Homology modeling meets site-directed mutagenesis: An ideal combination to elucidate the topology of 17ß-HSD2.

17ß-Hydroxysteroid dehydrogenase type 2 (17ß-HSD2) catalyzes the conversion of highly active estrogens and androgens into their less active forms using NAD+ as cofactor. Substrate and cofactor specificities of 17ß-HSD2 have been reported and potent 17ß-HSD2 inhibitors have been discovered in a ligand-based approach. However, the molecular basis and the amino acids involved in the enzymatic functionality are poorly understood, as no crystal structure of the membrane-associated 17ß-HSD2 exists. The functional properties of only few amino acids are known. The lack of topological information impedes structure-based drug design studies and limits the design of biochemical experiments. The aim of this work was the determination of the 17ß-HSD2 topology. For this, the first homology model of 17ß-HSD2 in complex with NAD+ and 17ß-estradiol was built, using a multi-fragment "patchwork" approach. To confirm the quality of the model, fifteen selected amino acids were exchanged one by one using site directed mutagenesis. The mutants' functional behavior demonstrated that the generated model was of very good quality and allowed the identification of several key amino acids involved in either ligand or internal structure stabilization. The final model is an optimal basis for further experiments like, for example, lead optimization.

Composing compound libraries for hit discovery--rationality-driven preselection or random choice by structural diversity?

AIMS: In order to identify new scaffolds for drug discovery, surface plasmon resonance is frequently used to screen structurally diverse libraries. Usually, hit rates are low and identification processes are time consuming. Hence, approaches which improve hit rates and, thus, reduce the library size are required. METHODS: In this work, we studied three often used strategies for their applicability to identify inhibitors of PqsD. In two of them, target-specific aspects like inhibition of a homologous protein or predicted binding determined by virtual screening were used for compound preselection. Finally, a fragment library, covering a large chemical space, was screened and served as comparison. RESULTS & CONCLUSION: Indeed, higher hit rates were observed for methods employing preselected libraries indicating that target-oriented compound selection provides a time-effective alternative.

Mechanistic details for anthraniloyl transfer in PqsD: the initial step in HHQ biosynthesis.

PqsD mediates the conversion of anthraniloyl-coenzyme A (ACoA) to 2-heptyl-4-hydroxyquinoline (HHQ), a precursor of the Pseudomonas quinolone signal (PQS) molecule. Due to the role of the quinolone signaling pathway of Pseudomonas aeruginosa in the expression of several virulence factors and biofilm formation, PqsD is a potential target for controlling this nosocomial pathogen, which exhibits a low susceptibility to standard antibiotics. PqsD belongs to the ß-ketoacyl-ACP synthase family and is similar in structure to homologous FabH enzymes in E. coli and Mycobacterium tuberculosis. Here, we used molecular dynamics simulations to obtain the structural position of the substrate ACoA in the binding pocket of PqsD, and semiempirical molecular orbital calculations to study the reaction mechanism for the catalytic cleavage of ACoA. Our findings suggest a nucleophilic attack of the deprotonated sulfur of Cys112 at the carbonyl carbon of ACoA and a switch in the protonation pattern of His257 whereby Nδ is protonated and the proton of Nε is shifted to the sulfur of CoA during the reaction. This is in agreement with the experimentally determined decreased catalytic activity of the Cys112Ser mutant, whereas the Cys112Ala, His257Phe, and Asn287Ala mutants are all inactive. ESI mass-spectrometric measurements of the Asn287Ala mutant show that anthraniloyl remains covalently bound to Cys112, thus further supporting the inference from our computed mechanism that Asn287 does not take part in the cleavage of ACoA. Since this mutant is inactive, we suggest instead that Asn287 must play an essential role in the subsequent formation of HHQ in vitro.

Discovery of novel bacterial RNA polymerase inhibitors: pharmacophore-based virtual screening and hit optimization.

