The aim of this proposal is the development and assay of new inhibitors of steriod hormone biosynthesis and studies on their mechanism of action. Information gained from the structure-activity relations and the specificity and mechanism of inhibition will promote our understanding of the molecular details of steroidogenic reactions and provide insight into the design of drugs for treatment of endocrine-related diseases and cancers in which steroid production plays a critical role. A major emphasis will be given to studies on the conversion of cholesterol to pregnenolone catalyzed by cytochrome P-450scc, and properties of this enzyme purified from bovine adrenal cortex will be studied. We propose to extend our recent studies on amine-containing and aryl-substituted active site-directed inhibitors. New derivatives will be synthesized and their interaction with P-450scc will be investigated spectroscopically and by the kinetics of inhibition. The experimental findings will be used together with conformational calculations to attempt to define a unique structure for the active site of this enzyme. We also propose to purify and study other steriod monooxygenases which participate in 11Beta, 17Alpha, and 21 hydroxylation. Active site-directed inhibitors will be used to map the active sites of these enzymes as well. Working with several P-450 enzymes we can test the specificity of the various inhibitors; it will also allow us to assess the generality of our findings with P-450scc. Finally, the cellular effectiveness and specificity of the different inhibitors will be determined using cultured cells from adrenal, testes and placenta. In the long term, well-characterized inhibitors would be useful for the control of steroid production in pregnancy, for regulating excess production associated with some pituitary, gonadal and adrenal tumors, and as antineoplastic agents by blocking the formation of steroids required for the growth of certain hormone-dependent cancers.
