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

Human 3β-hydroxysteroid dehydrogenase/isomerase type 1 (3β-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 3β-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 3β-HSD1 or 3β-HSD2, trilostane and epostane inhibit the DHEA-induced proliferation of MCF-7 3β-HSD1 cells with 12- to 16-fold lower IC50 values compared to the MCF-7 3β-HSD2 cells. The compounds also competitively inhibit purified human 3β-HSD1 with 12- to 16-fold lower Ki values compared to the noncompetitive Ki values measured for human 3β-HSD2. Using our structural model of 3β-HSD1, trilostane or 17β-acetoxy-trilostane was docked in the active site of 3β-HSD1, and Arg195 in 3β-HSD1 or Pro195 in 3β-HSD2 was identified as a potentially critical residue (one of 23 non-identical residues in the two isoenzymes). The P195R mutant of 3β-HSD2 were created, expressed and purified. Kinetic analyses of enzyme inhibition suggest that the high affinity, competitive inhibition of 3β-HSD1 by trilostane and epostane may be related to the presence of Arg195 in 3β-HSD1 vs. Pro195 in 3β-HSD2.