Substituted Aryl Benzylamines as Potent and Selective Inhibitors of 17ß-Hydroxysteroid Dehydrogenase Type 3.

17ß-Hydroxysteroid dehydrogenase type 3 (17ß-HSD3) is expressed at high levels in testes and seminal vesicles; it is also present in prostate tissue and involved in gonadal and non-gonadal testosterone biosynthesis. The enzyme is membrane-bound, and a crystal structure is not yet available. Selective aryl benzylamine-based inhibitors were designed and synthesised as potential agents for prostate cancer therapeutics through structure-based design, using a previously built homology model with docking studies. Potent, selective, low nanomolar IC50 17ß-HSD3 inhibitors were discovered using N-(2-([2-(4-chlorophenoxy)phenylamino]methyl)phenyl)acetamide (1). The most potent compounds have IC50 values of approximately 75 nM. Compound 29, N-[2-(1-Acetylpiperidin-4-ylamino)benzyl]-N-[2-(4-chlorophenoxy)phenyl]acetamide, has an IC50 of 76 nM, while compound 30, N-(2-(1-[2-(4-chlorophenoxy)-phenylamino]ethyl)phenyl)acetamide, has an IC50 of 74 nM. Racemic C-allyl derivative 26 (IC50 of 520 nM) was easily formed from 1 in good yield and, to determine binding directionality, its enantiomers were separated by chiral chromatography. Absolute configuration was determined using single crystal X-ray crystallography. Only the S-(+)-enantiomer (32) was active with an IC50 of 370 nM. Binding directionality was predictable through our in silico docking studies, giving confidence to our model. Importantly, all novel inhibitors are selective over the type 2 isozyme of 17ß-HSD2 and show <20% inhibition when tested at 10 µM. Lead compounds from this series are worthy of further optimisation and development as inhibitors of testosterone production by 17ß-HSD3 and as inhibitors of prostate cancer cell growth.

Order within disorder: aggrecan chondroitin sulphate-attachment region provides new structural insights into protein sequences classified as disordered.

Structural investigation of proteins containing large stretches of sequences without predicted secondary structure is the focus of much increased attention. Here, we have produced an unglycosylated 30 kDa peptide from the chondroitin sulphate (CS)-attachment region of human aggrecan (CS-peptide), which was predicted to be intrinsically disordered and compared its structure with the adjacent aggrecan G3 domain. Biophysical analyses, including analytical ultracentrifugation, light scattering, and circular dichroism showed that the CS-peptide had an elongated and stiffened conformation in contrast to the globular G3 domain. The results suggested that it contained significant secondary structure, which was sensitive to urea, and we propose that the CS-peptide forms an elongated wormlike molecule based on a dynamic range of energetically equivalent secondary structures stabilized by hydrogen bonds. The dimensions of the structure predicted from small-angle X-ray scattering analysis were compatible with EM images of fully glycosylated aggrecan and a partly glycosylated aggrecan CS2-G3 construct. The semiordered structure identified in CS-peptide was not predicted by common structural algorithms and identified a potentially distinct class of semiordered structure within sequences currently identified as disordered. Sequence comparisons suggested some evidence for comparable structures in proteins encoded by other genes (PRG4, MUC5B, and CBP). The function of these semiordered sequences may serve to spatially position attached folded modules and/or to present polypeptides for modification, such as glycosylation, and to provide templates for the multiple pleiotropic interactions proposed for disordered proteins.

Development of hormone-dependent prostate cancer models for the evaluation of inhibitors of 17beta-hydroxysteroid dehydrogenase type 3.

