{"paper_id":"a8050409-bb91-4dfa-a561-10f55fa1da7b","body_text":"REVIEW\nDesign and validation of specific inhibitors\nof 17 b-hydroxysteroid dehydrogenases for\ntherapeutic application in breast and\nprostate cancer, and in endometriosis\nJoanna M Day, Helena J Tutill, Atul Purohit and Michael J Reed\nDepartment of Endocrinology and Metabolic Medicine and Sterix Ltd, Imperial College London, St Mary’s Hospital, 2nd Floor, Mint\nWing, Winsland Street, London W2 1NY, UK\n(Correspondence should be addressed to M J Reed; Email: m.reed@imperial.ac.uk)\nAbstract\n17b-Hydroxysteroid dehydrogenases (17b-HSDs) are enzymes that are responsible for reduction\nor oxidation of hormones, fatty acids and bile acidsin vivo, regulating the amount of the active form\nthat is available to bind to its cognate receptor. All require NAD(P)(H) for activity. Fifteen\n17b-HSDs have been identified to date, and with one exception, 17b-HSD type 5 (17b-HSD5), an\naldo–keto reductase, they are all short-chain dehydrogenases/reductases, although overall\nhomology between the enzymes is low. Although named as 17b-HSDs, reflecting the major redox\nactivity at the 17b-position of the steroid, the activities of these 15 enzymes vary, with several of\nthe 17 b-HSDs able to reduce and/or oxidise multiple substrates at various positions. These\nactivities are involved in the progression of a number of diseases, including those related to steroid\nmetabolism. Despite the success of inhibitors of steroidogenic enzymes in the clinic, such as those\nof aromatase and steroid sulphatase, the development of inhibitors of 17b-HSDs is at a relatively\nearly stage, as at present none have yet reached clinical trials. However, many groups are now\nworking on inhibitors specific for several of these enzymes for the treatment of steroid-dependent\ndiseases, including breast and prostate cancer, and endometriosis, with demonstrable efficacy in\nin vivo disease models. In this review, the recent advances in the validation of these enzymes as\ntargets for the treatment of these diseases, with emphasis on 17 b-HSD1, 3 and 5, the\ndevelopment of specific inhibitors, the models used for their evaluation, and their progress\ntowards the clinic will be discussed.\nEndocrine-Related Cancer (2008) 15 665–692\nIntroduction\nSteroid-dependent diseases\nBreast cancer, a major cause of death in both European\nand American women, occurs most frequently in post-\nmenopausal women. After menopause, a low level of\noestrogen is produced, mainly from the local conver-\nsion of androstenedione (Adione; Fig. 1 a) to oestrone\n(E\n1; Fig. 1 b) by aromatase in adipose and normal and\nmalignant breast tissues, despite the cessation of\novarian function. This oestrogen has a crucial role in\nsupporting the growth of hormone-dependent breast\ncancer in these women. Much of the E\n1 formed by\naromatase is stored in an inactive form as E 1 sulphate,\nwhich can be reactivated within breast cells by steroid\nsulphatase ( Stanway et al . 2006 ). Presently, various\nmethodologies are used in the clinic to inhibit the\nstimulation of hormone-dependent breast cancer by\noestrogen. These include oestrogen receptor (ER)\nantagonists, such as tamoxifen (Heel et al. 1978, Jordan\n2003), and inhibitors of steroid synthesis, such as\nletrozole, an aromatase inhibitor (Bhatnagar et al. 1990,\nBhatnagar 2006, Scott & Keam 2006 ) or more recently\n667-COUMATE, a steroid sulphatase inhibitor (Purohit\net al. 2003, Stanway et al. 2006, 2007).\nTreatment of hormone-dependent prostate cancer\nby androgen ablation is initially usually successful,\nreducing primary tumour burden and increasing 5-year\nEndocrine-Related Cancer (2008) 15 665–692\nEndocrine-Related Cancer (2008) 15 665–692\n1351–0088/08/015–665 q 2008 Society for Endocrinology Printed in Great Britain\nDOI: 10.1677/ERC-08-0042\nOnline version via http://www.endocrinology-journals.org\nDownloaded from Bioscientifica.com at 06/06/2026 10:17:57PM\nvia free access\n\n\nsurvival rates. Presently, androgen ablation is achieved\nusing various approaches that include orchidectomy,\nandrogen receptor (AR) blockers such as bicalutamide\n(Fradet 2004), agonists of luteinising hormone-releasing\nhormone such as goserelin ( Akaza 2004 ), finasteride,\nan inhibitor of 5 a-reductase, the enzyme that converts\ntestosterone ( Fig. 1 d) to the more active androgen,\n5a-dihydrotestosterone (DHT; Fig. 1 e) ( Rittmaster\n1997), or a combination of the above.\nHowever, prostate cancer is the third highest cause\nof cancer-related death in men, because tumours are\noften unnoticed for several years, presenting at an\nadvanced stage in older men, by which time they have\noften progressed to hormone independency. At present,\nthere are many efforts to understand the mechanisms\nby which prostate cancer cells develop hormone\nindependence. These are thought to include AR\nup-regulation, an adaptation to the low levels of\nandrogen present during ablation therapy or mutations\nto the pathways involved in the activation of the AR,\nsuch as mutations in the receptor itself, or in its\nco-regulators, allowing enhanced activation by the\nlow-level androgens ( Mizokami et al. 2004) or ligands\nother than androgens to activate the proliferative\npathways ( Rau et al . 2005 , Pienta & Bradley 2006 ).\nIt has also been suggested that many cases of recurrent\nandrogen-independent prostate cancer may not\nactually be independent of androgen signalling, as\nhigh levels of testosterone and DHT have been found in\nthe prostates of patients with recurrence during\nablation therapy, suggesting that surgical or chemical\ncastration treatments may not result in complete\nremoval of active androgens, and that in situ formation\nof testosterone from adrenal production of androgens\nmay continue ( Titus et al . 2005 ).\nEndometriosis is one of the most common causes of\npelvic pain and infertility in women. In this condition,\nendometrial tissue grows abnormally outside the\nuterus, often in locations such as the ovaries, fallopian\ntubes and abdominal cavity. It causes adhesions and\nscarring, pain and heavy bleeding, and can damage\nreproductive organs, interfere with ovulation and\ninhibit implantation of the embryo. Although the\nspecific causes of endometriosis are still undeter-\nmined, it appears that inheritable defects ( Barlow &\nKennedy 2005 ) allow for the peritoneal implantation\nand survival of endometrial tissue displaced by\nretrograde menstruation, a process initially proposed\nby Sampson (1927) .\nTreatments for endometriosis include the use of\ncontraceptives, progestins and gonadotrophin-releasing\nhormone analogues (Farquhar 2007) to inhibit menstrua-\ntion, a source of much of the pain associated with\nendometriosis, and to suppress the growth of the\nendometriotic tissue. Additional treatments are also\nnecessary to relieve the effects of endometriosis,\nincluding drugs such as clomiphene citrate to improve\nfertility, and non-steroidal anti-inflammatory drugs\n(NSAIDs) and other analgesics to relieve the pain.\nHowever, although these treatments provide relief from\nthe symptoms of endometriosis, none provides a cure,\nand those which alter hormone balance can result in side\neffects such as hot flushes, weight gain and acne.\nThe clinical success of inhibitors of steroid synthesis\nor action in both hormone-dependent breast cancer\n(including tamoxifen, letrozole and 667-COUMATE)\nand hormone-dependent prostate cancer (including\nbicalutamide, goserelin and finasteride) has provided\njustification for this approach in the treatment of these\ncancers. As more is becoming understood about the\naltered expression of steroidogenic enzymes in\nendometriosis, and also in endometrial cancer, it is\nenvisaged that inhibitors of steroid action may also\nhave a role in the clinical treatment and possible\nFigure 1 Steroid structures. (a) 4-Androstene-3,17-dione\n(Adione; with carbon positions numbered), (b) oestrone (E1; with\nring positions labelled), (c) 17b-oestradiol (E2), (d) testosterone,\n(e) 5a-dihydrotestosterone (DHT), (f) 5-androstene-3b,17b-diol\n(Adiol) and (g) dehydroepiandrosterone (DHEA).\nJ M Day et al.: Inhibition of 17 b-HSDs\nwww.endocrinology-journals.org666\nDownloaded from Bioscientifica.com at 06/06/2026 10:17:57PM\nvia free access\n\n\nfuture cure of diseases of the endometrium ( Hompes &\nMijatovic 2007 ).\nIn all of these diseases, the clinical effect of the\ninhibition of the one step of hormone activation\nremains to be investigated ( Purohit et al . 2006 ). This\nis the reduction of the steroids at the 17 b-position,\ncatalysed by specific 17 b-hydroxysteroid dehydro-\ngenases (17 b-HSDs), to form the active steroid that\nbinds to its specific receptor to stimulate cell\nproliferation. In oestrogenic activation, this results in\nthe formation of active oestradiol (E\n2; Fig. 1c) from E1,\nand to a lesser degree, the production of androstenediol\n(Adiol; Fig. 1f) from dehydroepiandrosterone (DHEA;\nFig. 1 g). In androgenic activation, 17 b-HSDs reduce\nAdione to form testosterone, and this is finally\nmetabolised by 5 a-reductase enzymes ( Tindall &\nRittmaster 2008 ) to form DHT, the active androgen.\n17b-HSDs\n17b-HSDs are enzymes that are responsible for\nreduction or oxidation of hormones, fatty acids and\nbile acids in vivo . All require NAD(P)(H) for activity.\nFifteen 17 b-HSDs have been identified to date, and\nwith one exception, 17 b-HSD type 5 (17 b-HSD5), an\naldo–keto reductase (AKR), they are all short-chain\ndehydrogenases/reductases (SDRs).\nThe major substrates for these enzymes are\nhormones, and the reduction or oxidation of hormones\nby 17 b-HSDs regulates the amount of active steroid\navailable to bind to a particular receptor. Although\nnamed as 17 b-HSDs, reflecting the major redox\nactivity at the 17 b-position of the steroid, several of\nthe 17b-HSDs are able to convert multiple substrates at\nmultiple sites, such as at the 3 position on the steroid\nring. Most also have bidirectional capabilities, catalys-\ning either the oxidative or reductive reaction in the\npresence of NAD(P)\nC or NAD(P)H respectively, but\nin vivo appear to function unidirectionally. Although\nthey are generally of a similar size (250–350 amino\nacids) and contain highly conserved motifs, such as\nthose within the Rossman fold, overall homology\nacross the 17 b-HSDs is low ( Duax et al . 2000 , 2005,\nLukacik et al . 