Selective inhibition of human 3β-hydroxysteroid dehydrogenase type 1 as a potential treatment for breast cancer

Abstract

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 12-16-fold lower IC(50) values compared to the MCF-7 3β-HSD2 cells. Trilostane and epostane also competitively inhibit purified human 3β-HSD1 with 12-16-fold lower K(i) values compared to the noncompetitive K(i) values measured for human 3β-HSD2. Using our structural model of 3β-HSD1, 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. The R195P-1 mutant of 3β-HSD1 and the P195R-2 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 may be related to the presence of Arg195 in 3β-HSD1 versus Pro195 in 3β-HSD2. In addition, His156 in 3β-HSD1 may play a role in the higher affinity of 3β-HSD1 for substrates and inhibitors compared to 3β-HSD2 containing Try156. Structural modeling of the 3β-HSD1 dimer identified a possible interaction between His156 on one subunit and Gln105 on the other. Kinetic analyses of the H156Y-1, Q105M-1 and Q105M-2 support subunit interactions that contribute to the higher affinity of 3β-HSD1 for the inhibitor, epostane, compared to 3β-HSD2. Article from the Special issue on Targeted Inhibitors.

Fig. 1

Human 3β-hydroxysteroid dehydrogenase is expressed as two-tissue specific isoforms (3β-HSD1 and 3β-HSD2) as a key enzyme in the steroid biosynthetic pathways that produce estradiol, testosterone, cortisol and aldosterone. The enzymes that participate in the conversion of DHEA-S to 17β-estradiol in breast tumors are in red.

Fig. 2

Inhibition of the proliferation of genetically-engineered MCF-7 breast tumor cells by trilostane or letrozole. (A) The 3β-HSD inhibitor, trilostane (0-2.5 μM) slows the DHEA-stimulated (5 nM) proliferation of our recombinant MCF-7 Tet-off 3β-HSD1 aromatase cells in the absence (no dox, ■, solid line) and presence of 10 ng/ml doxycycline (dox, ▲, broken line) and recombinant MCF7 Tet-off 3β-HSD2 aromatase cells in the absence (no dox, ▼, solid line) and presence of doxycycline (dox, ◆, broken line). (B) The aromatase inhibitor, letrozole (0-20.0 nM), decreases the DHEA-stimulated (5 nM) proliferation of the recombinant MCF-7 Tet-off 3β-HSD1 aromatase cells in the absence (no dox, ■, solid line) or presence of 10 ng/ml doxycycline (dox, ▲, broken line) and of the recombinant MCF-7 Tet-off 3β-HSD2 aromatase cell lines in the absence (no dox, ▼, solid line) or presence of doxycycline (dox, ◆, broken line) as described in Experimental Procedures. Each value is the mean of 4 independent determinations ± SD. Assays are conducted as described in Experimental Procedures. Each value is the mean of 4 independent determinations ± SD.

Fig. 3

Docking of trilostane with our structural model of human 3β-HSD1. (A) The proposed interaction between the 17β-hydroxyl group of trilostane and the guanidinium group of the Arg195 residue of the wild-type enzyme (4.0 Å) and the apparent proximity of the anchoring hydroxyl group of the Ser124 residue to the 2α-cyanogroup of docked trilostane (3.4 Å) are shown. (B) Docking of trilostane with the R195P mutant of 3β-HSD1 (containing Pro195) shows a binding shift of the inhibitor compared to the orientation of trilostane docked with wild-type 3β-HSD1 containing Arg195 in Panel A. The illustrations of catalytic residues, Tyr154 and Ser124, and cofactor, NAD⁺, indicate that this view represents the active site of the enzyme. The protein backbone (green), carbon (black), oxygen (red) and nitrogen (blue) atoms plus estimated bond distances (magenta) are shown.

Fig. 4

Homology model of the human 3β-HSD1 dimer showing the hydrogen-binding residues, Gln105 and His156, in the subunit interface as well as the catalytic residues, Tyr154 and Lys158, in the active site. In the similar human 3β-HSD2 dimer interface, Tyr156 replaces His156 (not shown).