The bacterial RNA polymerase (RNAP) is a validated target for broad spectrum antibiotics. However, the efficiency of drugs is reduced by resistance. To discover novel RNAP inhibitors, a pharmacophore based on the alignment of described inhibitors was used for virtual screening. In an optimization process of hit compounds, novel derivatives with improved in vitro potency were discovered. Investigations concerning the molecular mechanism of RNAP inhibition reveal that they prevent the protein-protein interaction (PPI) between σ(70) and the RNAP core enzyme. Besides of reducing RNA formation, the inhibitors were shown to interfere with bacterial lipid biosynthesis. The compounds were active against Gram-positive pathogens and revealed significantly lower resistance frequencies compared to clinically used rifampicin.

Molecular basis of HHQ biosynthesis: molecular dynamics simulations, enzyme kinetic and surface plasmon resonance studies.

BACKGROUND: PQS (PseudomonasQuinolone Signal) and its precursor HHQ are signal molecules of the P. aeruginosa quorum sensing system. They explicate their role in mammalian pathogenicity by binding to the receptor PqsR that induces virulence factor production and biofilm formation. The enzyme PqsD catalyses the biosynthesis of HHQ. RESULTS: Enzyme kinetic analysis and surface plasmon resonance (SPR) biosensor experiments were used to determine mechanism and substrate order of the biosynthesis. Comparative analysis led to the identification of domains involved in functionality of PqsD. A kinetic cycle was set up and molecular dynamics (MD) simulations were used to study the molecular bases of the kinetics of PqsD. Trajectory analysis, pocket volume measurements, binding energy estimations and decompositions ensured insights into the binding mode of the substrates anthraniloyl-CoA and ß-ketodecanoic acid. CONCLUSIONS: Enzyme kinetics and SPR experiments hint at a ping-pong mechanism for PqsD with ACoA as first substrate. Trajectory analysis of different PqsD complexes evidenced ligand-dependent induced-fit motions affecting the modified ACoA funnel access to the exposure of a secondary channel. A tunnel-network is formed in which Ser317 plays an important role by binding to both substrates. Mutagenesis experiments resulting in the inactive S317F mutant confirmed the importance of this residue. Two binding modes for ß-ketodecanoic acid were identified with distinct catalytic mechanism preferences.

Structure optimization of 2-benzamidobenzoic acids as PqsD inhibitors for Pseudomonas aeruginosa infections and elucidation of binding mode by SPR, STD NMR, and molecular docking.

Pseudomonas aeruginosa employs a characteristic pqs quorum sensing (QS) system that functions via the signal molecules PQS and its precursor HHQ. They control the production of a number of virulence factors and biofilm formation. Recently, we have shown that sulfonamide substituted 2-benzamidobenzoic acids, which are known FabH inhibitors, are also able to inhibit PqsD, the enzyme catalyzing the last and key step in the biosynthesis of HHQ. Here, we describe the further optimization and characterization of this class of compounds as PqsD inhibitors. Structural modifications showed that both the carboxylic acid ortho to the amide and 3'-sulfonamide are essential for binding. Introduction of substituents in the anthranilic part of the molecule resulted in compounds with IC50 values in the low micromolar range. Binding mode investigations by SPR with wild-type and mutated PqsD revealed that this compound class does not bind into the active center of PqsD but in the ACoA channel, preventing the substrate from accessing the active site. This binding mode was further confirmed by docking studies and STD NMR.

Novel small molecule inhibitors targeting the "switch region" of bacterial RNAP: structure-based optimization of a virtual screening hit.

Rising resistance against current antibiotics necessitates the development of antibacterial agents with alternative targets. The "switch region" of RNA polymerase (RNAP), addressed by the myxopyronins, could be such a novel target site. Based on a hit candidate discovered by virtual screening, a small library of 5-phenyl-3-ureidothiophene-2-carboxylic acids was synthesized resulting in compounds with increased RNAP inhibition. Hansch analysis revealed π (lipophilicity constant) and σ (Hammet substituent constant) of the substituents at the 5-phenyl moiety to be crucial for activity. The binding mode was proven by the targeted introduction of a moiety mimicking the enecarbamate side chain of myxopyronin into the hit compound, accompanied by enhanced RNAP inhibitory potency. The new compounds displayed good antibacterial activities against Gram positive bacteria and Gram negative Escherichia coli TolC and a reduced resistance frequency compared to the established antibiotic rifampicin.

