Per- and polyfluoroalkyl substances inhibit human and rat 17ß-hydroxysteroid dehydrogenase 1: Quantitative structure-activity relationship and molecular docking analysis.

Per- and polyfluoroalkyl (PFAS) substances are enduring industrial materials. 17ß-Hydroxysteroid dehydrogenase isoform 1 (17ß-HSD1) is an estrogen metabolizing enzyme, which transforms estrone into estradiol in human placenta and rat ovary. Whether PFAS inhibit 17ß-HSD1 and what the structure-activity relationship (SAR) remains unexplored. We screened 18 PFAS for inhibiting human and rat 17ß-HSD1 in microsomes and studied their SAR and mode of action(MOA). Of the 11 perfluorocarboxylic acids (PFCAs), C8-C14 PFCAs at a concentration of 100 µM substantially inhibited human 17ß-HSD1, with order of C11 (half-maximal inhibition concentration, IC50, 8.94 µM) > C10 (10.52 µM) > C12 (14.90 µM) > C13 (30.97 µM) > C9 (43.20 µM) > C14 (44.83 µM) > C8 (73.38 µM) > others. Of the 7 per- and poly-fluorosulfonic acids (PFSAs), the potency was C8S (IC50, 14.93 µM) > C7S (80.70 µM) > C6S (177.80 µM) > others. Of the PFCAs, C8-C14 PFCAs at 100 µM markedly reduced rat 17ß-HSD1 activity, with order of C11 (IC50, 9.11 µM) > C12 (14.30 µM) > C10 (18.24 µM) > C13 (25.61 µM) > C9 (67.96 µM) > C8 (204.39 µM) > others. Of the PFSAs, the potency was C8S (IC50, 37.19 µM) > C7S (49.38 µM) > others. In contrast to PFOS (C6S), the partially fluorinated compound 6:2 FTS with an equivalent number of carbon atoms demonstrated no inhibition of human and rat 17ß-HSD1 activity at a concentration of 100 µM. The inhibition of human and rat enzymes by PFAS followed a V-shaped trend from C4 to C14, with a nadir at C11. Moreover, human 17ß-HSD1 was more sensitive than rat enzyme. PFAS inhibited human and rat 17ß-HSD1 in a mixed mode. Docking analysis revealed that they bind to the NADPH and steroid binding site of both 17ß-HSD1 enzymes. The 3D quantitative SAR (3D-QSAR) showed that hydrophobic region, hydrogen bond acceptor and donor are key factors in binding to 17ß-HSD1 active sites. In conclusion, PFAS exhibit inhibitory effects on human and rat 17ß-HSD1 depending on factors such as carbon chain length, degree of fluorination, and the presence of carboxylic acid or sulfonic acid groups, with a notable V-shaped shift observed at C11.

Chemical Synthesis and Biological Evaluation of 3-Substituted Estrone/Estradiol Derivatives as 17ß-Hydroxysteroid Dehydrogenase Type 1 Inhibitors Acting via a Reverse Orientation of the Natural Substrate Estrone.

Estradiol (E2) plays an important role in the progression of diseases such as breast cancer and endometriosis. Inhibition of 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1), the enzyme that catalyzes the last step in the biosynthesis of the estrogenic hormone E2, therefore constitutes an interesting approach for the treatment of these two estrogen-dependent diseases. In order to obtain new inhibitors of 17ß-HSD1, the impact of a m-carbamoylphenyloxy group at position three of an estrane nucleus was evaluated by preparing three derivatives of estrone (E1) and E2 using a microwave-assisted synthesis of diaryl ethers. Their inhibitory activity was addressed on two cell lines (T-47D and Z-12) representative of breast cancer and endometriosis, respectively, but unlike T-47D cells, Z-12 cells were not found suitable for testing potential 17ß-HSD1 inhibitors. Thus, the addition of the m-carbamoylphenyl group at C3 of E1 (compound 5) did not increase the inhibition of E1 to E2 transformation by 17ß-HSD1 present in T-47D cells (IC50 = 0.31 and 0.21 µM for 5 and E1, respectively), and this negative effect was more obvious for E2 derivatives 6 and 10 (IC50 = 1.2 and 1.3 µM, respectively). Molecular docking allowed us to identify key interactions with 17ß-HSD1 and to highlight these new inhibitors' actions through an opposite orientation than natural enzyme substrate E1's classical one. Furthermore, molecular modeling experiments explain the better inhibitory activity of E1-ether derivative 5, as opposed to the E2-ether derivatives 6 and 10. Finally, when tested on T-47D and Z-12 cells, compounds 5, 6 and 10 did not stimulate the proliferation of these two estrogen-dependent cell lines. In fact, they reduced it.