2006 ) and the intracellular location of\nthe enzymes is diverse. Different 17 b-HSDs have been\nfound specifically expressed in the cytosol\n(17b-HSD1), microsomes (17 b-HSD3), mitochondria\n(17b-HSD10) and peroxisomes (17 b-HSD4), and\nmany have specific expression patterns across tissues\nand organs. These observations, along with kinetic\nstudies, have demonstrated that although the enzymes\nhave multifunctional capabilities, most have prefer-\nential substrate usage and directionality in vivo .\nThe 17b-HSDs: nomenclature, substrate profile\nand expression patterns\n17b-HSD type 1 (17 b-HSD1/HSD17B1; Fig. 2a) is the\nmost well characterised of the 17 b-HSDs and catalyses\nthe reduction of E 1 to form active E 2 (Miettinen et al .\n1996, Peltoketo et al .1 9 9 6), and also the reduction of\nDHEA to form Adiol ( Lin et al.2 0 0 6). The reverse of\nthese reactions, the inactivation of E2 to E1 and Adiol to\nDHEA, as well as that of testosterone to Adione, is\nmediated by 17b-HSD2 (HSD17B2; Fig. 2). 17b-HSD2\nalso activates 20a-progesterone to form progesterone but\nmay also be involved in the oxidation of retinoids\n(Zhongyi et al.2 0 0 7). 17b-HSD3 (HSD17B3;Fig. 2b) is\nexpressed in the testes and catalyses the reduction of\nAdione to testosterone (Luu-The et al.1 9 9 5). It is not the\nonly 17 b-HSD thought to be responsible for the\nformation of testosterone in vivo ,a s1 7 b-HSD5\n(AKR1C3; Fig. 2 c), which is expressed more ubiqui-\ntously, forms the testosterone produced in other steroidal\ntissues, such as prostate, breast, ovary and endometrium\n(Pelletieret al.1 9 9 9, Ji et al.2 0 0 5). However, 17b-HSD5\nis a multifunctional enzyme, with additional 3 a-a n d\n20a- steroid reductase activities, including the conver-\nsion of DHT to 3a-androstanediol (Penning et al.2 0 0 0),\nand progesterone to 20 a-hydroxyprogesterone (Dufort\net al .1 9 9 9). It is also known as prostaglandin (PG) F\nsynthase (PGFS), its 11-ketoreductase activity preferen-\ntially reducing PGD 2 to 9 a,11b-PGF2 (Matsuura et al .\n1998), although it also forms 9 a,11a-PGF2 (PGF2a)\nfrom PGH2 (Komoto et al.2 0 0 4, Penning et al.2 0 0 6).\nThese 17 b-HSDs, 17 b-HSD1, 17 b-HSD2, 17 b-HSD3\nand 17b-HSD5, will be discussed in greater detail in later\nsections of this review.\nThe 17b-HSD4 enzyme (HSD17B4), also known as\nperoxisomal multifunctional protein-2 (MFP2), is a\n736 amino acid protein of w79 kDa. It comprises an\nN-terminal dehydrogenase domain, and a larger\nC-terminal domain, of around 45 kDa, which contains\nboth hydratase and lipid carrier moieties ( Breitling\net al. 2001, Huyghe et al. 2006). Although it can have\nE2 oxidoreductase activity ( Adamski et al . 1995 ),\nin vivo it is involved in peroxisomal fatty acid\nb-oxidation ( Breitling et al . 2001 ), with defects in its\nexpression causing D-specific MFP deficiency (Huyghe\net al. 2006). It is present in many tissues, with highest\nconcentrations in the liver, heart, prostate and testis,\nand is up-regulated in prostate cancer ( Zha et al. 2005).\n17b-HSD6, initially in humans designated as\n3(a/b)-hydroxysteroid epimerase (also known as\nRoDH-like 3 a-HSD/RL-HSD), is a 36 kDa enzyme\nwith both oxidoreductase and epimerase activities\ninvolved in androgen catabolism. The oxidoreductase\nEndocrine-Related Cancer (2008) 15 665–692\nwww.endocrinology-journals.org 667\nDownloaded from Bioscientifica.com at 06/06/2026 10:17:57PM\nvia free access\n\n\nactivity can oxidise 3 a-Adiol to form DHT ( Bauman\net al. 2006a), while the epimerase activity can convert\nandrosterone (ADT) to epiandrosterone ( Huang &\nLuu-The 2000 , 2001, Belyaeva et al . 2007 ). Despite\nthe major, and possibly the sole, site of action of this\nenzyme being at the 3 position of the androgens, it is\nclassified as 17 b-HSD6 in humans as it is 71.4%\nhomologous to rat hsd17b6 ( Huang & Luu-The 2000 )\nwhich does oxidise steroids at the 17 position ( Biswas\n& Russell 1997 ). Recently, data have indicated that\npolymorphisms in the HSD17B6 gene are associated\nwith errors in androgen metabolism in polycystic ovary\nsyndrome (PCOS; Jones et al . 2006 ).\nWhen prolactin receptor-associated protein (PRAP;\nDuan et al.1 9 9 6) was also identified as 17b-HSD7, it was\nthought to be involved predominantly in the reduction of\nE\n1 to E2 (Nokelainen et al.1 9 9 8, Krazeisen et al.1 9 9 9).\nHowever, sequence and promoter analysis indicate that\nthe major role of this enzyme may be as a 3-ketosteroid\nreductase in cholesterol biosynthesis, reducing zymos-\nterone at the 3 position to form zymosterol (Marijanovic´\net al.2 0 0 3, Ohnesorg et al.2 0 0 6). Despite this, a recent\nstudy has indicate that 17b-HSD7, along with 17b-HSD1,\n17b-HSD5 and other steroidogenic enzymes, is\nsignificantly up-regulated in ovarian tissue of patients\nwith ovarian endometriosis (Sˇmuc et al.2 0 0 7).\n17b-HSD8, also known as FabG (beta-ketoacyl-\n[acyl-carrierprotein] reductase, E. coli)-like (FABGL),\nHZ-K region expressed gene 6 (HKE6) and ring finger\nprotein 2 (RING2), is preferentially an oxidative\nenzyme. Although assays of the mouse enzyme\nin vitro have demonstrated that it can use both E\n2\nand testosterone as substrates ( Fomitcheva et al. 1998),\nsequence analysis again suggests that this enzyme may\nprimarily be involved in the regulation of fatty acid\nmetabolism ( Pletnev & Duax 2005 ). It is highly\nexpressed in murine kidney and spleen, with some\nexpression in the ovary and testes, but is significantly\ndown-regulated in mouse models of polycystic kidney\ndisease ( Fomitcheva et al . 1998 ). In humans, it is\nexpressed in tissues including the liver, pancreas,\nkidney and skeletal muscle ( Ando et al. 1996). A study\nof human genes expressed in the polymorphic human\nleukocyte antigen (HLA) region of chromosome 6 has\nindicated that 17b-HSD8 is also down-regulated in oral\ncavity tumour tissue compared with surrounding\nnormal tissue ( Reinders et al . 2007 ).\nMouse hsd17b9 has 17 b- and 3 a-HSD activities,\nrecognising both steroids and retinols as substrates\n(Napoli 2001). Although it has closest homology to rat\nhsd17b6, it is more homologous to members of the\nretinol dehydrogenase family than to other 17 b-HSDs\n(Su et al . 1999 ). A human 17 b-HSD9 homologue has\nnot been identified.\nHuman 17b-HSD10 was initially identified and named\nby several groups before its 17 b-HSD activity and\nhomology to 17b-HSD4 was recognised, resulting in its\nreclassification as 17b-HSD10 (He et al.1 9 9 9). It is also\n(a)\n(b)\n(c)\nFigure 2 Activity of (a) 17 b-HSD1, (b) 17 b-HSD3 and\n(c) 17b-HSD5 (AKR1C3/PGFS/20a-HSD/3a-HSD2).\nJ M Day et al.: Inhibition of 17 b-HSDs\nwww.endocrinology-journals.org668\nDownloaded from Bioscientifica.com at 06/06/2026 10:17:57PM\nvia free access\n\n\nknown as short-chain L-3-hydroxyacyl coenzyme\nA dehydrogenase II (SCHAD/HCD2/HADH2) and\nendoplasmic reticulum-associated binding protein\n(ERAB), as well as amyloid beta peptide-binding alcohol\ndehydrogenase (ABAD) and 2-methyl-3-hydroxy-\nbutyryl-CoA dehydrogenase (MHBD). 17b-HSD10 is a\nmitochondrial protein, initially isolated from rat liver\n(Luo et al.1 9 9 5) and subsequently from other species and\ntissues such as human brain (He et al.1 9 9 8). It has a wide\nsubstrate profile ( Nordling et al .2 0 0 1, Shafqat et al .\n2003), being involved in isoleucine degradation,\nb-oxidation of fatty acids and oxidation of steroids,\ninactivating E\n2, but converting 5 a-androstanediol to\nDHT (Yang et al.2 0 0 5a,b). It has been implicated in the\ndevelopment of Alzheimer’s disease as it is over-\nexpressed in neurones of patients with the disease, and\nassociates with the neurotoxic peptide amyloid- b (Yan\net al.1 9 9 7). It is expressed in other tissues including the\nprostate (Bauman et al.2 0 0 6a) and has been seen to be\noverexpressed in primary prostate cancer cell cultures\n(He et al.2 0 0 3).\n17b-HSD11 (HSD17B11), also known as Pan1b,\nretSDR2 and DHRS8, was initially isolated by a group\nattempting to find enzymes homologous to the\n11b-HSDs ( Li et al . 1998 ). It was found to have\noxidative 17b-steroid activity, metabolising 5 a-andro-\nstane-3a,17b-diol to the less androgenic ADT; but\ndespite also binding retinoids, it has no retinoid-\nmetabolising activity ( Brereton et al . 2001 ). It is\nexpressed in both steroidogenic and non-steroidogenic\ntissues, including the pancreas, kidney, liver, lung,\nsmall intestine and heart, and the adrenal glands, ovary,\nendometrium and Leydig cells, and its expression can\nbe down-regulated by the steroidogenic combination of\ncAMP with all- trans-retinoic acid ( Chai et al . 2003 ).\nHowever, a physiological role for the enzyme in lipid\nmetabolism is implicated: agonists of peroxisome\nproliferator-activated receptor- a (PPARa) induce a\nr a p i di n c r e a s ei n1 7b-HSD11 expression in the\nendoplasmic reticulum and lipid droplets of mouse\nliver and intestine ( Motojima 2004, Yokoi et al. 2007),\nand 17b-HSD11 has been identified as one of the three\nmajor proteins in lipid droplets of human liver cells\nthat accumulate in fatty liver disease, along with\nadipose differentiation-related protein and acyl-CoA\nsynthetase 3 ( Fujimoto et al . 2004 , 2006).\n3-ketoacyl reductase (KAR), a protein in the\nmembrane of the endoplasmic reticulum that uses\nNADPH to reduce 3-ketoacyl-CoA to 3-hydroxyacyl-\nCoA during the second step of fatty acid elongation\n(Moon & Horton 2003 ), has also been identified as\n17b-HSD12 and has high homology to 17 b-HSD3.\nSome recent studies have suggested that it may also be\nimportant in the reduction of E\n1 to form E 2 (Luu-The\net al .2 0 0 6, Blanchard & Luu-The 2007 ), although\nother studies have indicated that it does not efficiently\ncatalyse this reaction ( Day et al . 2008 ), despite its\nup-regulation in the tumours of breast cancer patients\n(Song et al . 2006 ). However, investigation of its\nexpression in normal human tissues indicated that it is\nhighly expressed in many active lipid-metabolising\ntissues, including liver, kidney, heart and skeletal\nmuscle, as well as in placenta and testis, with\nadditional expression in other steroidogenic tissues\nsuch as adrenal gland, ovary and prostate ( Sakurai\net al . 2006 ). This seems to support a primary role for\n17b-HSD12 as a regulator of lipid biosynthesis.\nPresently, little is known about 17 b-HSD13, 14\nand 15, as they have only recently been identified.\n17b-HSD13 is found at 4q22.1 and is also known as\nshort-chain dehydrogenase/reductase 9. It has 78%\nhomology with 17 b-HSD11, also located at 4q22.1,\nand is highly expressed in the liver, apparently within\nthe cytoplasm ( Liu et al . 