17beta-Hydroxysteroid dehydrogenases (17beta-HSDs) are responsible for the pre-receptor reduction/oxidation of steroids at the 17-position into active/inactive hormones, and the 15 known enzymes vary in their substrate specificity, localisation, and directional activity. 17beta-HSD Type 3 (17beta-HSD3) has been seen to be over-expressed in prostate cancer, and catalyses the reduction of androstenedione (Adione) to testosterone (T), which stimulates prostate tumour growth. Specific inhibitors of 17beta-HSD3 may have a role in the treatment of hormone-dependent prostate cancer and benign prostate hyperplasia, and also have potential as male anti-fertility agents. A 293-EBNA-based cell line with stable expression of transfected human 17beta-HSD3 was created and used to develop a whole cell radiometric TLC-based assay to assess the 17beta-HSD3 inhibitory potency of a series of compounds. STX2171 and STX2624 (IC(50) values in the 200-450nM range) were two of several active inhibitors identified. In similar TLC-based assays these compounds were found to be inactive against 17beta-HSD1 and 17beta-HSD2, indicating selectivity. A novel proof of concept model was developed to study the efficacy of the compounds in vitro using the androgen receptor positive hormone-dependent prostate cancer cell line, LNCaPwt, and its derivative, LNCaP[17beta-HSD3], transfected and selected for stable expression of 17beta-HSD3. The proliferation of the parental cell line was most efficiently stimulated by 5alpha-dihydrotestosterone (DHT), but the LNCaP[17beta-HSD3] cells were equally stimulated by Adione, indicating that 17beta-HSD3 efficiently converts Adione to T in this model. Adione-stimulated proliferation of LNCaP[17beta-HSD3] cells was inhibited in the presence of either STX2171 or STX2624. The compounds alone neither stimulated proliferation of the cells nor caused significant cell death, indicating that they are non-androgenic with low cytotoxicity. STX2171 inhibited Adione-stimulated growth of xenografts established from LNCaPwt cells in castrated mice in vivo. In conclusion, a primary screening assay and proof of concept model have been developed to study the efficacy of 17beta-HSD3 inhibitory compounds, which may have a role in the treatment of hormone-dependent cancer. Active compounds are selective for 17beta-HSD3 over 17beta-HSD1 and 17beta-HSD2, non-androgenic with low toxicity, and efficacious in both an in vitro proof of concept model and in an in vivo tumour model.

The design of novel 17beta-hydroxysteroid dehydrogenase type 3 inhibitors.

17beta-Hydroxysteroid dehydrogenase type 3 (17beta-HSD3) is expressed at high levels in the testes and seminal vesicles but has also been shown to be present in prostate tissue, suggesting its potential involvement in both gonadal and non-gonadal testosterone biosynthesis. The role of 17beta-HSD3 in testosterone biosynthesis makes this enzyme an attractive molecular target for small molecule inhibitors for the treatment of prostate cancer. Here we report the design of selective inhibitors of 17beta-HSD3 as potential anti-cancer agents. Due to 17beta-HSD3 being a membrane-bound protein a crystal structure is not yet available. A homology model of 17beta-HSD3 has been built to aid structure-based drug design. This model has been used with docking studies to identify a series of lead compounds that may give an insight as to how inhibitors interact with the active site. Compound 1 was identified as a potent selective inhibitor of 17beta-HSD3 with an IC(50)=700nM resulting in the discovery of a novel lead series for further optimisation. Using our homology model as a tool for inhibitor design compound 5 was discovered as a novel potent and selective inhibitor of 17beta-HSD3 with an IC(50) approximately 200nM.

STX140 is efficacious in vitro and in vivo in taxane-resistant breast carcinoma cells.