Halogen-Based 17ß-HSD1 Inhibitors: Insights from DFT, Docking, and Molecular Dynamics Simulation Studies.

The high expression of 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) mRNA has been found in breast cancer tissues and endometriosis. The current research focuses on preparing a range of organic molecules as 17ß-HSD1 inhibitors. Among them, the derivatives of hydroxyphenyl naphthol steroidomimetics are reported as one of the potential groups of inhibitors for treating estrogen-dependent disorders. Looking at the recent trends in drug design, many halogen-based drugs have been approved by the FDA in the last few years. Here, we propose sixteen potential hydroxyphenyl naphthol steroidomimetics-based inhibitors through halogen substitution. Our Frontier Molecular Orbitals (FMO) analysis reveals that the halogen atom significantly lowers the Lowest Unoccupied Molecular Orbital (LUMO) level, and iodine shows an excellent capability to reduce the LUMO in particular. Tri-halogen substitution shows more chemical reactivity via a reduced HOMO-LUMO gap. Furthermore, the computed DFT descriptors highlight the structure-property relationship towards their binding ability to the 17ß-HSD1 protein. We analyze the nature of different noncovalent interactions between these molecules and the 17ß-HSD1 using molecular docking analysis. The halogen-derived molecules showed binding energy ranging from -10.26 to -11.94 kcal/mol. Furthermore, the molecular dynamics (MD) simulations show that the newly proposed compounds provide good stability with 17ß-HSD1. The information obtained from this investigation will advance our knowledge of the 17ß-HSD1 inhibitors and offer clues to developing new 17ß-HSD1 inhibitors for future applications.

Synthesis of substituted 15ß-alkoxy estrone derivatives and their cofactor-dependent inhibitory effect on 17ß-HSD1.

17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is a key enzyme in the biosynthesis of 17ß-estradiol. Novel estrone-based compounds bearing various 15ß-oxa-linked substituents and hydroxy, methoxy, benzyloxy, and sulfamate groups in position C3 as potential 17ß-HSD1 inhibitors have been synthesized. In addition, in vitro inhibitory potentials measured in the presence of excess amount of NADPH or NADH were investigated. We observed substantial inhibitory potentials for several derivatives (IC50 < 1 µM) and increased binding affinities compared to unsubstituted core molecules. Binding and inhibition were found to be cofactor-dependent for some of the compounds and we propose structural explanations for this phenomenon. Our results may contribute to the development of new 17ß-HSD1 inhibitors, potential drug candidates for antiestrogen therapy of hormone-dependent gynecological cancers.

Synthesis and structure-activity relationships of 2- and/or 4-halogenated 13ß- and 13α-estrone derivatives as enzyme inhibitors of estrogen biosynthesis.

Ring A halogenated 13α-, 13ß-, and 17-deoxy-13α-estrone derivatives were synthesised with N-halosuccinimides as electrophile triggers. Substitutions occurred at positions C-2 and/or C-4. The potential inhibitory action of the halogenated estrones on human aromatase, steroid sulfatase, or 17ß-hydroxysteroid dehydrogenase 1 activity was investigated via in vitro radiosubstrate incubation. Potent submicromolar or low micromolar inhibitors were identified with occasional dual or multiple inhibitory properties. Valuable structure-activity relationships were established from the comparison of the inhibitory data obtained. Kinetic experiments performed with selected compounds revealed competitive reversible inhibition mechanisms against 17ß-hydroxysteroid dehydrogenase 1 and competitive irreversible manner in the inhibition of the steroid sulfatase enzyme.

Pd-Catalyzed microwave-assisted synthesis of phosphonated 13α-estrones as potential OATP2B1, 17ß-HSD1 and/or STS inhibitors.