2007 ). 17 b-HSD14 was\noriginally named retSDR3 as it was first discovered in\nthe retina, and is also known as DHRS10. It is a\ncytosolic enzyme with high levels of expression in the\nbrain, liver and placenta, and can oxidise E\n2,\ntestosterone and Adiol using NAD C as cofactor\n(Lukacik et al . 2007 ). Transfection of human MCF-7\nand SKBR3 breast cancer cell lines with 17 b-HSD14\nsignificantly decreased E 2 levels, and in an RT-PCR\nstudy of 131 breast tumours, patients with ER-positive\ntumours that highly expressed 17 b-HSD14 showed\nsignificantly better recurrence-free survival and breast\ncancer-specific survival prognoses ( Jansson et al .\n2006) indicating that 17 b-HSD14 may well have a\nrole in E\n2 inactivation in vivo .1 7 b-HSD15, the most\nrecently discovered of the 17 b-HSDs, may play a role\nin androgen biosynthesis ( Luu-The et al . 2008 ).\nInhibition of 17 b-HSD enzymes\nThe selectivity of each of the 17 b-HSD enzymes for\ntheir preferred substrates and directional redox activi-\nties, combined with their tissue-specific localisations,\ncontributes greatly to the fine tuning of the endocrine\nsystem. This selectivity of action also suggests that\nmany of the 17 b-HSDs would provide good targets\nfor modulation of the endocrine response in disease\nstates, especially in those diseases in which they or\nother steroidogenic enzymes are being abnormally\nexpressed.\nEarly work on inhibitors of these enzymes was\nreviewed by Penning & Ricigliano (1991) , again\nby Penning (1996) , and most recently and\nEndocrine-Related Cancer (2008) 15 665–692\nwww.endocrinology-journals.org 669\nDownloaded from Bioscientifica.com at 06/06/2026 10:17:57PM\nvia free access\n\n\ncomprehensively by Poirier (2003) . Several of these\nenzymes have now been validated as targets for the\ntreatment of endocrine-related diseases. Progress in\nthe design and development of inhibitors of specific\n17b-HSD enzymes for use in the treatment of various\ndisorders, including steroid-dependent diseases such as\nbreast and prostate cancer, and endometriosis, has\nadvanced greatly in recent years, and will be discussed\nin the course of the rest of this review.\n17b-HSD1 inhibition\nApplication of 17 b-HSD1 inhibitors in breast\ncancer and endometriosis\nOestrogens have a crucial role in supporting the growth\nof hormone-dependent breast cancer in post-menopau-\nsal women. Although both 17 b-HSD1 and 17 b-HSD2\nare present in healthy pre-menopausal subjects, several\nstudies have indicated that the ratio of 17 b-HSD1 to\n17b-HSD2 is increased in the tumours of post-\nmenopausal patients with hormone-dependent breast\ncancer ( Suzuki et al . 2000 , Miyoshi et al . 2001 ).\nThis results in an increased level of E\n2 that drives\nthe proliferation of the tumour tissue via the ER.\nSeveral studies have indicated that patients with\ntumours that have high 17 b-HSD1 expression have\nsignificantly shortened disease-free and overall survi-\nval (Gunnarsson et al. 2005, Salhab et al. 2006, Vihko\net al .2 0 0 6), suggesting that compounds which inhibit\nthe activity of this enzyme may be of therapeutic\nbenefit in the treatment of hormone-dependent breast\ncancer in post-menopausal patients ( Reed & Purohit\n1999, Purohit et al . 2006 , Sasano et al . 2006 ).\nIn endometriotic tissue, although data are conflict-\ning, there seems to be a change in the expression of\nsteroidogenic enzymes, resulting in the presence of a\nhigh concentration of E\n2 that stimulates proliferation of\nthe tissue ( Bulun et al . 2000 ). Aromatase, responsible\nfor the formation of E 1 from Adione, is negligible in\nnormal endometrium, but is up-regulated in endo-\nmetriotic tissue ( Gurates & Bulun 2003 ). It has been\nsuggested that the expression of 17 b-HSD2, the\nenzyme that inactivates E\n2 to E 1, is down-regulated\nin endometriosis ( Bulun et al . 2006 ), although in\ntwo studies of mRNA expression in the endo-\nmetriotic tissue down-regulation of 17 b-HSD2\nwas not seen ( Matsuzaki et al . 2006 , Carneiro et al .\n2007). A recent study, however, indicated that there\nis a down-regulation of 17 b-HSD2 mRNA expression\nin endometriotic samples, while both aromatase\nand 17 b-HSD1 are up-regulated in comparison\nwith normal endometrium ( Dassen et al .2 0 0 7 ).\nThe down-regulation of 17 b-HSD2 in endometriosis\nis thought to be due to a lack of progesterone receptor\nB (PR-B) expression and a very low level of PR-A\nexpression in the endometriotic tissue, resulting in the\nresistance to progesterone, which in normal endome-\ntrium stimulates the expression of 17 b-HSD2 ( Bulun\net al . 2006 ). In another recent RT-PCR study, the\nauthors found no change in the expression of\n17b-HSD2 mRNA between normal and endometriotic\ntissue (Sˇ muc et al. 2007), but did find an increase in the\nexpression of 17 b-HSD1, 17 b-HSD7, steroid sulpha-\ntase and ER b mRNA. It has also been suggested that\nthere is a link between a 17 b-HSD1 polymorphism,\nSer312Gly, and endometriotic risk and severity\n(Tsuchiya et al .2 0 0 5 ). This polymorphism has\npreviously been associated with higher E\n2 levels in\nsome women ( Setiawan et al . 2004).\nInhibitors of 17 b-HSD1\nAs both hormone-dependent breast cancer and endo-\nmetriosis are oestrogen-dependent diseases, with an\nincrease in the ratio of 17 b-HSD1 to 17 b-HSD2\nexpression implicated in many studies, it has been\nsuggested that this enzyme is a good target for\ninhibition in the treatment of both of these diseases.\nA recent 17 b-HSD1 transgenic mouse study indicated\nthat 17b-HSD1 is also capable of causing a significant\namount of androgen activation in vivo, suggesting that\n17b-HSD1 inhibitors may also have a role to play in\nwomen with diseases related to androgenic dysfunction\n(Saloniemi et al. 2007). There are now many different\ngroups working to find selective inhibitors of\n17b-HSD1.\nUntil recently, there have only been two major\nmethods by which the activity and inhibition of\n17b-HSD1 are assayed. In whole cells and lysates, as\nfor many other steroidal enzymes, 17 b-HSD1 activity\nis usually measured using radiometric assays, often\nwith tritiated substrate at physiological concentrations\n(Singh & Reed 1991 ). Substrate and product require\nextraction, followed by separation by either thin layer\nchromatography (TLC) or reverse phase high per-\nformance liquid chromatography (HPLC). In assays\nusing purified enzyme, however, the redox state of the\ncofactor, NAD(P)(H), can be used to determine the\nprogress of the reaction using spectrophotometric\nmeasurement at 340 nm ( Chin & Warren 1975 ). The\nradiometric TLC and HPLC methods allow for\nsensitive determination of changes in activity, but are\nvery time consuming; whereas the spectrophotometric\nmethod, though less sensitive, is faster, but cannot be\nused for analysis of 17 b-HSD1 activity in tissue\nJ M Day et al.: Inhibition of 17 b-HSDs\nwww.endocrinology-journals.org670\nDownloaded from Bioscientifica.com at 06/06/2026 10:17:57PM\nvia free access\n\n\nsamples as it requires purified enzyme. ELISA-based\nassays are also used for determination of steroid levels\nin blood and other tissues, but again there are problems\nwith the sensitivity requirements for their use.\nRecently, novel methods have been developed to\nimprove the determination of 17 b-HSD1 activity.\nThese include homogeneous proximity ( Kokko et al .\n2006) and fluorescence resonance energy transfer\n(FRET)-based assays ( Kokko et al . 2007 ) for high\nthroughput in vitro screening, and sensitive HPLC-\nbased methods for tissue sample analysis, such as that\nof Delvoux et al . (2007) , which can be used to\ndetermine the activity of several different steroido-\ngenic enzymes in one tissue sample.\nThe elucidation of the crystal structure of 17 b-HSD1\nprovided a good basis for the design of initial\ninhibitors. After optimisation of the crystallisation\nconditions for 17 b-HSD1 ( Zhu et al . 1993 , 1994), a\nhomodimer, structural data revealed a Rossman fold\nand an active site containing a Tyr-X-X-X-Lys\nsequence, both characteristic of short-chain dehydro-\ngenases ( Duax et al . 2000 ). 17 b-HSD1 also contains\nthree a-helices and a helix-turn-helix motif, which\nalong with a histidine residue, influence active site\navailability and thus substrate specificity ( Ghosh et al.\n1995). When crystallised in the presence of E\n2 (Azzi\net al . 1996 ), and also as a ternary complex in the\npresence of both E 2 and the cofactor NADP C (Breton\net al . 1996 ), data indicate that the histidine residue,\nHis221, and another residue, Glu 282, form hydrogen\nbonds with the hydroxyl group at the 3 position of E\n2,\nwhile two other residues in the narrow active site,\nSer142 and Tyr155, are involved in hydrogen bond\nformation with the oxygen atom at the 17 position of\nE\n2. There are further hydrophobic interactions between\nthe steroid and several other residues ( Lin et al. 1996,\nGhosh & Vihko 2001 ).\nMutational analysis of recombinant 17 b-HSD1\nindicated that Lys159 forms the catalytic triad with\nSer142 and Tyr155, and that all of these residues are\nessential for activity ( Puranen et al . 1997 ). In a\nmechanism postulated to be conserved across the\nSDR and AKR superfamilies ( Bennett et al. 1996), it is\nthought that Ser142 and Lys159 lower the pKa of the\nTyr155 hydroxyl proton for donation to the C17 keto\noxygen, while a hydride is transferred to the C17 a\nposition from the nicotinamide ring of the cofactor\n(Ghosh & Vihko 2001 ). Site-directed mutagenesis has\nalso helped to establish the importance of other\nresidues in substrate and cofactor recognition by\n17b-HSD1. Altering Ser12 for a positively charged\nresidue considerably increases the preference of\n17b-HSD1 for NADPH over NADH ( Huang et al .\n2001), whereas the substitution of Leu36 for a\nnegatively charged residue changes its preference\nfrom NADPH to NADH, resulting in a lower\nspecificity for E\n1 (Gangloff et al . 2001 ). Substitution\nof His221 for Ala lowers the affinity of the enzyme for\nE\n1 (Huang et al . 2001 ).\nCrystallisation studies with the enzyme in complex\nwith 20 a-hydroxyprogesterone, DHT and DHEA ( Lin\net al . 1999 , Han & Lin 2000 , Han et al . 2000 ),\nindicated that although all of these steroids can be\naccommodated in the binding site of 17 b-HSD1, they\nbind in the reverse orientation to E\n2 (Gangloff et al .