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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.

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.

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.

Many breast tumours are hormone-responsive and rely on estrogens for their sustained growth and development. The enzyme 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) is primarily responsible for the conversion of estrone (E1) into the most potent of the human estrogens 17beta-estradiol (E2). Here we report the syntheses, inhibitory activities and docking studies for a novel series of pyrazole amides which have been discovered with the aim of probing the structure activity relationships (SAR) for such a template and of using this template to mimic the potent inhibitor 1 (Fig. 1). Amides containing an aromatic pyridyl moiety have been found to give the best inhibition, indicating that the pyridyl group interacts beneficially in the active site. This work has shown that extension from this position on the pyrazole template is well tolerated and the optimization of such systems is under investigation.

17Beta-hydroxysteroid dehydrogenase Type 1 (17beta-HSD1) has a pivotal role in regulating the synthesis of oestradiol (E2) within breast tumours. In whole body studies in postmenopausal women with breast cancer the conversion of oestrone (E1) to E2 (4.4+/-1.1%) was much lower than the inactivation of E2 to E1 (17.3+/-5.0%). In contrast, an examination of in vivo oestrogen metabolism within breast tumours revealed that whereas little metabolism of E2 occurred, E1 was converted to E2 to a much greater extent in malignant (48+/-14%) than in normal (19+/-6%) breast tissue. Findings from these studies originally suggested that oestrogen metabolism within breast tumours may differ from the mainly oxidative direction found in most other body tissues and that the activity of 17beta-HSD1 might be regulated by tumour-derived factors. Several growth factors (e.g. IGF-I, IGF-II) and cytokines (e.g. IL-6, TNFalpha) have now been identified which can markedly stimulate the activity of 17beta-HSD1 and such a mechanism may account for the high concentrations of E2 found in most breast tumours. Cells of the immune system, which can infiltrate breast tumours, are thought to be a major source of the growth factors and cytokines which can modulate 17beta-HSD1 activity. Given the central role that 17beta-HSD1 has in regulating breast tumour E2 concentrations the development of potent inhibitors of this enzyme has recently attracted considerable attention. Our initial studies in this area explored the use of derivatives of E1 as inhibitors, with 2-ethyl- and 2-methoxy E1 being found to inhibit 17beta-HSD1 activity in T-47D breast cancer cells by 96+/-2 and 91+/-1% respectively at 10 microM, but with a lack of specificity. Using the E1 scaffold a number of potent, selective 17beta-HSD1 inhibitors have now been identified including E1- and 2-ethyl-E1 containing a side chain with a m-pyridylmethylamidomethyl functionality extending from the 16beta position of the steroid nucleus. At 10 microM these compounds both inhibited 17beta-HSD1 activity by >90%, however some inhibition of 17beta-HSD2 activity was exhibited by the E1 derivative (25%) but not the 2-ethyl analogue. It is now apparent that 17beta-HSD1 activity contributes to the high E2 concentrations found in most breast tumours. The identification of potent, selective novel 17beta-HSD1 inhibitors will allow their efficacy to be tested in in vitro and in vivo studies.

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.