Modulation of cytochromes P450 with xanthone-based molecules: from aromatase to aldosterone synthase and steroid 11ß-hydroxylase inhibition.

Imidazolylmethylflavones previously reported by us as aromatase inhibitors proved to be able to interact with aldosterone synthase (CYP11B2), a cytochrome P450 enzyme involved in the biosynthesis of the mineralcorticoid hormone aldosterone, and were used to obtain a pharmacophore model for this enzyme. Here, in the search for potential ligands for CYP11B2 and the related CYP11B1, a virtual screening of a small compounds library of our earlier synthesized aromatase inhibitors was performed and, according to the results and the corresponding biological data, led to the design and synthesis of a series of xanthones derivatives carrying an imidazolylmethyl substituent in position 1 and different substituents in position 4. Some very potent inhibitors were obtained; in particular, the 4-chlorine derivative was active in the low nanomolar or subnanomolar range on CYP11B2 and CYP11B1, respectively, proving that xanthone can be considered as an excellent scaffold, whose activity can be directed to different targets when appropriately functionalized.

Peptide-based investigation of the Escherichia coli RNA polymerase σ(70):core interface as target site.

The number of bacterial strains that are resistant against antibiotics increased dramatically during the past decades. This fact stresses the urgent need for the development of new antibacterial agents with novel modes of action targeting essential enzymes such as RNA polymerase (RNAP). Bacterial RNAP is a large multi-subunit complex consisting of a core enzyme (subunits: α(2)ßß'ω) and a dissociable sigma factor (σ(70); holo enzyme: α(2)ßß'ωσ(70)) that is responsible for promoter recognition and transcription initiation. The interface between core RNAP and σ(70) represents a promising binding site. Nevertheless, detailed studies investigating its druggability are rare. Compounds binding to this region could inhibit this protein-protein interaction and thus holo enzyme formation, resulting in inhibition of transcription initiation. Sixteen peptides covering different regions of the Escherichia coli σ(70):core interface were designed; some of them-all derived from σ(70) 2.2 region-led to a strong RNAP inhibition. Indeed, an ELISA-based experiment confirmed the most active peptide P07 to inhibit the σ(70):core interaction. Furthermore, an abortive transcription assay revealed that P07 impedes transcription initiation. In order to study the mechanism of action of P07 in more detail, molecular dynamics simulations and a rational amino acid replacement study were performed, leading to the conclusion that P07 binds to the coiled-coil region in ß' and that its flexible N-terminus inhibits the enzyme by interaction with the ß' lid-rudder-system (LRS). This work revisits the ß' coiled-coil as a hot spot for the protein-protein interaction inhibition and expands it by introduction of the LRS as target site.

Structural optimization of 2,5-thiophene amides as highly potent and selective 17ß-hydroxysteroid dehydrogenase type 2 inhibitors for the treatment of osteoporosis.

Inhibition of 17ß-HSD2 is an attractive mechanism for the treatment of osteoporosis. We report here the optimization of human 17ß-HSD2 inhibitors in the 2,5-thiophene amide class by varying the size of the linker (n equals 0 and 2) between the amide moiety and the phenyl group. While none of the phenethylamides (n = 2) were active, most of the anilides (n = 0) turned out to moderately or strongly inhibit 17ß-HSD2. The four most active compounds showed an IC50 of around 60 nM and a very good selectivity toward 17ß-HSD1, 17ß-HSD4, 17ß-HSD5, 11ß-HSD1, 11ß-HSD2 and the estrogen receptors α and ß. The investigated compounds inhibited monkey 17ß-HSD2 moderately, and one of them showed good inhibitory activity on mouse 17ß-HSD2. SAR studies allowed a first characterization of the human 17ß-HSD2 active site, which is predicted to be considerably larger than that of 17ß-HSD1.

