Structural basis for the selective inhibition of human 3beta-hydroxysteroid dehydrogenase 1 in human breast tumor MCF-7 cells

• Published in: Molecular and cellular endocrinology 2009-03, Vol.301 (1-2), p.174-182
• Main Authors: Thomas, James L, Bucholtz, Kevin M, Sun, Jingping, Mack, Vance L, Kacsoh, Balint
• Format: Article
• Language: English
• Subjects: 3-Hydroxysteroid Dehydrogenases - antagonists & inhibitors; 3-Hydroxysteroid Dehydrogenases - chemistry; Arginine - metabolism; Aromatase Inhibitors - pharmacology; Blotting, Western; Breast Neoplasms - enzymology; Breast Neoplasms - genetics; Cell Line, Tumor; Cell Proliferation - drug effects; Coenzymes - metabolism; Dehydroepiandrosterone - pharmacology; Dihydrotestosterone - analogs & derivatives; Dihydrotestosterone - pharmacology; Enzyme Inhibitors - pharmacology; Estradiol - pharmacology; Estrone - pharmacology; Female; Gene Expression Regulation, Enzymologic - drug effects; Gene Expression Regulation, Neoplastic - drug effects; Humans; Kinetics; Mutant Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - genetics; RNA, Messenger - metabolism; Substrate Specificity - drug effects
• ISSN: 0303-7207
• DOI: 10.1016/j.mce.2008.09.029

Human 3beta-hydroxysteroid dehydrogenase/isomerase type 1 (3beta-HSD1) is a critical enzyme in the conversion of DHEA to estradiol in breast tumors and may be a target enzyme for inhibition in the treatment of breast cancer in postmenopausal women. Human 3beta-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland in this population. In our recombinant human breast tumor MCF-7 Tet-off cells that express either 3beta-HSD1 or 3beta-HSD2, trilostane and epostane inhibit the DHEA-induced proliferation of MCF-7 3beta-HSD1 cells with 12- to 16-fold lower IC(50) values compared to the MCF-7 3beta-HSD2 cells. The compounds also competitively inhibit purified human 3beta-HSD1 with 12- to 16-fold lower K(i) values compared to the noncompetitive K(i) values measured for human 3beta-HSD2. Using our structural model of 3beta-HSD1, trilostane or 17beta-acetoxy-trilostane was docked in the active site of 3beta-HSD1, and Arg195 in 3beta-HSD1 or Pro195 in 3beta-HSD2 was identified as a potentially critical residue (one of 23 non-identical residues in the two isoenzymes). The P195R mutant of 3beta-HSD2 were created, expressed and purified. Kinetic analyses of enzyme inhibition suggest that the high affinity, competitive inhibition of 3beta-HSD1 by trilostane and epostane may be related to the presence of Arg195 in 3beta-HSD1 vs. Pro195 in 3beta-HSD2.