PURPOSE: The aim of these studies was to characterize the action of STX140 in a P-glycoprotein-overexpressing tumor cell line both in vitro and in vivo. In addition, its efficacy was determined against xenografts derived from patients who failed docetaxel therapy. EXPERIMENTAL DESIGN: The effects of STX140, Taxol, and 2-methoxyestradiol (2-MeOE2) on cell proliferation, cell cycle, and apoptosis were assessed in vitro in drug-resistant cells (MCF-7(DOX)) and the parental cell line (MCF-7(WT)). Mice bearing an MCF-7(DOX) tumor on one flank and an MCF-7(WT) tumor on the other flank were used to assess the in vivo efficacy. Furthermore, the responses to STX140 of three xenografts, derived from drug-resistant patients, were assessed. RESULTS: In this study, STX140 caused cell cycle arrest, cyclin B1 induction, and subsequent apoptosis of both MCF-7(DOX) and MCF-7(WT) cells. Taxol and 2-MeOE2 were only active in the MCF-7(WT) parental cell line. Although both STX140 and Taxol inhibited the growth of xenografts derived from MCF-7(WT) cells, only STX140 inhibited the growth of tumors derived from MCF-7(DOX) cells. 2-MeOE2 was ineffective at the dose tested against both tumor types. Two out of the three newly derived docetaxel-resistant xenografts, including a metastatic triple-negative tumor, responded to STX140 but not to docetaxel treatment. CONCLUSIONS: STX140 shows excellent efficacy in both MCF-7(WT) and MCF-7(DOX) breast cancer xenograft models, in contrast to Taxol and 2-MeOE2. The clinical potential of STX140 was further highlighted by the efficacy seen in xenografts recently derived from patients who had failed on taxane therapy.

Design and validation of specific inhibitors of 17beta-hydroxysteroid dehydrogenases for therapeutic application in breast and prostate cancer, and in endometriosis.

17beta-Hydroxysteroid dehydrogenases (17beta-HSDs) are enzymes that are responsible for reduction or oxidation of hormones, fatty acids and bile acids in vivo, regulating the amount of the active form that is available to bind to its cognate receptor. All require NAD(P)(H) for activity. Fifteen 17beta-HSDs have been identified to date, and with one exception, 17beta-HSD type 5 (17beta-HSD5), an aldo-keto reductase, they are all short-chain dehydrogenases/reductases, although overall homology between the enzymes is low. Although named as 17beta-HSDs, reflecting the major redox activity at the 17beta-position of the steroid, the activities of these 15 enzymes vary, with several of the 17beta-HSDs able to reduce and/or oxidise multiple substrates at various positions. These activities are involved in the progression of a number of diseases, including those related to steroid metabolism. Despite the success of inhibitors of steroidogenic enzymes in the clinic, such as those of aromatase and steroid sulphatase, the development of inhibitors of 17beta-HSDs is at a relatively early stage, as at present none have yet reached clinical trials. However, many groups are now working on inhibitors specific for several of these enzymes for the treatment of steroid-dependent diseases, including breast and prostate cancer, and endometriosis, with demonstrable efficacy in in vivo disease models. In this review, the recent advances in the validation of these enzymes as targets for the treatment of these diseases, with emphasis on 17beta-HSD1, 3 and 5, the development of specific inhibitors, the models used for their evaluation, and their progress towards the clinic will be discussed.

17beta-hydroxysteroid dehydrogenase Type 1, and not Type 12, is a target for endocrine therapy of hormone-dependent breast cancer.

Oestradiol (E2) stimulates the growth of hormone-dependent breast cancer. 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyse the pre-receptor activation/inactivation of hormones and other substrates. 17beta-HSD1 converts oestrone (E1) to active E2, but it has recently been suggested that another 17beta-HSD, 17beta-HSD12, may be the major enzyme that catalyses this reaction in women. Here we demonstrate that it is 17beta-HSD1 which is important for E2 production and report the inhibition of E1-stimulated breast tumor growth by STX1040, a non-oestrogenic selective inhibitor of 17beta-HSD1, using a novel murine model. 17beta-HSD1 and 17beta-HSD12 mRNA and protein expression, and E2 production, were assayed in wild type breast cancer cell lines and in cells after siRNA and cDNA transfection. Although 17beta-HSD12 was highly expressed in breast cancer cell lines, only 17beta-HSD1 efficiently catalysed E2 formation. The effect of STX1040 on the proliferation of E1-stimulated T47D breast cancer cells was determined in vitro and in vivo. Cells inoculated into ovariectomised nude mice were stimulated using 0.05 or 0.1 microg E1 (s.c.) daily, and on day 35 the mice were dosed additionally with 20 mg/kg STX1040 s.c. daily for 28 days. STX1040 inhibited E1-stimulated proliferation of T47D cells in vitro and significantly decreased tumor volumes and plasma E2 levels in vivo. In conclusion, a model was developed to study the inhibition of the major oestrogenic 17beta-HSD, 17beta-HSD1, in breast cancer. Both E2 production and tumor growth were inhibited by STX1040, suggesting that 17beta-HSD1 inhibitors such as STX1040 may provide a novel treatment for hormone-dependent breast cancer.