Novel 2- or 4-phosphonated 13α-estrone derivatives were synthesized via the Hirao reaction. Bromo regioisomers (2- or 4-) of 13α-estrone and its 3-benzyl or 3-methyl ether were reacted with diethyl phosphite or diphenylphosphine oxide using Pd(PPh3)4 as catalyst under microwave irradiation. The influence of the new compounds on the transport function of the organic anion transporting polypeptide OATP2B1 was investigated by measuring Cascade Blue uptake. Derivatives bearing a 3-benzyl ether function displayed substantial submicromolar OATP2B1 inhibitory activity. The inhibitory effects of the compounds on human placental steroid sulfatase (STS) and 17ß-hydroxysteroid dehydrogenase type 1 isozyme (17ß-HSD1) were investigated by in vitro radiosubstrate incubation methods. None of the test compounds inhibited the STS markedly. The structure-activity relationship evaluation revealed that 2-substituted 3-hydroxy derivatives are able to inhibit the 17ß-HSD1 enzyme with submicromolar IC50 values. Dual OATP2B1 and 17ß-HSD1 inhibitors have been identified.

Synthesis of novel 13α-estrone derivatives by Sonogashira coupling as potential 17ß-HSD1 inhibitors.

Novel 13α-estrone derivatives were synthesized by Sonogashira coupling. Transformations of 2- or 4-iodo regioisomers of 13α-estrone and its 3-methyl ether were carried out under different conditions in a microwave reactor. The 2-iodo isomers were reacted with para-substituted phenylacetylenes using Pd(PPh3)4 as catalyst and CuI as a cocatalyst. Coupling reactions of 4-iodo derivatives could be achieved by changing the catalyst to Pd(PPh3)2Cl2. The product phenethynyl derivatives were partially or fully saturated. Compounds bearing a phenolic OH group furnished benzofurans under the conditions used for the partial saturation. The inhibitory effects of the compounds on human placental 17ß-hydroxysteroid dehydrogenase type 1 isozyme (17ß-HSD1) were investigated by an in vitro radiosubstrate incubation method. Certain 3-hydroxy-2-phenethynyl or -phenethyl derivatives proved to be potent 17ß-HSD1 inhibitors, displaying submicromolar IC50 values.

Addressing cytotoxicity of 1,4-biphenyl amide derivatives: Discovery of new potent and selective 17ß-hydroxysteroid dehydrogenase type 2 inhibitors.

Four different classes of new 17ß-hydroxysteroid dehydrogenase type 2 (17ß-HSD2) inhibitors were synthesized, in order to lower the cytotoxicity exhibited by the lead compound A, via disrupting the linearity and the aromaticity of the biphenyl moiety. Compounds 3, 4, 7a and 8 displayed comparable or better inhibitory activity and selectivity, as well as a lower cytotoxic effect, compared to the reference compound A. The best compound 4 (IC50=160nM, selectivity factor=168, LD50≈25µM) turned out as new lead compound for inhibition of 17ß-HSD2.

Synthesis and in vitro pharmacological evaluation of N-[(1-benzyl-1,2,3-triazol-4-yl)methyl]-carboxamides on d-secoestrone scaffolds.

An efficient synthesis of several N-[(1-benzyl-1,2,3-triazol-4-yl)methyl]carboxamides in the 13ß- and 13α-d-secoestrone series is reported. Novel triazoles were synthesized via the Cu(I)-catalyzed azide-alkyne cycloaddition of steroidal alkynyl carboxamides and p-substituted benzyl azides. Each of the products was evaluated in vitro by means of MTT assays for antiproliferative activity against a panel of human adherent cancer cell lines (HeLa, MCF-7, A431 and A2780). Some of them exhibited activities similar to those of the reference agent cisplatin. On change of the substitution pattern of the benzyl group of the azide, great differences in the cell growth-inhibitory properties were observed. The p-alkylbenzyl-substituted triazoles selectively exerted high cytostatic action against A2780 cells, with IC50 values of 1 µM. We investigated the potential inhibitory action exerted on the human 17ß-HSD1 activity of the new secosteroids. Three triazoles effectively suppressed the estrone to 17ß-estradiol conversion with IC50 values in low micromolar range.