\n2003), and it is Leu149 that is involved in the\ndiscrimination between E 2 and C19 steroids, resulting\nin a far greater affinity for E 2. Val225 has been shown\nto act on the a-face of E 2, in concert with Leu149 on\nthe b-face, to sandwich the A-ring in place and\nsterically hinder binding of C19 steroids. The\ncarboxamide group of NADP(H) forms a hydrogen\nbond with the peptidic amide group of Val188,\nstabilising the binding of the cofactor, but in C19\nsteroid complexes this bonding is disturbed, destabilis-\ning the ternary structure ( Shi & Lin 2004 ).\nModelling studies of 17b-HSD1 place E\n2 in the same\nbinding area within the cleft as the crystallographic\ndata, with His221 and Glu282 forming hydrogen bonds\nto the C3-OH end of E\n2, and Ser142 and Tyr155\nbinding to C17-OH, and also interacting with the\ncofactor. These mechanisms of selectivity over C19\nsteroids, steric hindrance at the A-ring and hydrogen\nbonding of the hydroxyl groups at either end of the\nmolecule, are also seen in the binding of E\n2 to ER a\n(Nahoum et al . 2003 ). Modelling has also confirmed\nthat cofactor and substrate may bind in either order\n(Zhorov & Lin 2000 ), as was seen in early kinetic\nactivity studies ( Betz 1971 ).\nBefore 17b-HSD1 was successfully crystallised, the\ninhibitory potency of other steroids and similar\nnon-steroidal compounds was assessed to study the\nbinding characteristics and specificity of the enzyme\n(Blomquist et al . 1984 ). Substrate and cofactor\nanalogues were developed to explore the involve-\nment of amino acid residues of the active site in the\nbinding mechanism. These included alkylating\nagents such as 3-chloroacetylpyridine-adenine dinu-\ncleotide, an NAD\nC analogue ( Fig. 3 a; Biellmann\net al . 1976 ), 16 a-bromoacetoxyoestradiol 3-methyl\nether, an E 2 analogue ( Fig. 3 b; Chin & Warren 1975 )\nand 6 b-bromoacetoxyprogesterone, a progesterone\nanalogue ( Fig. 3 c; Thomas & Strickler 1983 ).\nC16,C17-substituted pyrazole and isoxazole E 1 deriva-\ntives (Fig. 3d) have also been shown to be competitive\ninhibitors of 17 b-HSD1 ( Sweet et al . 1991).\nEndocrine-Related Cancer (2008) 15 665–692\nwww.endocrinology-journals.org 671\nDownloaded from Bioscientifica.com at 06/06/2026 10:17:57PM\nvia free access\n\n\nLater, equilin ( Fig. 3e), an equine oestrogen that is a\nmajor component of Premarin, used in hormone\nreplacement therapy, was also shown to inhibit\n17b-HSD1 (IC 50!1 mM), and the crystal structure of\nthe 17 b-HSD1 homodimer in a ternary complex with\nNADPC and equilin was solved ( Sawicki et al. 1999).\nEquilin binds at the substrate binding site, with the\nsubstrate entry loop in a closed conformation. In the\nequilin complex, the 17-keto group is a greater distance\nfrom the C4 atom of the cofactor due to the C7 ZC8\ndouble bond of equilin, and this results in inhibition of\nthe catalytic hydride transfer.\nMany flavonoids and other phytoestrogens also\ninhibit 17 b-HSD1 ( Ma¨kela¨ et al . 1998 , Hoffre´n et al .\n2001). Flavonoids with a hydroxyl group in position\n7 of the A-ring, which mimics the D-ring of steroids,\nsuch as apigenin, chrysin, genistein and naringenin,\ninhibit 17 b-HSD1, apigenin being most potent\n(IC\n50!1 mM; Fig. 3f; Le Bail et al. 1998). Derivatives\nof flavonoids, the chalcones, also inhibit 17 b-HSD1\nand aromatase when a hydroxyl substitution is present\non position 4 of the A-ring (e.g. 4-hydroxychalcone,\nIC\n50Z16 mM; Fig. 3g), equivalent to position 7 of the\nflavonoids ( Le Bail et al . 2001 ). Phytoestrogens have\nalso been found to inhibit fungal Cochliobolus lunatus\n17b-HSD (Kristan et al. 2005, Sova et al. 2006), used\nas a model enzyme for the SDR family, as do cinnamic\nacid ( Fig. 3 h) and its derivatives ( Gobec et al . 2004 ,\nKristan et al . 2006 , Sova et al . 2006 ), suggesting that\nthey would be good inhibitors of 17 b-HSD1. Two\nresidues that appear crucial for binding of inhibitors in\nthe fungal enzyme active site, Asn154 and Tyr212, can\nbe matched by their counterparts, Arg258 and Tyr218,\nin 17 b-HSD1. The cofactor NADP also occupies the\nsame position in both enzymes. However, there are no\nresidues in the fungal enzyme which correspond to\nGlu282 and His221 of human 17 b-HSD1, and these\nappear to determine selectivity for 4\n0-hydroxyflavones.\nUnfortunately, although many of these steroids and\nphytoestrogens are potent inhibitors of 17 b-HSD1,\nthey are not useful as therapeutic inhibitors as they are\noften oestrogenic, or are not specific for 17 b-HSD1\ninhibition, having inhibitory effects on other steroidal\nenzymes and receptors ( Deluca et al . 2005 ). These\ninclude other 17 b-HSDs, for example, 17 b-HSD5\n(Brozˇicˇ et al . 2006 ); aromatase, as the 7-hydroxy\ngroup of the flavonoids mentioned above is also\nessential for aromatase inhibition ( Le Bail et al .\n2001); 3 b-HSD ( Arlt et al . 2004 ) and the ER, often\nresulting in stimulatory effects ( Usui 2006 , Turner\net al . 2007 ). However, as we have seen, their use has\nbeen invaluable in the understanding of the mechanism\nof catalysis and inhibition of 17 b-HSD1.\nFrom these studies, several important factors have\nbeen determined for the design of potent inhibitors: a\nplanar hydrophobic ring core structure, such as E\n2,\nlacking a C19 group, to fit into the narrow hydrophobic\nbinding region; b-oriented electron withdrawing\ngroups to form hydrogen bonds with catalytically\nessential amino acids such as Tyr155 and Ser142;\na-oriented hydrophobic groups at C17 or C16 to block\nthe cofactor from binding; and the availability of space\nto accommodate substituents at the 7 a-position of the\nsteroid ( Han et al . 2000 , Owen & Ahmed 2004 ,\nAlho-Richmond et al . 2006 ).\nA series of compounds designed as pure anti-\noestrogens for the treatment of hormone-dependent\nbreast cancer had additional 17 b-HSD1 inhibitory\nproperties in in vivo mouse studies (Labrie et al. 1992).\nThe competitive inhibitors, steroidal derivatives pos-\nsessing both a 7 a-undecanamide group and either a\nhalogen atom at C16 or a double bond at C14–C15 or\nFigure 3 17b-HSD1 inhibition. (a) 3-chloroacetylpyridine-\nadenine dinucleotide, (b) 16a-bromoacetoxy-E2 3-methyl\nether, (c) 6b-bromoacetoxyprogesterone, (d) 16,17-pyrazole-/-\nisoxazole-E1, (e) equilin, (f) apigenin, (g) 4-hydroxychalcone,\n(h) cinnamic acid, (i) EM139, (j) 6b-(thiaheptanamide)-E2, (k)\nEM1745, (l) 16b-m-carbamoyl benzyl-E2, (m) C50-pyridylethy-\nlamide-16,17-pyrazole-E1, (n) STX1040 (2-ethyl-16b-m-pyridyl\nmethyl amido-methyl-E1), (o) non-steroidal STX1040 mimic, (p)\npyrimidinone core and (q) 3-benzyl-2-(2-bromo-3,4,5-tri-\nmethoxy-phenyl)-8-hydroxy-3H-benzo[4,5]thieno[2,3-d]pyri-\nmidin-4-one.\nJ M Day et al.: Inhibition of 17 b-HSDs\nwww.endocrinology-journals.org672\nDownloaded from Bioscientifica.com at 06/06/2026 10:17:57PM\nvia free access\n\n\nC15–C16, inhibited both E 1-stimulated uterine growth,\nan anti-oestrogenic effect, and the conversion of E 1 to\nE2,a n dAt oT ,i nt h em i c e ,w h e nd o s e da t\nconcentrations as low as 3 mg twice daily. 17 b-HSD1\nwas crystallised with one of these dual-site inhibitors,\nEM139 (Fig. 3i), which although larger than E\n2, binds\nat the same position ( Zhu et al . 1999 ). These first\ndual-site inhibitors had only weak 17 b-HSD1 inhibi-\ntory activity but were optimised with the aims of\nimproving 17 b-HSD1 inhibitory potency and speci-\nficity and reducing intrinsic oestrogenicity, while also\nmaintaining the ER antagonist activity ( Tremblay &\nPoirier 1998 ).\nMany steroid derivatives, including those of E\n2,E 1,\nprogesterone and Adione substituted at C16 with\nreactive halogenated functional groups, such as\nbromoacetoxy and bromoacetamido groups, were\nfound to be irreversible inhibitors of 17 b-HSD1 as\nthe halogenated group covalently bonds with an amino\nacid residue, permanently inactivating the enzyme\n(Poirier et al . 1998 ). Several oestratriene derivatives\nwith fluorine substitutions at C17 inhibit 17 b-HSD1\nwith micromolar potencies, but most have activities\nagainst other 17 b-HSD enzymes, including types 2, 5\nand 7 ( Deluca et al . 2006 ). 16 a-iodopropyl and\nbromopropyl substituted E\n2 derivatives are potent\ninhibitors, with IC 50 values of 420 nM ( Sam et al .\n1998) and 460 nM ( Sam et al . 1998 , Tremblay &\nPoirier 1998 ) respectively in assays using partially\npurified 17 b-HSD1 from human placenta.\nReversible E 2 derivatives with b-oriented thia-\nalkanamide side chains at C6 show enhanced\ninhibitory activity, with the most potent, 6 b-\n(thiaheptanamide) E\n2 (Fig. 3 j), having an IC 50 of\n170 nM in assays using 17 b-HSD1 partially purified\nfrom human placenta ( Poirier et al . 1998 ). This\ncompound, however, was found to be oestrogenic,\nand attempts to improve it by removing the 3-hydroxy\ngroup, changing the 6 b-substitution for a 6 a-sub-\nstitution, changing the amide group of the side chain\nfor a methyl or changing the thio-ether for an ether\nbond, all reduced the potency of the compound, despite\nthe ether bond improving the oestrogenic profile\n(Tremblay et al . 2005 ).\nThese structure-activity relationships (SAR) studies\nindicate that 17b-HSD1 potency and specificity may be\noptimised by the rational design of compounds that\ninteract with both the cofactor-binding and substrate-\nbinding regions of the enzyme. A series of E\n2\nderivatives with 16 b-propylaminoacyl substitutions\nwere designed containing hydrophilic and hydrophobic\nmoieties to interact with the cofactor- and substrate-\nbinding regions of the enzyme respectively ( Tremblay\net al .2 0 0 1). Although these compounds are non-\noestrogenic, having no interaction with the ER, they\nalso fail to inhibit 17 b-HSD1. Derivatives of gossypol,\nwhich potently inhibits la ctate dehydrogenase by\ntargeting the Rossmann fold, inhibit 17 b-HSD1 in the\nmicromolar range, also by binding in the Rossmann\nfold from the cofactor binding site across to the\nsubstrate-binding region ( Brown et al . 2003 ). Active\nsite modelling suggested that their potency and\nspecificity may be improved by the incorporation of\na substrate analogue into their structure.