Effects of mutations and glycosylations on STS activity: a site-directed mutagenesis study.

Steroid sulphatase (STS) catalyses the formation of active steroids from inactive steroid sulphates. High levels of intra-tumoural STS mRNA are associated with a poor prognosis in post-menopausal patients with oestrogen receptor positive breast cancer. In this study, analysis of the mutated STS protein showed that N- and C-terminal truncated STS constructs are inactive. Histidine 136, located inside the active site, is crucial for STS activity whereas proline 212, which allows the protein turn into the membrane, is not. Mutations in glycosylation sites asparagine 47 and 259 decreased STS activity while asparagine 333 and 459 mutations did not affect it. However, immunoblot studies revealed that all four N-linked sites are glycosylated to some extent. In addition, a polyclonal antibody raised in rabbits against human STS was developed and characterised. These data increase our knowledge of the STS enzyme structure and may help design new STS inhibitors.

Novel inhibitors of 17beta-hydroxysteroid dehydrogenase type 1: templates for design.

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyze the interconversion between the oxidized and reduced forms of androgens and estrogens at the 17 position. The 17beta-HSD type 1 enzyme (17beta-HSD1) catalyzes the reduction of estrone (E1) to estradiol and is expressed in malignant breast cells. Inhibitors of this enzyme thus have potential as treatments for hormone dependent breast cancer. Syntheses and biological evaluation of novel non-steroidal inhibitors designed to mimic the E1 template are reported using information from potent steroidal inhibitors. Of the templates investigated biphenyl ethanone was promising and led to inhibitors with IC(50) values in the low micromolar range.

The role of 17beta-hydroxysteroid dehydrogenases in modulating the activity of 2-methoxyestradiol in breast cancer cells.

The bis-sulfamoylated derivative of 2-methoxyestradiol (2-MeOE2), 2-methoxyestradiol-3,17-O,O-bis-sulfamate (2-MeOE2bisMATE), has shown potent antiproliferative and antiangiogenic activity in vitro and inhibits tumor growth in vivo. 2-MeOE2bisMATE is bioavailable, in contrast to 2-MeOE2 that has poor bioavailability. In this study, we have examined the role of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type 2 in the metabolism of 2-MeOE2. In MDA-MB-231 cells, which express high levels of 17beta-HSD type 2, and in MCF-7 cells transfected with 17beta-HSD type 2, high-performance liquid chromatography analysis showed that a significant proportion of 2-MeOE2 was metabolized to inactive 2-methoxyestrone. Furthermore, MCF-7 cells transfected with 17beta-HSD type 2 were protected from the cytotoxic effects of 2-MeOE2. In contrast, no significant metabolism of 2-MeOE2bisMATE was detected in transfected cells and 17beta-HSD type 2 transfection did not offer protection against 2-MeOE2bisMATE cytotoxicity. This study may go some way to explaining the poor bioavailability of 2-MeOE2, as the gastrointestinal mucosa expresses high levels of 17beta-HSD type 2. In addition, this study shows the value of synthesizing sulfamoylated derivatives of 2-MeOE2 with C17-position modifications as these compounds have improved bioavailability and potency both in vitro and in vivo.

Cytokines in human breast cyst fluid.