Comparative investigation of the in vitro inhibitory potencies of 13-epimeric estrones and D-secoestrones towards 17ß-hydroxysteroid dehydrogenase type 1.

The inhibitory effects of 13-epimeric estrones, D-secooxime and D-secoalcohol estrone compounds on human placental 17ß-hydroxysteroid dehydrogenase type 1 isozyme (17ß-HSD1) were investigated. The transformation of estrone to 17ß-estradiol was studied by an in vitro radiosubstrate incubation method. 13α-Estrone inhibited the enzyme activity effectively with an IC50 value of 1.2 µM, which indicates that enzyme affinity is similar to that of the natural estrone substrate. The 13ß derivatives and the compounds bearing a 3-hydroxy group generally exerted stronger inhibition than the 13α and 3-ether counterparts. The 3-hydroxy-13ß-D-secoalcohol and the 3-hydroxy-13α-D-secooxime displayed an outstanding cofactor dependence, i.e. more efficient inhibition in the presence of NADH than NADPH. The 3-hydroxy-13ß-D-secooxime has an IC50 value of 0.070 µM and is one of the most effective 17ß-HSD1 inhibitors reported to date in the literature.

Synthesis and Biological Evaluation of Triazolyl 13α-Estrone-Nucleoside Bioconjugates.

2'-Deoxynucleoside conjugates of 13α-estrone were synthesized by applying the copper-catalyzed alkyne-azide click reaction (CuAAC). For the introduction of the azido group the 5'-position of the nucleosides and a propargyl ether functional group on the 3-hydroxy group of 13α-estrone were chosen. The best yields were realized in our hands when the 3'-hydroxy groups of the nucleosides were protected by acetyl groups and the 5'-hydroxy groups were modified by the tosyl-azide exchange method. The commonly used conditions for click reaction between the protected-5'-azidonucleosides and the steroid alkyne was slightly modified by using 1.5 equivalent of Cu(I) catalyst. All the prepared conjugates were evaluated in vitro by means of MTT assays for antiproliferative activity against a panel of human adherent cell lines (HeLa, MCF-7 and A2780) and the potential inhibitory activity of the new conjugates on human 17ß-hydroxysteroid dehydrogenase 1 (17ß-HSD1) was investigated via in vitro radiosubstrate incubation. Some protected conjugates displayed moderate antiproliferative properties against a panel of human adherent cancer cell lines (the protected cytidine conjugate proved to be the most potent with IC50 value of 9 µM). The thymidine conjugate displayed considerable 17ß-HSD1 inhibitory activity (IC50 = 19 µM).

Effects of organochlorine pesticides on human and rat 17ß-hydroxysteroid dehydrogenase 1 activity: Structure-activity relationship and in silico docking analysis.

The objective of this study was to examine the effect of 11 organochlorine pesticides on human and rat 17ß-Hydroxysteroid dehydrogenase 1 (17ß-HSD1) in human placental and rat ovarian microsome and on estradiol production in BeWo cells. The results showed that the IC50 values for endosulfan, fenhexamid, chlordecone, and rhothane on human 17ß-HSD1 were 21.37, 73.25, 92.80, and 117.69 µM. Kinetic analysis revealed that endosulfan acts as a competitive inhibitor, fenhexamid as a mixed/competitive inhibitor, chlordecone and rhothane as a mixed/uncompetitive inhibitor. In BeWo cells, all insecticides except endosulfan significantly decreased estradiol production at 100 µM. For rats, the IC50 values for dimethomorph, fenhexamid, and chlordecone were 11.98, 36.92, and 109.14 µM. Dimethomorph acts as a mixed inhibitor, while fenhexamid acts as a mixed/competitive inhibitor. Docking analysis revealed that endosulfan and fenhexamid bind to the steroid-binding site of human 17ß-HSD1. On the other hand, chlordecone and rhothane binds to a different site other than the steroid and NADPH-binding site. Dimethomorph binds to the steroid/NADPH binding site, and fenhexamid binds to the steroid binding site of rat 17ß-HSD1. Bivariate correlation analysis showed a positive correlation between IC50 values and LogP for human 17ß-HSD1, while a slight negative correlation was observed between IC50 values and the number of HBA. ADMET analysis provided insights into the toxicokinetics and toxicity of organochlorine pesticides. In conclusion, this study identified the inhibitory effects of 3-4 organochlorine pesticides and binding mechanisms on human and rat 17ß-HSD1, as well as their impact on hormone production.