Exploring the PDE5 H-pocket by ensemble docking and structure-based design and synthesis of novel ß-carboline derivatives.

By studying the co-crystal information of interactions between PDE5 and its inhibitors, forty new tetrahydro-ß-carbolines based-analogues were synthesized, and tested for their PDE5 inhibition. Some compounds were as active as tadalafil in inhibiting PDE5 and of better selectivity profile particularly versus PDE11A, the nature of the terminal ring and its nitrogen substituent are the main determinants of selectivity. Ensemble docking confirmed the role of H-loop closed conformer in activity versus its occluded and open forms. Conformational studies showed the effect of bulkiness of the terminal ring N-alkyl substituent on the formation of stable enzyme ligands conformers. The difference in potencies of hydantoin and piperazinedione analogues, together with the necessity of C-5/C-6 R-absolute configuration has been revealed through molecular docking.

Influence of DNA template choice on transcription and inhibition of Escherichia coli RNA polymerase.

In recent decades, quantitative transcription assays using bacterial RNA polymerase (RNAP) have been performed under widely diverse experimental conditions. We demonstrate that the template choice can influence the inhibitory potency of RNAP inhibitors. Furthermore, we illustrate that the sigma factor (σ(70)) surprisingly increases the transcription efficiency of templates with nonphysiological nonprokaryotic promoters. Our results might be a useful guideline in the early stages of using RNAP for drug discovery.

Hydroxybenzothiazoles as new nonsteroidal inhibitors of 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1).

17ß-estradiol (E2), the most potent estrogen in humans, known to be involved in the development and progession of estrogen-dependent diseases (EDD) like breast cancer and endometriosis. 17ß-HSD1, which catalyses the reduction of the weak estrogen estrone (E1) to E2, is often overexpressed in breast cancer and endometriotic tissues. An inhibition of 17ß-HSD1 could selectively reduce the local E2-level thus allowing for a novel, targeted approach in the treatment of EDD. Continuing our search for new nonsteroidal 17ß-HSD1 inhibitors, a novel pharmacophore model was derived from crystallographic data and used for the virtual screening of a small library of compounds. Subsequent experimental verification of the virtual hits led to the identification of the moderately active compound 5. Rigidification and further structure modifications resulted in the discovery of a novel class of 17ß-HSD1 inhibitors bearing a benzothiazole-scaffold linked to a phenyl ring via keto- or amide-bridge. Their putative binding modes were investigated by correlating their biological data with features of the pharmacophore model. The most active keto-derivative 6 shows IC50-values in the nanomolar range for the transformation of E1 to E2 by 17ß-HSD1, reasonable selectivity against 17ß-HSD2 but pronounced affinity to the estrogen receptors (ERs). On the other hand, the best amide-derivative 21 shows only medium 17ß-HSD1 inhibitory activity at the target enzyme as well as fair selectivity against 17ß-HSD2 and ERs. The compounds 6 and 21 can be regarded as first benzothiazole-type 17ß-HSD1 inhibitors for the development of potential therapeutics.

Computational investigation of the binding mode of bis(hydroxylphenyl)arenes in 17ß-HSD1: molecular dynamics simulations, MM-PBSA free energy calculations, and molecular electrostatic potential maps.

17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) catalyzes the last step of the estrogen biosynthesis, namely the reduction of estrone to the biologically potent estradiol. As such it is a potentially attractive drug target for the treatment of estrogen-dependent diseases like breast cancer and endometriosis. 17ß-HSD1 belongs to the bisubstrate enzymes and exists as an ensemble of conformations. These principally differ in the region of the ßFαG'-loop, suggesting a prominent role in substrate and inhibitor binding. Although several classes of potent non-steroidal 17ß-HSD1 inhibitors currently exist, their binding mode is still unclear. We aimed to elucidate the binding mode of bis(hydroxyphenyl)arenes, a highly potent class of 17ß-HSD1 inhibitors, and to rank these compounds correctly with respect to their inhibitory potency, two essential aspects in drug design. Ensemble docking experiments resulted in a steroidal binding mode for the closed enzyme conformations and in an alternative mode for the opened and occluded conformers with the inhibitors placed below the NADPH interacting with it synergically via π-π stacking and H-bond formation. Both binding modes were investigated by MD simulations and MM-PBSA binding free energy estimations using as representative member for this class compound 1 (50 nM). Notably, only the alternative binding mode proved stable and was energetically more favorable, while when simulated in the steroidal binding mode compound 1 was displaced from the active site. In parallel, ab initio studies of small NADPH-inhibitor complexes were performed, which supported the importance of the synergistic interaction between inhibitors and cofactor.