Gross cystic breast disease is a common benign disorder in which palpable cysts occur in the breast and are normally treated by aspiration of the contents. The cysts are classified as either Type 1, containing a high level of potassium ions and a low level of sodium ions, or as Type 2, with low potassium and high sodium ion concentrations. Steroid sulphatase activity in MDA-MB-231 and MCF-7 cell lines is regulated by exogenous breast cyst fluid (BCF), possibly because of cytokines in the BCF. A screening method was used to determine the range of cytokines in eight BCFs, four of each type. This was an array system, which uses antibodies immobilised on a membrane to qualitatively detect 79 different cytokines or growth factors. Nine cytokines were detected well above background levels: all were found in both types of BCF, but only epidermal growth factor (EGF) was higher in Type 1. All the other factors were higher in Type 2 BCF. Two of these cytokines, IL-6 and EGF, have previously been suggested to affect steroid sulphatase expression and several (MIP-1beta, IL-8, NAP-2) are known to affect MCF-7 cell chemotaxis. In addition two cytokines were measured by ELISA in 57 BCFs, and both IL-1beta and IL-13 were found in BCF, with significantly higher amounts of IL-1beta in Type 1 than Type 2 BCF (35.5+/-4.4 pg/ml versus 9.9+/-2.9 pg/ml).

Modification of estrone at the 6, 16, and 17 positions: novel potent inhibitors of 17beta-hydroxysteroid dehydrogenase type 1.

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyze the interconversion between the oxidized and reduced forms of androgens and estrogens at the 17 position. The 17beta-HSD type 1 enzyme (17beta-HSD1) catalyzes the reduction of estrone to estradiol and is expressed in malignant breast cells. Inhibitors of this enzyme thus have potential as treatments for hormone dependent breast cancer. Here we report the syntheses and biological evaluation of novel inhibitors based on the estrone or estradiol template. These have been investigated by modification at the 6, 16 or 17 positions or combinations of these in order to gain information about structure-activity relationships by probing different areas in the enzyme active site. Activity data have been incorporated into a QSAR with predictive power, and the X-ray crystal structures of compounds 15 and 16c have been determined. Compound 15 has an IC50 of 320 nM for 17beta-HSD1 and is selective for 17beta-HSD1 over 17beta-HSD2. Three libraries of amides are also reported that led to the identification of inhibitors 19e and 20a, which have IC50 values of 510 and 380 nM respectively, and 20 h which, having an IC50 value of 37 nM, is the most potent inhibitor of 17beta-HSD1 reported to date. These amides are also selective for 17beta-HSD1 over 17beta-HSD2.

17Beta-hydroxysteroid dehydrogenase Type 1 and Type 2: association between mRNA expression and activity in cell lines.

17Beta-hydroxysteroid dehydrogenases (17beta-HSDs) are a family of enzymes that regulate steroid availability within a tissue by catalysing the interconversion of active and inactive forms. Type 1 is up-regulated in many breast tumours, and is responsible for the reduction of oestrone to active oestradiol which stimulates cell proliferation within the tumour. Type 2 oxidises many active steroids to their inactive forms, including oestradiol to oestrone. In this study, we have compared the mRNA expression and enzyme activities of Type 1 and Type 2 in MCF-7, MDA-MB-231, T47D, JEG3 and 293-EBNA cell lines. Also studied were two cell lines stably expressing transfected Type 1 cDNA. RT-PCR indicated that little Type 1 mRNA is expressed in two of the breast cancer cell lines, MCF-7 and MDA-MB-231, and in 293-EBNA cells, but that expression is much higher in the T47D breast cancer cell line, and in the choriocarcinoma cell line, JEG3. However, a higher level of expression of Type 1 is seen in the transfected cell lines MCF-7.8H and 293-EBNA[His617beta-HSD1]. Activity assays show that there is high association between mRNA expression and enzyme activity. Assays indicate that, with the exception of MDA-MB-231 cells, Type 2 activity is low in these lines. The study of the basal activities of these enzymes will be used in future studies investigating the regulation of the enzymes by endogenous and exogenous factors. An understanding of their regulation in both healthy and malignant tissues may lead to future therapeutic intervention at the regulatory level.

The effects of 2-substituted oestrogen sulphamates on the growth of prostate and ovarian cancer cells.

