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

ABSTRACT: 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.

SUBMITTER: Thomas JL

PROVIDER: S-EPMC2667100 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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

Thomas James L JL Bucholtz Kevin M KM Sun Jingping J Mack Vance L VL Kacsoh Balint B

Molecular and cellular endocrinology 20081008 1-2

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 ...[more]

PMID: 18955108

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Project description:17?-Hydroxysteroid dehydrogenase type 1 (17?-HSD1) catalyzes the reduction of estrone to estradiol, which is the most potent estrogen in humans. Inhibition of 17?-HSD1 and thereby reducing the intracellular estradiol concentration is thus a promising approach for the treatment of estrogen dependent diseases. In the past, several steroidal and non-steroidal inhibitors of 17?-HSD1 have been described but so far there is no cocrystal structure of the latter in complex with 17?-HSD1. However, a distinct knowledge of active site topologies and protein-ligand interactions is a prerequisite for structure-based drug design and optimization. An elegant strategy to enhance this knowledge is to compare inhibition values obtained for one compound toward ortholog proteins from various species, which are highly conserved in sequence and differ only in few residues. In this study the inhibitory potencies of selected members of different non-steroidal inhibitor classes toward marmoset 17?-HSD1 were determined and the data were compared with the values obtained for the human enzyme. A species specific inhibition profile was observed in the class of the (hydroxyphenyl)naphthols. Using a combination of computational methods, including homology modelling, molecular docking, MD simulation, and binding energy calculation, a reasonable model of the three-dimensional structure of marmoset 17?-HSD1 was developed and inhibition data were rationalized on the structural basis. In marmoset 17?-HSD1, residues 190 to 196 form a small ?-helix, which induces conformational changes compared to the human enzyme. The docking poses suggest these conformational changes as determinants for species specificity and energy decomposition analysis highlighted the outstanding role of Asn152 as interaction partner for inhibitor binding. In summary, this strategy of comparing the biological activities of inhibitors toward highly conserved ortholog proteins might be an alternative to laborious x-ray or site-directed mutagenesis experiments in certain cases. Additionally, it facilitates inhibitor design and optimization by offering new information on protein-ligand interactions.

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.

Dataset Information

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

Publications

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

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