Structural basis for species specific inhibition of 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1): computational study and biological validation.

17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) catalyzes the reduction of estrone to estradiol, which is the most potent estrogen in humans. Inhibition of 17ß-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 17ß-HSD1 have been described but so far there is no cocrystal structure of the latter in complex with 17ß-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 17ß-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 17ß-HSD1 was developed and inhibition data were rationalized on the structural basis. In marmoset 17ß-HSD1, residues 190 to 196 form a small α-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.

Fine-tuning the selectivity of aldosterone synthase inhibitors: structure-activity and structure-selectivity insights from studies of heteroaryl substituted 1,2,5,6-tetrahydropyrrolo[3,2,1-ij]quinolin-4-one derivatives.

Pyridine substituted 3,4-dihydro-1H-quinolin-2-ones (e.g., 1-3) constitute a class of highly potent and selective inhibitors of aldosterone synthase (CYP11B2), a promising target for the treatment of hyperaldosteronism, congestive heart failure, and myocardial fibrosis. Among these, ethyl-substituted 3 possesses high selectivity against CYP1A2. Rigidification of 3 by incorporation of the ethyl group into a 5- or 6-membered ring affords compounds with a pyrroloquinolinone or pyridoquinolinone molecular scaffold (e.g., 4 and 5). It was found that these molecules are even more potent and selective CYP11B2 inhibitors than their corresponding open-chain analogues. Moreover, pyrroloquinolinone 4 exhibits no inhibition of the six most important hepatic CYP enzymes as well as a bioavailability in the range of the marketed drug fadrozole. The SAR studies disclose that subtle changes in the heterocyclic moiety are responsible for either a strong or a weak inhibition of the highly homologous 11ß-hydroxylase (CYP11B1). These results are not only important for fine-tuning the selectivity of CYP11B2 inhibitors but also for the development of selective CYP11B1 inhibitors that are of interest for the treatment of Cushing's syndrome and metabolic syndrome.

17ß-Hydroxysteroid dehydrogenases (17ß-HSDs) as therapeutic targets: protein structures, functions, and recent progress in inhibitor development.

17ß-Hydroxysteroid dehydrogenases (17ß-HSDs) are oxidoreductases, which play a key role in estrogen and androgen steroid metabolism by catalyzing final steps of the steroid biosynthesis. Up to now, 14 different subtypes have been identified in mammals, which catalyze NAD(P)H or NAD(P)(+) dependent reductions/oxidations at the 17-position of the steroid. Depending on their reductive or oxidative activities, they modulate the intracellular concentration of inactive and active steroids. As the genomic mechanism of steroid action involves binding to a steroid nuclear receptor, 17ß-HSDs act like pre-receptor molecular switches. 17ß-HSDs are thus key enzymes implicated in the different functions of the reproductive tissues in both males and females. The crucial role of estrogens and androgens in the genesis and development of hormone dependent diseases is well recognized. Considering the pivotal role of 17ß-HSDs in steroid hormone modulation and their substrate specificity, these proteins are promising therapeutic targets for diseases like breast cancer, endometriosis, osteoporosis, and prostate cancer. The selective inhibition of the concerned enzymes might provide an effective treatment and a good alternative to the existing endocrine therapies. Herein, we give an overview of functional and structural aspects for the different 17ß-HSDs. We focus on steroidal and non-steroidal inhibitors recently published for each subtype and report on existing animal models for the different 17ß-HSDs and the respective diseases. Article from the Special issue on Targeted Inhibitors.