\nE\n2-adenosine-based compounds were designed to\nspecifically target both the substrate and cofactor binding\nsites. The most potent of these hybrid inhibitors is\nEM1745 (Fig. 3k), a reversible competitive inhibitor with\nan IC\n50 of 52 nM, in which the steroid is linked to the\nadenosine moiety by a 16 b-oriented side chain\ncontaining eight methylene groups, allowing it to bind\nto Leu96 and Val196 (Qiu et al.2 0 0 2). Two compounds,\nwhich each have only one of the components of\nEM1745, 16 b-nonyl-E\n2 and 5-nonanoyl- O-adenosine,\ndo not inhibit the enzyme (Poirier et al.2 0 0 5). Simplified\nversions of these hybrids, containing adenosine\nmimics to improve the stability and bioavailability\nof these compounds, are less potent than EM1745\n(Be´rube´ & Poirier 2004 ), as are C16-substituted aryl\nE\n1 and E 2 derivatives, such as 16 b-benzyl E 2, which\nhas an IC50 of w800 nM using purified enzyme (Poirier\net al .2 0 0 6). However, further modification of this\nstructure resulted in 16 b-m-carbamoylbenzyl-E2,a n\ninhibitor with an IC 50 of 44 nM ( Fig. 3 l) in which the\nm-carbamoylbenzyl group mimics the nicotinamide\nring of the cofactor. Although this compound is\nweakly oestrogenic (Laplante et al.2 0 0 8), its oestrogenic\nprofile is improved by modifications at the C2, C3\nand C7 positions; however, these substitutions lead to a\ndecrease in potency.\nOther inhibitors have also been designed to bind across\nthe substrate binding site towards that of the cofactor\nby substituting the steroid scaffold at the C16 position,\nboth alone and in combination with C2, C6 and\nC17 substitutions. One class of compounds resulting\nfrom this approach is the E-ring pyrazole amides (e.g.\nC5\n0-pyridylethylamide-16,17-pyrazole-E 1; Fig. 3 m),\ninhibitors that selectively inhibit 17 b-HSD1 with sub-\nmicromolar IC50 values in a whole cell assay ( Fischer\net al .2 0 0 5, Allan et al .2 0 0 6a). Of the others, which\ninclude C16-substituted alkenyl, alkyl and carboxyl,\nC6-oxo and C6 and C16 and C17-oxime E\n1 and E 2\nderivatives, the most ac tive inhibitors are those\ncontaining an m-methylene carboxamide functionality\nextending from the C16b position (Lawrence et al.2 0 0 5,\nAllan et al.2 0 0 6b, Vicker et al.2 0 0 6).\nEndocrine-Related Cancer (2008) 15 665–692\nwww.endocrinology-journals.org 673\nDownloaded from Bioscientifica.com at 06/06/2026 10:17:57PM\nvia free access\n\n\nThe most potent of the m-methylene carboxamide\ncompounds, with an IC 50 of 27 nM in a whole cell\nassay, is STX1040 (2-ethyl-16 b-m-pyridyl methyl\namido-methyloestrone; Fig. 3 n; Lawrence et al .\n2005). Fig. 4 shows STX1040 docked in place of E 2\nin the 17 b-HSD1 crystal structure, protein database\n(PDB) entry ‘IFDT’, in complex with NADP C using\nthe docking programme GOLD (Jones et al. 1997). The\nhigh potency of STX1040 may be explained by its\ninteractions with the cofactor. In 1FDT, the nicotina-\nmide carbonyl and amide nitrogen of the cofactor form\nhydrogen bonds with Val188 and Thr140 respectively.\nIn the inhibitor complex, it appears that there may be\nan interaction between the nicotinamide amide moiety\nand the amide carbonyl of the 16 b side chain. The\npyridyl nitrogen of the 16 b side chain may interact\nwith an oxygen atom 3.16 A ˚ away in the phosphate\ngroup of the cofactor. A C2 ethyl group, included to\neliminate oestrogenicity as it interferes with hydro-\nphobic interactions with the ER ( Vicker et al . 2006 ),\nalso contributes to inhibitory activity by interacting\nwith Leu262 and Phe259. Hydrophobic interactions\nwith Leu149, Val225, Phe226 and Phe259, observed\nwhen substrate is docked into the enzyme, are\nmaintained in the inhibitor complex.\nNon-steroidal E\n1 mimics are also active as\n17b-HSD1 inhibitors ( Allan et al . 2008 ). In these\ninhibitors, the E 1 moiety is replaced by aryl ring-\ncontaining scaffolds that retain both a hydroxyl group\nequivalent to that at the 3 position of E\n1 and a ketone\nfunctionality at the 17-position equivalent. The most\npotent of these, with IC\n50 values of 3.7 and 1.7 mMi na\nwhole cell assay, are the biphenyl ethanones and\nbiphenyl indanones respectively. Substitution of the\nbiphenyl ethanone scaffold to form mimics of\nSTX1040 results in improved activity, with the most\npotent compound having an IC\n50 of 1.8 mM( Fig. 3 o).\nThe potency of STX1040 was assayed using T47D\ncells to measure 17 b-HSD1 inhibition, while the high\nselectivity of STX1040 for the 17b-HSD1 enzyme over\n17b-HSD2 was determined using MDA-MB-231 cells,\nas these breast cancer cell lines have high 17 b-HSD1\nand 17 b-HSD2 activities respectively ( Day et al .\n2006a, Purohit et al . 2006 ). The T47D cell line was\nalso used to develop an in vitro proof of concept assay\nfor the inhibition of E 1-stimulated proliferation of\nhormone-dependent breast cancer cells by 17 b-HSD1\ninhibitors such as STX1040, as it is an ER-positive\nbreast cancer cell line whose proliferation is dependent\non oestrogens in the medium. For this reason, it is also\nan appropriate cell line to use to establish whether such\ninhibitors are oestrogenic in vitro (Day et al . 2006 b,\n2008, Laplante et al . 2008 ). Using this model, and\ndosing E\n1 from 1 nM to 1 mM, STX1040 at 5 mM\nis seen to be non-oestrogenic while significantly\ninhibiting the E\n1-dependent proliferation of T47D\ncells ( Day et al . 2006 b, 2008).\nThe low inter-species homology of the 17 b-HSD1\nenzyme has made the development of animal models\nfor the assessment of the inhibitors difficult. Mouse\n17b-HSD1 is only 63% homologous to human\n17b-HSD1 at the amino acid level, and this is reflected\nin its different substrate affinity, as, in addition to E\n1 to\nE2 activity, it also converts Adione to testosterone\n(Nokelainen et al . 1996 ). The rat enzyme has 93%\nhomology to that of the mouse, but only 68% to human\n17b-HSD1 ( Ghersevich et al . 1994 ). STX1040 has an\nIC\n50 of w100 mM against the rat enzyme when it is\nexpressed in 293-EBNA (Invitrogen, Paisley, UK)\ncells (unpublished results), indicating that inhibition by\nSTX1040 is specific to the human enzyme. In female\nrodents, the major expression of 17 b-HSD1 is in the\novary ( Peltoketo et al . 1999 ), with low levels in the\nuterus and some expression in the sebaceous glands of\nthe skin ( Nokelainen et al. 1996, Pelletier et al. 2004).\nTo limit endogenous steroid enzyme expression and\noestrogen synthesis ovariectomised mice are often\nused in animal models of steroidogenic enzyme\nFigure 4 STX1040 docked in place of E2 in the 17b-HSD1 crystal structure (reproduced with permission from: Day et al. 2008).\nJ M Day et al.: Inhibition of 17 b-HSDs\nwww.endocrinology-journals.org674\nDownloaded from Bioscientifica.com at 06/06/2026 10:17:57PM\nvia free access\n\n\ninhibition, resulting in similar oestrogen levels to those\nof post-menopausal women ( Yue et al . 1994 ). Human\ntumour xenograft growth can then be stimulated by\nexogenous doses of steroid, dependent on expression of\nthe required enzyme by the human tumour cells for\nconversion to the more active steroid.\nSTX1040 was shown to be efficacious in vivo using a\nnovel ovariectomised nude mouse model based on the\nprinciples above ( Day et al . 2006 b, 2008). As T47D\ncells had been successful inin vitro model development,\nthis cell line was also used to develop the in vivo model\nin which growth of the T47D tumours was stimulated by\nlow doses of E\n1, dependent on the human 17 b-HSD1\nexpressed by the tumours for conversion to E2, the more\nactive oestrogen. Tumours stimulated with E 1, either\ndaily by s.c. injection (0.05–0.1 mg/day) or by use of a\ntime-release pellet (0.1–0.28 mg/day), as unconjugated\noestrogens have a short half-life in vivo (Ruder et al .\n1972), were significantly larger than those of the control\nanimals. Treatment of the mice with STX1040 caused\na significant decrease in their tumour volumes and\nplasma E\n2 levels in comparison with those dosed with\nE1 alone. STX1040 was confirmed to be non-oestro-\ngenic in vivo using a standard rat uterotrophic model.\nDespite the number of groups working towards the\ndevelopment of 17 b-HSD1 inhibitors for clinical use,\nonly one other group has reported activity in vivo\nusing inhibitors optimised from those with core\nstructures which mimic those of the natural inhibitors,\ncoumestrol and kaempfe ride. From various non-\nsteroidal flavone-, tetrahydrochromanoquinoline-,\nchromenone- and pyrimidinone-based structures, the\npyrimidinone core was selected for further optimisation\n(Fig. 3 p; Messinger et al . 2006 ) as selectivity and\nsolubility problems were observed with the other\nclasses of compounds. Several compounds were\nselected for synthesis and evaluation from those\nmodelled using published crystal structures. The most\npotent of these ( Fig. 3q) has an IC\n50 of 5 nM in assays\nusing purified recombinant 17 b-HSD1, although in\nwhole cell assays, its efficacy is much lower than that of\nSTX1040 (inhibition at 1 mM!67%). It is selective for\n17b-HSD1 over 17 b-HSD2, is non-oestrogenic and is\nefficacious in vivo using an intact nude mouse model\nbearing MCF-7 cells transfected with 17 b-HSD1\n(Husen et al. 2006a,b).\nAlthough there are differences between the two\nin vivo models, in both studies treatment of the mice\nwith E\n1 caused mouse uterine weight to increase\nsignificantly. Use of the inhibitors, however, although\nresulting in a decrease in E\n1-stimulated tumour size,\ndid not significantly decrease the E 1-stimulated uterine\nweight. Despite this, E 2 levels in the T47D tumour\nstudy ( Day et al . 2006 b, 2008) were shown to be\nsignificantly decreased after treatment with STX1040,\nsuggesting that tumour 17 b-HSD1 expression is a\nmajor E\n2 source in these models. The lack of an effect\nof the 17 b-HSD1 inhibitors on uterine weight in both\nmodels may therefore be due to either the higher\nsensitivity of the uterus than the tumour to circulating\noestrogens or maximal stimulation of uterine growth\nover the initial few weeks.\nThe demonstration of E\n1-dependent tumour growth\ninhibition in vivo by 17 b-HSD1 inhibitors provides\njustification for the many years of research into\ninhibition of the enzyme, and suggests that it may\nindeed be a valid target for the treatment of hormone-\ndependent breast cancer. Although models for the use\nof these inhibitors in other hormone-dependent\ndiseases, such as endometriosis, are yet to be\ndeveloped, the observed decrease in the plasma level\nof active hormone after STX1040 treatment of animals\ncarrying the human enzyme indicates that these\ninhibitors may well be effective for the treatment of\nthese other diseases of hormone metabolism. The\nsuccessful in vivo application of inhibitors of\n17b-HSD1, the most studied of the 17 b-HSDs, also\nsuggests that inhibition of other 17 b-HSDs known to\nbe involved in disease states may well prove clinically\nbeneficial in the future.