Inhibitory effects of parabens on human and rat 17ß-hydroxysteroid dehydrogenase 1: Mechanisms of action and impact on hormone synthesis.

Parabens are commonly used preservatives in cosmetics, food, and pharmaceutical products. The objective of this study was to examine the effect of nine parabens on human and rat 17ß-hydroxysteroid dehydrogenase 1 (17ß-HSD1) in human placental and rat ovarian cytosols, as well as on estradiol synthesis in BeWo cells. The results showed that the IC50 values for these compounds varied from methylparaben with the weakest inhibition (106.42 µM) to hexylparaben with the strongest inhibition (2.05 µM) on human 17ß-HSD1. Mode action analysis revealed that these compounds acted as mixed inhibitors. For rats, the IC50 values ranged from the weakest inhibition for methylparaben (no inhibition at 100 µM) to the most potent inhibition for hexylparaben (0.87 µM), and they functioned as mixed inhibitors. Docking analysis indicated that parabens bind to the region bridging the NADPH and steroid binding sites of human 17ß-HSD1 and the NADPH binding site of rat 17ß-HSD1. Bivariate correlation analysis demonstrated negative correlations between LogP, molecular weight, heavy atoms, and apolar desolvation energy, and the IC50 values of these compounds. In conclusion, this study identified the inhibitory effects of parabens and their binding mechanisms on human and rat 17ß-HSD1, as well as their impact on hormone synthesis.

Bisphenol analogues inhibit human and rat 17ß-hydroxysteroid dehydrogenase 1: 3D-quantitative structure-activity relationship (3D-QSAR) and in silico docking analysis.

Bisphenols, estrogenic endocrine-disrupting chemicals, disrupt at least one of three endocrine pathways (estrogen, androgen, and thyroid). 17ß-Hydroxysteroid dehydrogenase 1 (17ß-HSD1) is a steroidogenic enzyme that catalyzes the activation of estradiol from estrone in human placenta and rat ovary. However, whether bisphenols inhibit 17ß-HSD1 and the mode of action remains unclear. This study we screened 17 bisphenols for inhibiting human 17ß-HSD1 in placental microsomes and rat 17ß-HSD1 in ovarian microsomes and determined 3D-quantitative structure-activity relationship (3D-QSAR) and mode of action. We observed some bisphenols with substituents were found to significantly inhibit both human and rat 17ß-HSD1 with the most potent inhibition on human enzyme by bisphenol H (IC50 = 0.90 µM) when compared to bisphenol A (IC50 = 113.38 µM). Rat enzyme was less sensitive to the inhibition of bisphenols than human enzyme with bisphenol H (IC50 = 32.94 µM) for rat enzyme. We observed an inverse correlation between IC50 and hydrophobicity (expressed as Log P). Docking analysis showed that they bound steroid-binding site of 17ß-HSD1. The 3D-QSAR models demonstrated that hydrophobic region, hydrophobic aromatic, ring aromatic, and hydrogen bond acceptor are key factors for the inhibition of steroid synthesis activity of 17ß-HSD1.

New insights into the substrate inhibition of human 17ß-hydroxysteroid dehydrogenase type 1.

Human type 1 17ß-hydroxysteroid dehydrogenase (17ß-HSD1),a member of the short-chain dehydrogenase/reductase family, catalyzes the last step in the bioactivation of the most potent estrogen estradiol with high specificity and is thus involved in estrogen-dependent diseases. As an oxidoreductase, 17ß-HSD1 can utilize both triphosphate and diphosphate cofactors in reaction at the molecular level, but more specific with triphosphate cofactor. The NADPH is much higher than NADP+ in living cells leading to preliminary reduction action. The enzyme also showed substrate-induced inhibition unprecedented in other members of 17ß-HSDs. Our previous study elucidated the structural mechanism of substrate inhibition is due to the reversely bound estrone (E1) in the substrate-binding pocket of the enzyme resulting in a dead-end complex. However, the effect of the cofactor preference on the substrate inhibition of the enzyme is not yet clear. In the present study, we solved the ternary crystal structures of 17ß-HSD1 in complex with E1 and cofactor analog NAD+ . Combined with molecular dynamics simulation using the enzyme with NADH/NADPH and different oriented E1 (normally oriented, E1N; reversely oriented, E1R), such ternary structure provides a complete picture of enzyme-substrate-cofactor interactions. The results reveal that different cofactors and substrate binding mode affect the allosteric effect between the two subunits of the enzyme. And the results from MD simulations confirmed that His221 plays a key role in the formation of dead-end complex in NADPH complex, and the absence of stable interaction between His221 and E1R in the NADH complex should be the main reason for its lack of substrate inhibition.

