared. Kinetic and mechanistic data needed for the structure/activity correlations will be obtained from measurements of rates of HSD inactivation under a variety of experimental conditions. Structural data will be obtained from carbon-13 nuclear magnetic reasonance (NMR) experiments done with HSDs inactivated by enzyme-generated acetylenic ketones that contain less than 99% carbon-13 enrichment in both carbon atoms of the acetylenic bond. Solution and/or solid state NMR experiments are proposed. Structural conclusions based on carbon-13 chemical shift data from these NMR experiments will be made possible by comparison of the observed chemical shifts with those found in appropriate model adducts.