The human endogenous metabolite 2-methoxyoestradiol (2-MeOE2) has been shown to inhibit the proliferation of breast cancer cells. We have previously shown that sulphamoylation of a series of 2-substituted oestrogens greatly enhances their ability to inhibit breast cancer cell proliferation and induce apoptosis. In this study, we have investigated the ability of a number of 2-substituted oestrogens and their sulphamoylated derivatives to inhibit the proliferation of two prostate cancer cell lines, an ovarian cancer cell line and its drug-resistant derivatives. 2-Methoxyoestrone, 2-ethyloestrone and 2-ethyloestradiol had little effect on the growth of the cell lines tested (IC(50)>10 microM). 2-MeOE2 did inhibit the growth of the cells (IC(50)<10 microM), but to a lesser extent than any of the sulphamoylated derivatives tested (IC(50)<1.0 microM). Cells treated with the sulphamoylated derivatives became detached and rounded, displaying a characteristic apoptotic appearance. FACS analysis revealed induced G(2)/M cell cycle arrest. Treatment of cells and subsequent drug removal indicated that the effects of the drugs on the cells were irreversible. Immunoblot analysis indicated that apoptosis may be induced by phosphorylation of BCL-2. From these studies, 2-substituted oestrogen sulphamates are emerging as a potent new class of drug that may be effective against AR+/AR- prostate and ovarian tumours, and against tumours that are resistant to conventional chemotherapeutic regimens.

STX140, but not paclitaxel, inhibits mammary tumour initiation and progression in C3(1)/SV40 T/t-antigen transgenic mice.

Despite paclitxael's clinical success, treating hormone-refractory breast cancer remains challenging. Paclitaxel has a poor pharmacological profile, characterized by a low therapeutic index (TIX) caused by severe dose limiting toxicities, such as neutropenia and peripheral neuropathy. Consequently, new drugs are urgently required. STX140, a compound previously shown to have excellent efficacy against many tumors, is here compared to paclitaxel in three translational in vivo breast cancer models, a rat model of peripheral neuropathy, and through pharmacological testing. Three different in vivo mouse models of breast cancer were used; the metastatic 4T1 orthotopic model, the C3(1)/SV40 T-Ag model, and the MDA-MB-231 xenograft model. To determine TIX and pharmacological profile of STX140, a comprehensive dosing regime was performed in mice bearing MDA-MD-231 xenografts. Finally, peripheral neuropathy was examined using a rat plantar thermal hyperalgesia model. In the 4T1 metastatic model, STX140 and paclitaxel significantly inhibited primary tumor growth and lung metastases. All C3(1)/SV40 T-Ag mice in the control and paclitaxel treated groups developed palpable mammary cancer. STX140 blocked 47% of tumors developing and significantly inhibited growth of tumors that did develop. STX140 treatment caused a significant (P<0.001) survival advantage for animals in early and late intervention groups. Conversely, in C3(1)/SV40 T-Ag mice, paclitaxel failed to inhibit tumor growth and did not increase survival time. Furthermore, paclitaxel, but not STX140, induced significant peripheral neuropathy and neutropenia. These results show that STX140 has a greater anti-cancer efficacy, TIX, and reduced neurotoxicity compared to paclitaxel in C3(1)/SV40 T-Ag mice and therefore may be of significant benefit to patients with breast cancer.

STX2171, a 17ß-hydroxysteroid dehydrogenase type 3 inhibitor, is efficacious in vivo in a novel hormone-dependent prostate cancer model.