An irreversible inhibitor of 17ß-hydroxysteroid dehydrogenase type 1 inhibits estradiol synthesis in human endometriosis lesions and induces regression of the non-human primate endometriosis.

Endometriosis is a gynecological disorder affecting about 10% of women and can lead to invalidating painful symptoms and infertility. Since there is no current definitive cure for this disease, new therapeutic options are necessary. 17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is involved in the production of estradiol (E2), the most potent estrogen in women, and of 5-androstene-3ß,17ß-diol (5-diol), a weaker estrogen than E2, but whose importance increases after menopause. 17ß-HSD1 is therefore a pharmacological target of choice for the treatment of estrogen-dependent diseases such as endometriosis. We developed a targeted-covalent (irreversible) and non-estrogenic inhibitor of 17ß-HSD1, a molecule named PBRM, and herein evaluated its efficiency for the treatment of endometriosis. In a cell-free assay containing estrone (E1), the natural substrate of 17ß-HSD1, PBRM was able to block the formation of E2 in a collection of 50 human endometriosis lesions from a different clinical feature type, location, and phase. When given orally by gavage at 15 mg/kg to baboons, the resulting plasmatic concentration of PBRM was found to be sufficiently high (up to 125 ng/mL) for an efficacy study in a non-human primate (baboon) endometriosis model. After 2 months of treatment, the number of lesions/adhesions decreased in 60% of animals (3/5) in the PBRM-treated group, compared to the placebo group which showed an increase in the number of lesion/adhesions in 60% (3/5) of animals. Indeed, the total number of lesions/adhesions decreased in treated group (-6.5 or -19% when excluding one animal) while it increased in the control group receiving a placebo (+11%). Analysis of specific endometriotic lesions revealed that PBRM decreased the number of red lesions (-67%; 8/12) and white lesions (-35%; 11/31), but not of blue-black lesions. Similarly, PBRM decreased the surface area of dense adhesions and filmy adhesions, as compared to placebo. Also, PBRM treatment did not significantly affect the number of menstrual days. Finally, this targeted covalent inhibitor showed no adverse effects and no apparent toxicity for the duration of the treatment. These data indicate that 17ß-HSD1 inhibitor PBRM is a promising candidate for therapy targeting endometriosis and supports the need of additional efforts toward clinical trials.

Steroidal ferrocenes as potential enzyme inhibitors of the estrogen biosynthesis.

The potential inhibitory effect of diverse triazolyl-ferrocene steroids on key enzymes of the estrogen biosynthesis was investigated. Test compounds were synthesized via copper-catalyzed cycloaddition of steroidal azides and ferrocenyl-alkynes using our efficient methodology published previously. Inhibition of human aromatase, steroid sulfatase (STS) and 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) activities was investigated with in vitro radiosubstrate incubations. Some of the test compounds were found to be potent inhibitors of the STS. A compound bearing ferrocenyl side chain on the C-2 displayed a reversible inhibition, whereas C-16 and C-17 derivatives displayed competitive irreversible binding mechanism toward the enzyme. 17α-Triazolyl-ferrocene derivatives of 17ß-estradiol exerted outstanding inhibitory effect and experiments demonstrated a key role of the ferrocenyl moiety in the enhanced binding affinity. Submicromolar IC50 and Ki parameters enroll these compounds to the group of the most effective STS inhibitors published so far. STS inhibitory potential of the steroidal ferrocenes may lead to the development of novel compounds able to suppress in situ biosynthesis of 17ß-estradiol in target tissues.

