Structure/function of the inhibition of human 3β-hydroxysteroid dehydrogenase type 1 and type 2 by trilostane

The human type 1 (placenta, breast tumors) and type 2 (gonads, adrenals) isoforms of 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) are key enzymes in biosynthesis of all active steroid hormones. Human 3β-HSD1 is a critical enzyme in the conversion of DHEA to estradiol in breast tumors and may be a major target enzyme for the treatment of breast cancer. 3β-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland. The goals of this project are to evaluate the role of the 2α-cyano group on trilostane (2α-cyano-4α,5α-epoxy-17β-ol-androstane-3-one) and determine which amino acids may be critical for 3β-HSD1 specificity. Trilostane without the 2α-cyano group, 4α,5α-epoxy-testosterone, was synthesized. Using our structural model of 3β-HSD1, trilostane or 4α,5α-epoxy-testosterone was docked in the active site using Autodock 3.0, and the potentially critical residues (Met187 and Ser124) were identified. The M187T and S124T mutants of 3β-HSD1 were created, expressed and purified. Dixon analyses of the inhibition of wild-type 3β-HSD1, 3β-HSD2, M187T and S124T by trilostane and 4α,5α-epoxy-testosterone suggest that the 2α-cyano group of trilostane is anchored by Ser124 in both isoenzymes. Kinetic analyses of cofactor and substrate utilization as well as the inhibition kinetics of M187T and the wild-type enzymes suggest that the 16-fold higher-affinity inhibition of 3β-HSD1 by trilostane may be related to the presence of Met187 in 3β-HSD1 and Thr187 in 3β-HSD2. This structure/function information may lead to the production of more highly specific inhibitors of 3β-HSD1 to block the hormone-dependent growth of breast tumors.