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

ABSTRACT: The human type 1 (placenta, breast tumors) and type 2 (gonads, adrenals) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) are key enzymes in biosynthesis of all active steroid hormones. Human 3beta-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. 3beta-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 2alpha-cyano group on trilostane (2alpha-cyano-4alpha,5alpha-epoxy-17beta-ol-androstane-3-one) and determine which amino acids may be critical for 3beta-HSD1 specificity. Trilostane without the 2alpha-cyano group, 4alpha,5alpha-epoxy-testosterone, was synthesized. Using our structural model of 3beta-HSD1, trilostane or 4alpha,5alpha-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 3beta-HSD1 were created, expressed and purified. Dixon analyses of the inhibition of wild-type 3beta-HSD1, 3beta-HSD2, M187T and S124T by trilostane and 4alpha,5alpha-epoxy-testosterone suggest that the 2alpha-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 3beta-HSD1 by trilostane may be related to the presence of Met187 in 3beta-HSD1 and Thr187 in 3beta-HSD2. This structure/function information may lead to the production of more highly specific inhibitors of 3beta-HSD1 to block the hormone-dependent growth of breast tumors.

SUBMITTER: Thomas JL

PROVIDER: S-EPMC2580795 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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

Thomas James L JL Mack Vance L VL Glow Jason A JA Moshkelani Delaram D Terrell J Ross JR Bucholtz Kevin M KM

The Journal of steroid biochemistry and molecular biology 20080503 1-2

The human type 1 (placenta, breast tumors) and type 2 (gonads, adrenals) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) are key enzymes in biosynthesis of all active steroid hormones. Human 3beta-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. 3beta-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland. The goals of this project are to ...[more]

PMID: 18524572

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Project description:The human type 1 (placenta, breast tumors, and prostate tumors) and type 2 (adrenals and gonads) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1 and 3beta-HSD2) are encoded by two distinct genes that are expressed in a tissue-specific pattern. Our recent studies have shown that His156 contributes to the 14-fold higher affinity that 3beta-HSD1 exhibits for substrate and inhibitor steroids compared with human 3beta-HSD2 containing Tyr156 in the otherwise identical catalytic domain. Our structural model of human 3beta-HSD localizes His156 or Tyr156 in the subunit interface of the enzyme homodimer. The model predicts that Gln105 on one enzyme subunit has a higher probability of interacting with His156 on the other subunit in 3beta-HSD1 than with Tyr156 in 3beta-HSD2. The Q105M mutant of 3beta-HSD1 (Q105M1) shifts the Michaelis-Menten constant (Km) for 3beta-HSD substrate and inhibition constants (Ki) for epostane and trilostane to the much lower affinity profiles measured for wild-type 3beta-HSD2 and H156Y1. However, the Q105M2 mutant retains substrate and inhibitor kinetic profiles similar to those of 3beta-HSD2. Our model also predicts that Gln240 in 3beta-HSD1 and Arg240 in 3beta-HSD2 may be responsible for the 3-fold higher affinity of the type 1 isomerase activity for substrate steroid and cofactors. The Q240R1 mutation increases the isomerase substrate Km by 2.2-fold to a value similar to that of 3beta-HSD2 isomerase and abolishes the allosteric activation of isomerase by NADH. The R240Q2 mutation converts the isomerase substrate, cofactor, and inhibitor kinetic profiles to the 4-14-fold higher affinity profiles of 3beta-HSD1. Thus, key structural reasons for the substantially higher affinities of 3beta-HSD1 for substrates, coenzymes, and inhibitors have been identified. These structure and function relationships can be used in future docking studies to design better inhibitors of the 3beta-HSD1 that may be useful in the treatment of hormone-sensitive cancers and preterm labor.

Project description:In postmenopausal women, human 3beta-hydroxysteroid dehydrogenase type 1 (3beta-HSD1) is a critical enzyme in the conversion of DHEA to estradiol in breast tumors, while 3beta-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland. The goals of this project are to determine if Arg195 in 3beta-HSD1 vs. Pro195 in 3beta-HSD2 in the substrate/inhibitor binding site is a critical structural difference responsible for the higher affinity of 3beta-HSD1 for inhibitor and substrate steroids compared to 3beta-HSD2 and whether Asp61, Glu192 and Thr8 are fingerprint residues for cofactor and substrate binding using site-directed mutagenesis. The R195P-1 mutant of 3beta-HSD1 and the P195R-2 mutant of 3beta-HSD2 have been created, expressed, purified and characterized kinetically. Dixon analyses of the inhibition of the R195P-1 mutant, P195R-2 mutant, wild-type 3beta-HSD1 and wild-type 3beta-HSD2 by trilostane has produced kinetic profiles that show inhibition of 3beta-HSD1 by trilostane (K(i)=0.10microM, competitive) with a 16-fold lower K(i) and different mode than measured for 3beta-HSD2 (K(i)=1.60microM, noncompetitive). The R195P-1 mutation shifts the high-affinity, competitive inhibition profile of 3beta-HSD1 to a low-affinity (trilostane K(i)=2.56microM), noncompetitive inhibition profile similar to that of 3beta-HSD2 containing Pro195. The P195R-2 mutation shifts the low-affinity, noncompetitive inhibition profile of 3beta-HSD2 to a high-affinity (trilostane K(i)=0.19microM), competitive inhibition profile similar to that of 3beta-HSD1 containing Arg195. Michaelis-Menten kinetics for DHEA, 16beta-hydroxy-DHEA and 16alpha-hydroxy-DHEA substrate utilization by the R195P-1 and P195R-2 enzymes provide further validation for higher affinity binding due to Arg195 in 3beta-HSD1. Comparisons of the Michaelis-Menten values of cofactor and substrate for the targeted mutants of 3beta-HSD1 (D61N, D61V, E192A, T8A) clarify the functions of these residues as well.

Project description:The enzyme 3beta-hydroxysteroid dehydrogenase/isomerase (3betaHSD) is required for the biosynthesis of all active steroid hormones. It exists as multiple isoforms in humans and rodents, each a product of a distinct gene. Two isoforms, 3betaHSD I and II, are expressed in a tissue-specific manner in humans. 3betaHSD I is the only isoform expressed in the placenta, where it is required for the biosynthesis of progesterone and thus essential for the maintenance of pregnancy. We recently identified two transcription factors, activating protein-2gamma (AP-2gamma) and the homeodomain protein, distaless-3 (Dlx-3), that are expressed in both human and mouse trophoblast cells that were shown to be required for trophoblast-specific expression of the orthologous murine 3betaHSD, 3betaHSD VI. Although we identified specific binding sites for AP-2gamma and Dlx-3 in the distal promoter of the human 3betaHSD I gene, HSD3B1, it was found that these transcription factors were not involved in determining placental-specific expression of human 3betaHSD I. Instead, a 53-bp placental-specific enhancer element located between -2570 and -2518 of the HSD3B1 promoter was identified. Within this 53-bp element, two potential placental transcription factor binding sites were found. EMSAs with a 20-bp oligonucleotide containing these two potential placental-specific binding sites identified one of the binding sites specific for the transcription enhancer factor (TEF)-5, which is highly expressed in human placenta and in placental choriocarcinoma-derived JEG-3 cells and the other overlapping binding site, specific for a GATA-like protein. Site-specific mutations in either the TEF-5 binding site or in the GATA binding site, each resulted in complete loss of enhancer activity. The data indicate that TEF-5 and the GATA-like protein act in a coordinate manner to determine the placental-specific expression of the human 3betaHSD I enzyme and therefore are critical for placental progesterone production required for the maintenance of pregnancy.

Dataset Information

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

Publications

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

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