17ß-Hydroxysteroid dehydrogenases (17ß-HSDs) catalyse the 17-position reduction/oxidation of steroids. 17ß-HSD type 3 (17ß-HSD3) catalyses the reduction of the weakly androgenic androstenedione (adione) to testosterone, suggesting that specific inhibitors of 17ß-HSD3 may have a role in the treatment of hormone-dependent prostate cancer and benign prostate hyperplasia. STX2171 is a novel selective non-steroidal 17ß-HSD3 inhibitor with an IC(50) of ∼200 nM in a whole-cell assay. It inhibits adione-stimulated proliferation of 17ß-HSD3-expressing androgen receptor-positive LNCaP(HSD3) prostate cancer cells in vitro. An androgen-stimulated LNCaP(HSD3) xenograft proof-of-concept model was developed to study the efficacies of STX2171 and a more established 17ß-HSD3 inhibitor, STX1383 (SCH-451659, Schering-Plough), in vivo. Castrated male MF-1 mice were inoculated s.c. with 1×10(7) cells 24 h after an initial daily dose of testosterone propionate (TP) or vehicle. After 4 weeks, tumours had not developed in vehicle-dosed mice, but were present in 50% of those mice given TP. One week after switching the stimulus to adione, mice were dosed additionally with the vehicle or inhibitor for a further 4 weeks. Both TP and adione efficiently stimulated tumour growth and increased plasma testosterone levels; however, in the presence of either 17ß-HSD3 inhibitor, adione-dependent tumour growth was significantly inhibited and plasma testosterone levels reduced. Mouse body weights were unaffected. Both inhibitors also significantly lowered plasma testosterone levels in intact mice. In conclusion, STX2171 and STX1383 significantly lower plasma testosterone levels and inhibit androgen-dependent tumour growth in vivo, indicating that 17ß-HSD3 inhibitors may have application in the treatment of hormone-dependent prostate cancer.

Overexpression of 17ß-hydroxysteroid dehydrogenase type 1 increases the exposure of endometrial cancer to 17ß-estradiol.

CONTEXT: The local interconversions between estrone (low activity) and 17ß-estradiol (potent compound) by 17ß-hydroxysteroid dehydrogenases (17ß-HSDs) can lead to high 17ß-estradiol generation in endometrial cancer (EC). OBJECTIVE: Examine the balance between the 17ß-HSDs reducing estrone to 17ß-estradiol (types 1, 5, 12, and 7) and those oxidizing 17ß-estradiol to estrone (2, 4, and 8), in EC. PATIENTS AND METHODS: Reducing and oxidizing 17ß-HSD activities (HPLC) and mRNA level (RT-PCR) were assessed in normal post-menopausal (n = 16), peritumoral endometrium (normal tissue beside cancer, n = 13), and 58 EC (29 grade 1, 18 grade 2, 11 grade 3). RESULTS: Grade 1 EC displayed a shifted estrone reduction/17ß-estradiol oxidation balance in favor of 17ß-estradiol compared with controls. This was more pronounced among estrogen receptor-α (ER-α)-positive biopsies. Type 1 17ß-HSD mRNA (HSD17B1 gene expression, real time PCR) and protein levels (immunohistochemistry) were higher in ER-α-positive grade 1 EC than controls. The mRNA coding for types 4, 5, 7, 8, and 12 17ß-HSD did not vary, whereas that coding for type 2 17ß-HSD was increased in high-grade lesions compared with controls. Three-dimensional ex vivo EC explant cultures demonstrated that 17ß-HSD type 1 generated 17ß-estradiol from estrone and increased tumor cell proliferation. Additional in vitro studies using EC cells confirmed that in the presence of 17ß-HSD type 1, estrone induced estrogen signaling activation similarly to 17ß-estradiol. Therefore, estrone was reduced to 17ß-estradiol. CONCLUSIONS: Type 1 17ß-HSD increases 17ß-estradiol exposure in grade 1 EC, thus supporting tumor growth. This enzyme represents a potential therapeutic target.

The development of steroid sulfatase inhibitors for hormone-dependent cancer therapy.