\n17b-HSD3 inhibition\nApplication of 17 b-HSD3 inhibitors in prostate\ncancer\nDHT is the main intracellular androgen in the\nprostate and stimulates the growth of hormone-\ndependent prostate tumours via its interaction\nwith the AR. It is formed from testosterone by\n5a-reductases 1 and 2. 17 b-HSD3, microsomally\nexpressed almost exclusively in the testes ( Geissler\net al. 1994, Luu-The et al. 1995), specifically converts\nnon-androgenic Adione ( Laplante & Poirier 2008 )t o\nactive circulating testosterone in the presence of\nNADPH. It has not been reported to have any other\nactivities. However, it is not the only enzyme that\nprovides testosterone to the body, as 17 b-HSD5\n(AKR1C3), whose activities and inhibition will be\ndiscussed later in this review, converts Adione to\ntestosterone in the prostate and other tissues.\nA defect in the expression of 17 b-HSD3 causes the\nautosomal recessive genetic disorder, male pseudoher-\nmaphroditism ( Geissler et al . 1994 ), in which the\nindividual is usually reared as a female, often having\nbeen born with female external genitalia and the\nEndocrine-Related Cancer (2008) 15 665–692\nwww.endocrinology-journals.org 675\nDownloaded from Bioscientifica.com at 06/06/2026 10:17:57PM\nvia free access\n\n\nabsence of a prostate, des pite having testes and\nWolffian duct-derived male internal genitalia\n(Andersson & Moghrabi 1997 ). Diagnosis is assisted\nby the presence of a high Adione to testosterone ratio,\ndistinguishing the disorder from the clinically similar\nandrogen insensitivity syndrome. The phenotype can\nvary in severity, even in individuals with the same\nmutation ( Lee et al . 2007 ), but subjects often become\nvirilised at puberty. The mechanism by which this\noccurs is not fully understood, although it has been\nsuggested that either this is due to incomplete\nimpairment of testes testosterone production, or due\nto peripheral conversion of Adione to testosterone\n(Andersson et al . 1996 ), perhaps by the action of\n17b-HSD5.\nAlthough 17b-HSD3 is expressed almost exclusively\nin the testes, there have been some reports of its\nexpression in other tissues. One report indicated that\nexpression of 17b-HSD3 mRNA increased over 30-fold\nin cancerous prostate biopsies (Koh et al.2 0 0 2). In this\nstudy, the authors also found a corresponding decrease\nin 17 b-HSD2 mRNA expression, indicating that the\nreductive formation of testosterone is favoured, but\nfound no change in the expression of AKR1C3 mRNA.\nExpression of 17 b-HSD3 was up-regulated in an\nAR-positive prostate cell line, LNCaP, after it was\ntreated for 48 h with dutasteride (Biancolellaet al.2 0 0 7),\nan inhibitor of 5a-reductases 1 and 2. A polymorphism in\nthe HSD17B3 gene, G289S, has also been linked to an\nincreased susceptibility to prostate cancer ( Margiotti\net al .2 0 0 2). Microarray and subsequent RT-PCR and\nfunctional analysis indicated that 17b-HSD3, along with\n17b-HSD12, is also expressed in human blood platelets\nand megakaryocytes. While 17b-HSD12 is up-regulated\nO25-fold in essential thrombocythemia, a rare myelo-\nproliferative disorder, 17 b-HSD3 is down-regulated\nw4.5-fold (Gnatenko et al.2 0 0 5).\nInhibitors of 17 b-HSD3\nBecause of its unique expression and substrate\nspecificity, 17 b-HSD3 provides a good target for the\ninhibition of testosterone formation in the treatment of\nandrogen-dependent diseases such as hormone-depen-\ndent prostate cancer. Although its crystal structure has\nnot yet been solved, 17 b-HSD3 has undergone various\nmutational analyses as pseudohermaphroditism results\nfrom the effects of deleterious mutations on 17 b-HSD3\nactivity (Lee et al. 2007). This has given an insight into\nthe catalytic importance of various residues, such as\nARG80, which are involved in the binding and\nselectivity of the enzyme for the cofactor, NADPH\n(McKeever et al . 2002 ).\nThe first demonstrated inhibition of 17 b-HSD3\nactivity was in canine testicular microsomes by\ntwo steroids, 4-estrene-3,17-dione ( Fig. 5 a) and\n5-androstene-3,17-dione (Fig. 5b), which are structurally\nvery similar to the substrate, Adione ( Pittaway 1983).\nThese studies indicated that a non-aromatic A-ring and\nC17 carbonyl group were important for inhibition. Using\nhuman testicular tissue, the similarly structured atames-\ntane (1-methyl-3,17-dione-androsta-1,4-diene;Fig. 5c),\na potent aromatase inhibitor, was also shown to inhibit\n17b-HSD3 (Lombardo et al.1 9 9 3).\nAs expression of 17 b-HSD3 is specific to the testes,\nattempts have been made to find a more readily\navailable source of the enzyme for the screening of\nFigure 5 17b-HSD3 inhibitors. (a) 4-estrene-3,17-dione,\n(b) 5-androstene-3,17-dione, (c) atamestane, (d) 1,4-androsta-\ndiene-3,6,17-trione, (e) androsterone (ADT), (f) 3b-phenyl-\nmethyl-ADT, (g) 3b-amidomethyl-ADT derivatives, (h) 3-\ncarbamate-ADT derivatives, (i) 3R-spiro-{3\n0-[300-N-morpholino-\n200-(3000-cyclopentylpropionyloxy) propyl]-20-oxo-oxazolidin-50-\nyl}-5a-androstan-17-one, (j) 3-O-benzyl-androsterone, (k) BMS-\n856, (l) 8/9-substituted tetrahydrodibenzazocine (THB), (m)\ndiphenyl-p-benzoquinone, (n) umbelliferone, (o) 1-(4-hydroxy-\nphenyl)-nonan-1-one, (p) tributyltin chloride (TBT) and triphe-\nnyltin chloride (TPT).\nJ M Day et al.: Inhibition of 17 b-HSDs\nwww.endocrinology-journals.org676\nDownloaded from Bioscientifica.com at 06/06/2026 10:17:57PM\nvia free access\n\n\npotential inhibitors of 17b-HSD3. Microsomes isolated\nfrom rat testicular tissue were tested, but the enzyme\nwas found to differ from the human enzyme in\noptimum reaction pH, in sensitivity to inhibition by\ncandidate compounds, and in substrate specificity,\nefficiently reducing E\n1 as well as Adione (Le Lain et al.\n2001). Most groups working on the inhibition of\n17b-HSD3 now use enzyme from cells transfected with\n17b-HSD3 cDNA, in purified form, as microsomes, or\nin whole cell assays. Recently, the pig 17 b-HSD3\nenzyme has been sequenced, cloned and expressed. As\nit has a similar substrate profile to the human enzyme\nand a higher amino acid homology to the human\nenzyme, at 82%, than that of rats and mice,\nmicrosomes from pig testicular homogenates may\nalso have the potential to be used as a ready source\nof 17 b-HSD3 for inhibitor studies ( Ohno et al .2 0 0 6).\nUsing transfected cell microsomes, 1,4-androsta-\ndiene-3,6,17-trione ( Fig. 5 d) potently and selectively\ninhibited 17 b-HSD3 activity ( Luu-The et al . 1995 ).\nAnother steroid, the weak androgen ADT ( Fig. 5 e),\nwas found to be twice as potent as the substrate Adione,\nwith an IC\n50 of 330 nM in transfected cell microsomes.\nSeveral libraries of ADT derivatives were synthesised,\nand their inhibitory potencies and androgenicity\ncompared ( Tche´dam Ngatcha et al .2 0 0 0 , 2005,\nMaltais et al.2 0 0 1, 2002). ADT derivatives substituted\nat the 16 position, although proving non-androgenic,\ndemonstrated only weak inhibition of 17 b-HSD3\nactivity (Tche´dam Ngatcha et al. 2002). 3b-Substituted\nalkyl and aryl derivatives of ADT were potent\ninhibitors, with IC\n50 values of 57–147 nM, and were\nfound to be specific for 17b-HSD3 over 17b-HSD1 and\n17b-HSD5. 3 a-Ether-3b-substituted ADT derivatives\nhad a lower inhibitory activity than the 3 b-substituted\nADT analogues, with the exception of 3 b-phenylethyl-\n3a-methyl-O-ADT and 3 b-phenylethyl-ADT, which\nhad IC 50 values of 73 and 99 nM respectively. The\nmost potent of these 3 b derivatives was 3 b-phenyl-\nmethyl-ADT (IC50Z57 nM; Fig. 5f; Tche´dam Ngatcha\net al. 2000, 2005), but unfortunately it was found to be\nas androgenic as DHT. However, a somewhat less\npotent (IC\n50Z227 nM) ADT derivative synthesised\nusing parallel solid-phase techniques, 3 b-peptido-3a-\nhydroxy-5a-androstan-17-one, was found to be tenfold\nless androgenic ( Maltais et al . 2001 ).\nFurther understanding of the SAR for the ADT\nderivatives was achieved using 3 b-amidomethyl-\nADT ( Fig. 5 g) and 3-carbamate-ADT ( Fig. 5 h)\nlibraries generated by liquid-phase parallel syntheses.\nLong alkyl chains at R\n1 and R 2 were found to be\nbadly tolerated, with preference for a shorter R 2\nchain than that at R 1, and tertiary amides were more\nactive than secondary amines. Adding polarity to the\nchains did not generally improve their inhibitory\npotential, but adding rigidity at R\n1 did. The most\npotent of nearly 300 amidomethyl compounds, with\nan IC\n50 of only 35 nM, was 3 b-[(N-adamantylmethyl-\nN-butanoyl)aminomethyl]-3 a-hydroxy-5a-androstan-\n17-one, but this compound was found to be almost as\nandrogenic as 3 b-phenylmethyl-ADT. The most\npotent of the 25 3-carbamate-ADT compounds was\n3R-spiro-{3\n0-[3 00-N-morpholino-2 00-(3 000-cyclopentyl\npropionyloxy)-propyl]-2 0-oxo-oxazolidin-5 0-yl}-5 a-\nandrostan-17-one ( Fig. 5 i), with an IC 50 of 74 nM,\nand this compound was not significantly androgenic,\neven at 1 mM( Maltais et al . 2002 ).\nBisubstrate compounds from the same group,\nincorporating components that bind in both the\nsubstrate and cofactor binding sites, similar to the\nhybrid inhibitors developed for the inhibition of\n17b-HSD1, were also tested as inhibitors of\n17b-HSD3. Activity in homogenated cells overexpres-\nsing 17 b-HSD3 was 78% inhibited by 1 mM Adione\nsubstituted at the 17 a-position with a 12 methylene\nspacer esterified to adenosine, although this compound\nwas less potent in whole cells ( Be´rube´ et al . 2006 ).\nAttempts to improve the potency of this class of\ncompound by phosphorylation of the adenosine group,\nto mimic the preferred cofactor NADPH over NADH,\ndid not prove successful, suggesting that either this\nmoiety is not interacting with the cofactor binding site,\npossibly due to prior binding of the cofactor itself, or\nthat its phosphate group interaction is not optimised\n(Be´rube´ & Poirier 2007 ).\nA 3-substituted ADT derivative, 3- O-benzylandros-\nterone ( Fig. 5 j), was used by Bristol Myers Squibb\n(BMS) to initiate a large-scale screening programme\n(Spires et al. 2005). Inhibition of 17b-HSD3 activity in\ntransfected cell microsomes was assayed using a\n96-well format scintillation proximity assay (SPA)\nwith a testosterone-specific antibody for higher\nthroughput of compounds than the usual radiolabelled\nTLC-based assay ( Luu-The et al . 1995 ). 3- O-benzy-\nlandrosterone and 18 b-glycyrrhetinic acid both had\ninhibitory IC\n50so f w90 nM in the SPA assay.\nInhibition in whole cells was measured using an\nAR stimulation-dependent chemiluminescent secreted\nalkaline phosphatase (SEAP) reporter assay in\nAR-positive cell lines transfected stably with\n17b-HSD3 and transiently with a prostate serum\nantigen (PSA)-SEAP reporter plasmid. The inhibitory\nIC\n50 values for 3- O-benzylandrosterone and 18 b-gly-\ncyrrhetinic acid were, as expected, much higher in this\nwhole cell assay, at 1.6 and 3.8 mM respectively\n(Spires et al . 