Crystal structures of human 17ß-hydroxysteroid dehydrogenase type 1 complexed with estrone and NADP+ reveal the mechanism of substrate inhibition.

Human 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) catalyses the last step in estrogen activation and is thus involved in estrogen-dependent diseases (EDDs). Unlike other 17ß-HSD members, 17ß-HSD1 undergoes a significant substrate-induced inhibition that we have previously reported. Here we solved the binary and ternary crystal structures of 17ß-HSD1 in complex with estrone (E1) and cofactor analog NADP+ , demonstrating critical enzyme-substrate-cofactor interactions. These complexes revealed a reversely bound E1 in 17ß-HSD1 that provides the basis of the substrate inhibition, never demonstrated in estradiol complexes. Structural analysis showed that His221 is the key residue responsible for the reorganization and stabilization of the reversely bound E1, leading to the formation of a dead-end complex, which exists widely in NADP(H)-preferred enzymes for the regulation of their enzymatic activity. Further, a new inhibitor is proposed that may inhibit 17ß-HSD1 through the formation of a dead-end complex. This finding indicates a simple mechanism of enzyme regulation in the physiological background and introduces a pioneer inhibitor of 17ß-HSD1 based on the dead-end inhibition model for efficiently targeting EDDs. DATABASES: Coordinates and structure factors of 17ß-HSD1-E1 and 17ß-HSD1-E1-NADP+ have been deposited in the Protein Data Bank with accession code 6MNC and 6MNE respectively. ENZYMES: 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) EC 1.1.1.62.

Mimicking postmenopausal steroid metabolism in breast cancer cell culture: Differences in response to DHEA or other steroids as hormone sources.

Following menopause virtually 100% of estrogens are synthesized in peripheral target tissues from precursor steroids of adrenal origin. These steroids are the unique source of sex steroids in these women. This positions some steroid metabolizing enzymes as primary targets for novel therapies for estrogen receptor-positive (ER+) breast cancer. However, previous research on the steroid-converting enzymes has been performed using their direct substrate as a hormone source, depending on the facility where studied and the robust signal obtained. These experiments may not always provide an accurate reflection of physiological and post-menopausal conditions. We suggest providing dehydroepiandrosterone (DHEA) as an intracrinological hormone source, and comparing the role of steroid-converting enzymes using DHEA and their direct substrates when an extensive mechanistic understanding is required. Here, we present a comparative study of these enzymes with the provision of DHEA and the direct substrates, estrone (E1) or dihydrotestosterone (DHT), or additional steroids as hormone sources, in breast cancer cells. Enzyme knockdown by respective specific siRNAs and observations on the resulting differences in biological function were carried out. Cell biology studies showed no difference in biological function for 17ß-HSD1 and 17ß-HSD7 when cultured with different steroid hormones: cell proliferation and estradiol levels decreased, whereas DHT accumulated; cyclinD1, PCNA, and pS2 were down-regulated after knocking down these two enzymes, although the quantitative results varied. However, culture medium supplementation was found to have a marked impact on the study of 3α-HSD3. We demonstrated that provision of different steroids as a substrate or hormone sources may promote modified biological effects: provision of DHEA is the preferred choice to mimic postmenopausal steroid metabolism in cell culture.

Synthesis of A-ring halogenated 13α-estrone derivatives as potential 17ß-HSD1 inhibitors.

13α-Estrone and its 3-methyl or benzyl ether were halogenated in ring A with N-bromo- or N-iodosuccinimide or 1,3-dibromo-5,5-dimethylhydantoin as electrophile triggers. The chemo- and regioselectivities of the reactions depended greatly on the nature of the substituent on C-3. Bromination of the ethers led to 2- and 4-regioisomers. Bis-halogenation occurred only in the case of the phenolic derivative. Iodination and bromination resulted in similar products, except that the 3-benzyl ether could not be iodinated under the applied conditions. The potential inhibitory action of the new halogenated 13α-estrones on human 17ß-hydroxysteroid dehydrogenase 1 activity was investigated via in vitro radiosubstrate incubation. Some compounds proved to be effective inhibitors, with IC50 values in the submicromolar range.