Insights in 17beta-HSD1 enzyme kinetics and ligand binding by dynamic motion investigation.

BACKGROUND: Bisubstrate enzymes, such as 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), exist in solution as an ensemble of conformations. 17beta-HSD1 catalyzes the last step of the biosynthesis of estradiol and, thus, it is a potentially attractive target for breast cancer treatment. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the conformational transitions of its catalytic cycle, a structural analysis of all available crystal structures was performed and representative conformations were assigned to each step of the putative kinetic mechanism. To cover most of the conformational space, all-atom molecular dynamic simulations were performed using the four crystallographic structures best describing apoform, opened, occluded and closed state of 17beta-HSD1 as starting structures. With three of them, binary and ternary complexes were built with NADPH and NADPH-estrone, respectively, while two were investigated as apoform. Free energy calculations were performed in order to judge more accurately which of the MD complexes describes a specific kinetic step. CONCLUSIONS/SIGNIFICANCE: Remarkably, the analysis of the eight long range trajectories resulting from this multi-trajectory/-complex approach revealed an essential role played by the backbone and side chain motions, especially of the betaF alphaG'-loop, in cofactor and substrate binding. Thus, a selected-fit mechanism is suggested for 17beta-HSD1, where ligand-binding induced concerted motions of the FG-segment and the C-terminal part guide the enzyme along its preferred catalytic pathway. Overall, we could assign different enzyme conformations to the five steps of the random bi-bi kinetic cycle of 17beta-HSD1 and we could postulate a preferred pathway for it. This study lays the basis for more-targeted biochemical studies on 17beta-HSD1, as well as for the design of specific inhibitors of this enzyme. Moreover, it provides a useful guideline for other enzymes, also characterized by a rigid core and a flexible region directing their catalysis.

The role of fluorine substitution in biphenyl methylene imidazole-type CYP17 inhibitors for the treatment of prostate carcinoma.

It has been established that the growth of most prostate carcinomas depends on androgen stimulation. The inhibition of cytochrome P450-17 (CYP17) to block androgen biosynthesis is therefore regarded as a promising approach to therapy. Based on our previously identified lead compound Ref 1, a series of fluorine-substituted biphenyl methylene imidazoles were designed, synthesized, and evaluated as CYP17 inhibitors to elucidate the influence of fluorine on in vitro and in vivo activity. It was found that meta-fluoro substitution at the C ring improved activity, whereas ortho substitution decreased potency. Docking studies performed with our human CYP17 homology model suggest the presence of multipolar interactions between fluorine and Arg109, Lys231, His235, and Glu305. As expected, introduction of fluorine also prolonged the half-life in plasma. The SARs obtained confirm the reliability of the protein model; compound 9 (IC(50)=131 nM) was identified as a strong CYP17 inhibitor, showing potent activity in rat, high bioavailability, and a long plasma half-life: 12.8 h.

Novel estrone mimetics with high 17beta-HSD1 inhibitory activity.

17Beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) catalyzes the reduction of estrone into estradiol, which is the most potent estrogen in humans. Lowering intracellular estradiol concentration by inhibition of this enzyme is a promising new option for the treatment of estrogen-dependent diseases like breast cancer and endometriosis. Combination of ligand- and structure-based design resulted in heterocyclic substituted biphenylols and their aza-analogs as new 17beta-HSD1 inhibitors. The design was based on mimicking estrone, especially focusing on the imitation of the D-ring keto group with (substituted) heterocycles. Molecular docking provided insights into plausible protein-ligand interactions for this class of compounds. The most promising compound 12 showed an inhibitory activity in the high nanomolar range and very low affinity for the estrogen receptors alpha and beta. Thus, compound 12 is a novel tool for the elucidation of the pharmacological relevance of 17beta-HSD1 and might be a lead for the treatment of estrogen-dependent diseases.