2005 ).\nEndocrine-Related Cancer (2008) 15 665–692\nwww.endocrinology-journals.org 677\nDownloaded from Bioscientifica.com at 06/06/2026 10:17:57PM\nvia free access\n\n\nOf over 200 000 compounds screened by BMS, a\nseries of anthranilamide-based compounds substituted\nat position 1 with an amide-linked chlorophenol\ngroup were identified as inhibitors of 17 b-HSD3\nactivity, and these were most potent when substituted\nat the 4 position, further carboxamide substitutions at\nthe 2 position (e.g. BMS-856; Fig. 5 k; IC\n50 values of\n60 and 300 nM respectively in the enzyme and whole\ncell assays). Dibenzothiazocines (DBTs) and tetrahy-\ndrodibenzothiazocines (THBs) were also discovered to\nbe highly active in these assays ( Fink et al . 2006 ).\nAlthough DBTs were most potent, especially when\nsubstituted with chlorine at position 2 or 3, the THBs\nwere chosen for further SAR study as the aryl sulphur\natom of the DBT compounds renders them susceptible\nto metabolic degradation. Unlike the DBTs, sub-\nstitution of the THBs at the A-ring did not improve\ntheir potency, although substitution at positions 8 and 9\nof the C-ring did (Fig. 5l), with an aryl ring substitution\nat position 8 providing significant improvement.\nOrtho-substitution of the 8-aryl-substituted ring with\nelectron donating groups, including methyl ester,\nmethyl ketone and trifluoromethyl and aldehyde\ngroups, resulted in the highest potencies in both the\nenzyme and cellular assays (IC\n50 values of around 20\nand 500 pM respectively). However, no data on the\nselectivity of these inhibitors for 17 b-HSD3 over other\n17b-HSDs has been published.\nScreening of a range of compounds by another group\n(Le Lain et al . 2001) revealed that other non-steroidal\ncompounds, such as diphenyl-p-benzoquinone, phenyl-\np-benzoquinone ( Fig. 5 m), 7-hydroxyflavone, baica-\nlein and biochanin A, with IC\n50 values of 2.7, 5.7, 9.0,\n9.3 and 10.8 mM respectively, are also able to inhibit\n17b-HSD3. The 7-hydroxycoumarins, umbelliferone\n(Fig. 5 n) and 4-methylumbelliferone are more potent\ninhibitors of human testes microsomal 17 b-HSD3,\nwith IC 50 values of 1.4 and 1.9 mM respectively ( Le\nLain et al. 2002). However, many of these compounds,\nsuch as the flavones, as already discussed in regard to\n17b-HSD1 inhibition, are non-selective for 17 b-HSD3\ninhibitory activity, having effects on other 17 b-HSDs,\nother steroidal enzymes and on steroid receptors.\n4-Hydroxyphenyl ketones with attached straight\nchain alkyl groups inhibit 17 b-HSD3 activity in rat\ntesticular microsomes with IC\n50 values in the low\nmicromolar range. The best of those tested was\n1-(4-hydroxyphenyl)-nonan-1-one (IC\n50Z2.86 mM;\nFig. 5 o; Lota et al . 2006 ), containing an octyl chain,\nthought to mimic the steroid backbone of Adione while\nthe 4-hydroxy group forms hydrogen bonds at the\nactive site. In this study, baicalein was shown to be far\nless potent (IC\n50 value of 186 mM) than in the human\nmicrosome study discussed above (IC 50Z9.3 mM; Le\nLain et al . 2001), suggesting that the alkyl-substituted\n4-hydroxyphenyl ketones may prove more potent if\ntested on the human enzyme.\nOrganotin compounds are known to be endocrine-\ndisrupting marine pollutants. Two of these compounds,\ntributyltin chloride and triphenyltin chloride (TBT and\nTPT respectively; Fig. 5 p), were found to be potent\ninhibitors of 17b-HSD3 in microsomes from pig testes,\nwith IC\n50 values of 7.2 and 2.6 mM respectively. The\ncompounds did not affect expression of the enzyme,\nsuggesting that these effects are due to direct inhibition\nof 17b-HSD3 activity. Surprisingly, however, in whole\ncultured Leydig cells, the potency of both compounds\nincreased w60-fold to an IC\n50 of 114 nM for TBT and\n48 nM for TPT. The unusual increased activity in the\nwhole cell assay was suggested to be either due to the\nfat solubility of organotins resulting in their high\nconcentration throughout the membrane structure of\nLeydig cells, in which 17 b-HSD3 is located, or due to\neffects on other enzymes involved in the production of\ntestosterone, such as those involved in transcription\nand signal transduction ( Ohno et al . 2005 ).\nDespite the many groups now working on inhibitors\nof 17 b-HSD3, the crystal structure of the enzyme has\nnot been published and there are no formal reports of\nthe efficacy of these inhibitors in in vivo models.\nGroups such as Schering–Plough (WO2004060488)\nand Sterix Ltd (WO2007003934) have published\npatents pertaining to the development of 17 b-HSD3\ninhibitors, with Schering–Plough indicating efficacy of\nnon-steroidal inhibitors in mouse Shionogi tumour\n(Minesita & Yamaguchi 1965 ) and monkey models in\nconference abstracts (American Association of Cancer\nResearch annual meeting 2005); however, data from\nthese studies have not yet been published in peer-\nreviewed journals.\nThe development of successful cost-effective\nin vivo models to ascertain the efficacy of 17 b-HSD3\ninhibitors may prove complex due to the low\nhomology between the human and mouse enzymes.\nAlthough use of the Shionogi model, a transplantable\nandrogen-dependent AR-positive mouse breast tumour\n(Minesita & Yamaguchi 1965 ) may give an indication\nof efficacy, the effect would depend on inhibition of\ntestosterone formation by mouse 17 b-HSD3, and thus\nwould not truly reflect the use of inhibitors against\nt h eh u m a ne n z y m e .D e s p i t et h em o r ea c c u r a t e\nrepresentation of the human situation by primate\nmodels, the cost and regulations involved in their use\nare prohibitive for most groups and for screening of\nmore than a few compounds. To overcome the problem\nof the lack of homology between human and rodent\nJ M Day et al.: Inhibition of 17 b-HSDs\nwww.endocrinology-journals.org678\nDownloaded from Bioscientifica.com at 06/06/2026 10:17:57PM\nvia free access\n\n\n17b-HSD3, the development of models using castrated\nmice with human 17 b-HSD3-expressing androgen-\ndependent tumour xenografts may prove useful.\n17b-HSD5 inhibition\nApplication of 17 b-HSD5 inhibitors in prostate\ncancer\n17b-HSD5 (AKR1C3/PGFS) also catalyses the\nreduction of Adione to form testosterone, but unlike\n17b-HSD3 it is expressed far more ubiquitously in\ntissues including the pros tate, breast, ovary and\nendometrium ( Pelletier et al . 1999 , Penning et al .\n2000, Ji et al . 2005 ). It has additional 3 a-a n d\n20a-steroid reductase activities ( Dufort et al . 1999 ,\nPenning et al . 2000 ), including the protective inacti-\nvation of deoxycorticosterone in mineralocorticoid\ntissues ( Sharma et al . 2006 ). However, its other\nmajor activity is as PGFS: it has PGD\n2 11-ketor-\neductase activity that reduces PGD 2 to 9 a,11b-PGF2\n(Matsuura et al . 1998 ) and also PGH 2 9,11-endoper-\noxide reductase activity that reduces unstable PGH 2 to\nPGF2a (Komoto et al . 2004 ). AKR1C3 is the only\n17b-HSD that is not a short-chain dehydrogenase. It is\nan AKR and is highly homologous to three other AKR\nenzymes, AKR1C1, AKR1C2 and AKR1C4. Although\nmultifunctional, AKR1C2 and AKR1C4 primarily\nhave 3 a-HSD activities, while the major activity of\nAKR1C1 is as a 20 a-HSD ( Penning et al . 2004).\n17b-HSD5/AKR1C3 has been seen to be up-regu-\nlated in prostate cancer. Elevated expression of\n17b-HSD5 protein has been demonstrated in the\nepithelium of malignant prostate tissue as well as in\nnon-neoplastic processes such as benign prostatic\nhyperplasia and inflammation ( Bauman et al . 2006 b,\nFung et al . 2006 ). Its expression is increased in\nadvanced prostate cancer ( Nakamura et al . 2005 ) and\nhas been correlated with Gleason grade ( Wako et al .\n2008), a measure of tumour aggressiveness. In a\nmicroarray analysis of bone marrow metastases of\nhormone-independent prostate cancer, the expression\nof 17 b-HSD5 was more than fivefold higher than in\nprimary androgen-dependent tumours, and this was\nconfirmed by both RT-PCR and immunoblotting\n(Stanbrough et al .2 0 0 6 ). Remarkably however,\nalthough other androgen synthesis genes were also\nup-regulated, and expression of the AR was 5.8-fold\nthat in primary tumours, there was no correlation\nbetween the increased expression of 17 b-HSD5 and\nAR in the samples, suggesting the involvement of\ndifferent mechanisms of androgen independence.\nAlthough there are no reports of increased\nexpression of 17 b-HSD5 in the ovaries of hyperan-\ndrogenised individuals with PCOS, in one study a\nsingle nucleotide polymorphism in the AKR1C3\npromoter that increases its affinity to nuclear transcrip-\ntion factors, SNP-71G was found in around 10% of\naffected individuals (Qin et al. 2006). Conversely, in a\nseparate study, there was no association between\nPCOS or testosterone levels and the occurrence of\neither SNP-71G or four other AKR1C3 polymorphisms\n(Goodarzi et al . 2008 ). There was also no association\nbetween four common AKR1C3 polymorphisms and\nprecocious puberty, a hyperandrogenic condition\nthought to be a risk factor for PCOS ( Petry et al .\n2007). 17b-HSD5 is also expressed in normal (Pelletier\net al . 1999 , 2001) and malignant breast tissue ( Amin\net al. 2006). Its expression is significantly up-regulated\nin breast cancer and is associated with a poor prognosis\n(Vihko et al . 2005 ).\nHowever, as previously mentioned, 17 b-HSD5 is\nalso known as PGFS. It is thought that it may also exert\na proliferative signal in cancer by reducing PGD\n2 to\n9a,11b-PGF2.9 a,11b-PGF2 is a proliferative PG that\nstimulates the MAP kinase pathway, preventing the\nformation of the PPARg ligand, PGJ\n2, a PG involved in\ndifferentiation signalling ( Desmond et al . 2003 ).\nInhibitors of 17 b-HSD5\nThe earliest patents for the inhibition of AKR1C1–\nAKR1C4 (US5439943, US5399790, US5187187,\nUS5118621 and US5068250) were published by the\nUniversity of Pennsylvania from 1992–1995. Novel\nnon-steroidal suicide inhibitors were described for\npotential therapeutic use in potentiating the action of\nandrogens, in androgen replacement therapy for\nhypogonadism of pituitary and testicular origin, and\nin the maintenance of male fertility. Suicide substrates\nmimic the physiological substrate and are transformed\nby the enzyme to highly reactive alkylating agents\nwhich then inactivate it by forming a covalent bond at\nthe active site. They can be highly selective as they are\nthemselves inactive until they are transformed by the\ntarget enzyme. These patents described the activity of\nmonocyclic-aromatic allylic and acetylenic alcohols as\nhighly selective inhibitors that are transformed by the\n3a-HSD activity to monocyclic-aromatic vinyl and\nacetylenic ketones that alkylate the pyridine nucleotide\nbinding site of the enzyme (e.g. 1-(4\n0-nitrophenyl)-2-\npropen-1-ol is transformed to 1-(4 0-nitrophenyl)-2-\npropen-1-one by the action of the 3 a-HSDs; Fig. 6 a).