Steroid sulfatase (STS) regulates the hydrolysis of steroid sulfates to their unconjugated forms. Estrone sulfate and dehydroepiandrosterone sulfate can be hydrolyzed by STS to estrone and dehydroepiandrosterone, respectively, with these steroids being the precursors for the synthesis of more biologically active estrogens or androgens. A number of potent STS inhibitors have now been developed including STX64, which entered a phase I trial for the treatment of postmenopausal women with advanced metastatic hormone-dependent breast cancer. The results from this phase I trial were encouraging, suggesting that STS inhibitors may also have a role in the treatment of other hormone-dependent cancers including those of the endometrium, ovary, and prostate. In this paper the potential use of STS inhibitors to treat these hormone-dependent cancers is reviewed. In addition, results from in vitro studies show that Ishikawa endometrial cancer cells, OVCAR-3 ovarian cancer cells, and LNCaP prostate cancer cells all possess significant STS activity. Furthermore, STS activity in these cells can be almost completely inhibited by STX64 or the second-generation STS inhibitor, STX213. Results from these investigations therefore suggest that STS inhibitors could have therapeutic potential for the treatment of a range of hormone-dependent cancers.

Focused libraries of 16-substituted estrone derivatives and modified e-ring steroids: inhibitors of 17beta-hydroxysteroid dehydrogenase type 1.

17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), an oxidoreductase which has a preferential reductive activity using NADPH as cofactor, converts estrone to estradiol and is expressed in many steroidogenic tissues including breast and in malignant breast cells. As estradiol stimulates the growth and development of hormone-dependent breast cancer, inhibition of the final step of its synthesis is an attractive target for the treatment of this disease. The parallel synthesis of novel focused libraries of 16-substituted estrone derivatives and modified E-ring pyrazole steroids as new potent 17beta-HSD1 inhibitors is described. Substituted 3-O-sulfamoylated estrone derivatives were used as templates and were immobilised on 2-chlorotrityl chloride resin to give resin-bound scaffolds with a multi-detachable linker. Novel focused libraries of 16-substituted estrone derivatives and new modified E-ring steroids were assembled from these immobilised templates using solid-phase organic synthesis and solution-phase methodologies. Among the derivatives synthesised, the most potent 17beta-HSD1 inhibitors were 25 and 26 with IC50 values in T-47D human breast cancer cells of 27 and 165 nm, respectively. Parallel synthesis resulting in a library of C5'-linked amides from the pyrazole E-ring led to the identification of 62 with an IC50 value of 700 nM. These potent inhibitors of 17beta-HSD1 have a 2-ethyl substituent which will decrease their estrogenic potential. Several novel 17beta-HSD1 inhibitors emerged from these libraries and these provide direction for further template exploration in this area. A new efficient diastereoselective synthesis of 25 has also been developed to facilitate supply for in vivo evaluation, and an X-ray crystal structure of this inhibitor is presented.

Alternative splicing in the aggrecan G3 domain influences binding interactions with tenascin-C and other extracellular matrix proteins.

The proteoglycans aggrecan, versican, neurocan, and brevican bind hyaluronan through their N-terminal G1 domains, and other extracellular matrix proteins through the C-type lectin repeat in their C-terminal G3 domains. Here we identify tenascin-C as a ligand for the lectins of all these proteoglycans and map the binding site on the tenascin molecule to fibronectin type III repeats, which corresponds to the proteoglycan lectin-binding site on tenascin-R. In the G3 domain, the C-type lectin is flanked by epidermal growth factor (EGF) repeats and a complement regulatory protein-like motif. In aggrecan, these are subject to alternative splicing. To investigate if these flanking modules affect the C-type lectin ligand interactions, we produced recombinant proteins corresponding to aggrecan G3 splice variants. The G3 variant proteins containing the C-type lectin showed different affinities for various ligands, including tenascin-C, tenascin-R, fibulin-1, and fibulin-2. The presence of an EGF motif enhanced the affinity of interaction, and in particular the splice variant containing both EGF motifs had significantly higher affinity for ligands, such as tenascin-R and fibulin-2. The mRNA for this splice variant was shown by reverse transcriptase-PCR to be expressed in human chondrocytes. Our findings suggest that alternative splicing in the aggrecan G3 domain may be a mechanism for modulating interactions and extracellular matrix assembly.