\nSince then, as 17 b-HSD5 has been shown to have\nactivities other than as a 3 a-HSD, interest in its\nEndocrine-Related Cancer (2008) 15 665–692\nwww.endocrinology-journals.org 679\nDownloaded from Bioscientifica.com at 06/06/2026 10:17:57PM\nvia free access\n\n\ninhibition for anti-cancer therapy has been growing.\nSeveral environmental dietary phytoestrogens, such as\nzearalenone, coumestrol, quercetin and biochanin A\n(IC50Z2–14 mM; Fig. 6 b–e), have been shown to\ninhibit 17b-HSD5 (Krazeisen et al. 2001). The potency\nof these compounds increases with increasing number\nof hydroxylated sites, apparently due to binding to the\nhydrophilic cofactor-binding pocket of the enzyme.\nDerivatives of trans-cinnamic acids, a/b-unsaturated\nplant carboxylic acids that are natural precursors of\nstructurally related 17 b-HSD5-inhibiting flavonoids,\nhave also been evaluated as 17 b-HSD5 inhibitors\n(Brozˇicˇ et al .2 0 0 6 ). The best inhibitor in the\nseries tested was a-methylcinnamic acid ( Fig. 6 f;\nIC\n50Z6.4 mM). Parallel tests indicated that these\ncompounds have no inhibitory effects on fungal\n17b-HSD, suggesting that they may be selective for\n17b-HSD5 over the 17 b-HSDs from the short-chain\ndehydrogenase family. A C17-difluorinated oestra-\ntriene derivative, J2404 ( Fig. 6g), inhibited 17 b-HSD5\nby 72% at 2 mM, with no effect on the other SDR\n17b-HSD enzymes tested, including types 1, 2, 4 and 7\n(Deluca et al . 2006 ).\n17b-HSD5 is also found in the brain, where it is\nbelieved to be involved with AKR1C1 and AKR1C2 in\nthe metabolism of neurosteroids, which act on the\ng-aminobutyric acid type A (GABA\nA) receptors.\nBenzodiazepines are sed atives that modulate the\nactivity of the GABA A receptors. However, several\nof them have been found to inhibit AKR1C1–AKR1C3\n(17b-HSD5), and one, cloxazolam ( Fig. 6h) is a potent\nand specific inhibitor of 17 b-HSD5 with an IC\n50 of\n2.5 mM( Usami et al . 2002 ).\nStudies of the catalytic mechanism of the enzyme by\nPenning et al .( 2 0 0 1 ), based on studies of a 69%\nhomologous rat 3 a-HSD (AKR1C9), indicated that an\nordered bi-mechanism operates in which the substrate\nbinds to the enzyme once the pyridine nucleotide\ncofactor, NADPH, is bound. After the reduction of the\nsubstrate through an oxyanion transition state, the\nproduct is released before the release of the cofactor.\nThis mechanism suggests that steroidal-based inhibitors\nthat compete with the steroid product are desirable as\nthey would act as uncompetitive inhibitors. This led to\nthe suggestion that transition state analogues, such\nas steroid carboxylates and pyrazoles, and mechanism-\nbased inactivators, such as 3 a-, 17 b-o r2 0 a-spiro-\noxiranyl steroids ( Fig. 6i–k) and oxiranyl non-steroids,\nmay be potent 17 b-HSD5 inhibitors. Endorecherche,\nInc. (Quebec, Canada) has published several patents\npertaining to the inhibition of 17b-HSD5 by steroidal and\nspirolactone compounds for the treatment of androgen-\ndependent diseases including prostate cancer (e.g.\nUS2004082556, AU2004200173, MXPA00008868,\nZA9901924, EP1321146).\nOne of the most potent of the Endorecherche, Inc.\ncompounds is an E\n2 derivative, EM1404 ( Fig. 6 l), a\nstrong competitive inhibitor with an IC50 of 3.2G1.5 nM\nusing 100 nM Adione as substrate, a higher potency than\nthat of any other reported inhibitor. The lactone ring of\nEM1404 is located at the base of the substrate binding\nsite, and the amide group is oriented towards the surface\nof the enzyme, despite the fact that the inhibitor occupies\nonly part of the binding cavity (Qiu et al.2 0 0 7). Although\nthis compound is specific for 17 b-HSD5 over\n17b-HSD1, 17b-HSD2 and 17 b-HSD3 (IC\n50O10 mM;\npatent WO9946279), its specificity over other members\nof the AKR1C family and cyclooxygenase 1 and 2\n(COX1/COX2) remains to be assessed.\n17b-HSD5 is also inhibited by NSAIDs, including\nindomethacin ( Fig. 6m) and flufenamic acid ( Fig. 6n),\nFigure 6 17b-HSD5 inhibitors and substrate analogues.\n(a) 1-(4 0-nitrophenyl)-2-propen-1-ol to 1-(4 0-nitrophenyl)-2-\npropen-1-one that alkylates the enzyme, (b) zearalenone,\n(c) coumestrol, (d) quercetin, (e) biochanin A,\n(f) a-methylcinnamic acid, (g) J2404, (h) cloxazolam,\n(i) 3 a-spiro-oxirane, (j) 17 b-spiro-oxirane, (k) 20 a-spiro-\noxirane, (l) EM1404, (m) indomethacin, (n) flufenamic acid,\n(o) N-(4-chlorobenzoyl)-melatonin, (p) rutin, (q) bimatoprost,\n(r) Ex144 and (s) 9,10-phenanthrenequinone.\nJ M Day et al.: Inhibition of 17 b-HSDs\nwww.endocrinology-journals.org680\nDownloaded from Bioscientifica.com at 06/06/2026 10:17:57PM\nvia free access\n\n\nat similar concentrations and the same order of potency\nas for the inhibition of COX1/COX2 ( Bauman et al .\n2005, Byrns et al . 2008 ). In HL-60 promyelocytic\nleukaemia cells, the inhibition of 17 b-HSD5 by\nindomethacin is thought to lead to a decrease in\nproliferation and a corresponding increase in differen-\ntiation via the PPAR g signalling pathway due to the\npresence of higher concentrations of PGJ\n2 and lower\nconcentrations of PGF 2a (Desmond et al .2 0 0 3).\nUsing the extensive SAR knowledge generated\nduring the development of COX1 and COX2\ninhibitors, derivatives of NSAIDs with increased\n17b-HSD5 potency and selectivity for 17 b-HSD5\ninhibition over COX1 and COX2 inhibition are being\ndeveloped, with structures based on N-phenylanthra-\nnilic acids, cholanic acids, and on N-acylanthranilic\nacids, 2-benzoylbenzoic acids, benzophenones, and\nphenoxybenzoic acid ( Bauman et al . 2005 , Gobec\net al . 2005 , Penning et al . 2006 ). One of the most\nspecific is N-(4-chlorobenzoyl)-melatonin ( Fig. 6 o), a\nderivative of indomethacin that inhibits the reduction\nof 5 mM Adione to testosterone by 17 b-HSD5 with\nsimilar potency (IC\n50Z11.4 mM) to indomethacin\n(IC50Z8.5 mM), but which does not inhibit COX1,\nCOX2, AKR1C1 or AKR1C2 ( Byrns et al . 2008 ).\nThe rational design of these inhibitors has been\ngreatly assisted by the elucidation of the crystal\nstructure of 17 b-HSD5, complexed as a ternary\nstructure with the cofactor NADP(H), and with either\nthe substrates PGD\n2 (Komoto et al . 2004 ) or Adione\n(Qiu et al . 2004 ), the product testosterone ( Qiu et al .\n2004) or potential inhibitors, such as the NSAIDs,\nindomethacin and flufenamic acid ( Lovering et al .\n2004), rutin ( Fig. 6 p; Komoto et al . 2004 ), a PGF 2a\nanalogue, bimatoprost ( Fig. 6 q; Komoto et al . 2006 )\nand EM1404 ( Qiu et al . 2007 ).\nA patent for the use of N-sulphonylindole deriva-\ntives as selective 17 b-HSD5 inhibitors was recently\npublished by Astellas Pharma Inc. (Tokyo, Japan,\nWO2007100066). Derivatives in which a carbon atom\nin the indole group is substituted by a carboxy group, a\ncarboxy-substituted lower alkyl group or a carboxy-\nsubstituted lower alkenyl group were found to potently\ninhibit 17 b-HSD5 with selectivity over 17 b-HSD3\ninhibition (IC\n50O10 mM). The most potent of these\ninhibitors have IC50 values below 100 nM (e.g. Ex144;\nFig. 6 r).\nIn all of these studies, the potency of the inhibitors has\nbeen assayed using cell lines transfected with 17b-HSD5.\nThe expressed enzyme has then either been assayed\nin situ in whole cell assays using radiolabelled steroid\nsubstrate or purified for direct enzyme assay, measuring\nthe decrease in absorbance at 340 nm as NADPH is\noxidised to NADP\nC, using either steroid substrate or\n9,10-phenanthrenequinone (Fig. 6 s) as a test substrate.\nThe purified enzyme has also been used for crystal-\nlisation studies, as previously mentioned, to determine\nthe structure of the active site and the residues that are\nimportant to its activity and inhibition. Although there\nare several groups presently working on these inhibitors,\nthere have been no reports of the development of in vivo\nmodels of 17b-HSD5 inhibition. This may well be due to\nthe ubiquitous expression of 17 b-HSD5 throughout the\ntissues, its substrate plasticity and the initial lack of\nspecificity of these inhibitors. However, in order to\nunderstand the contribution of the various catalytic\nactivities of 17b-HSD5 to the development of hormone-\ndependent prostate cancer and its progression to hormone\nindependence, and to other cancers, analysis of the effect\nof inhibition of 17 b-HSD5 in vivo is necessary. Until\nthese models have been developed and the effects of these\ninhibitors on both androgen and PG production have been\ntested in vivo , the value of the development of these\ninhibitors and their future application in the treatment of\nprostate or other cancers cannot be fully established.\nInhibition of other 17 b-HSDs\nApplications and inhibitor development\n17b-HSD2 is the enzyme responsible for the oxidation\nof active E2 and testosterone to their inactive forms, E 1\nand Adione. As it is involved in inactivation of active\nsteroids, there has been less interest in the development\nof 17 b-HSD2 inhibitors than in those of 17 b-HSD1,\n17b-HSD3 and 17 b-HSD5. However, partly for use as\na research tool and partly as inhibitors of 17 b-HSD2\nmay prove beneficial for conditions in which the\nconcentration of active steroid is too low, some effort\nhas been made to find inhibitors of this enzyme. In\n2002, Bayer Pharmaceuticals published patent\nWO0226706 for the use of pyrrolidinones, pyrroli-\ndin-thiones and 1-methyl-4-phenylpyrrolidin-2-ones\n(Fig. 7 a i) in the treatment of osteoporosis. Another\ngroup has explored the use of 17-spiro-lactones\nattached to either an E\n2 nucleus ( Sam et al . 2000 ,\nBydal et al . 2004 ) or other steroid nuclei ( Tremblay\net al. 1999, Poirier et al. 2001)a s1 7b-HSD2 inhibitors.\nThese compounds are selective for 17 b-HSD2 inhi-\nbition over 17 b-HSD1 and 17 b-HSD3, with IC 50\nvalues in the nM range. However, the most active of\nthese compounds, the spiro- d-lactone C17 b-O/C17a-\nd-lactone ( Fig. 7 a ii), which has an IC 50 of 6 nM for\n17b-HSD2 inhibition, also inhibits 17 b-HSD5 with an\nIC50 of w10 nM and has some oestrogenicity ( Bydal\net al . 2004 ).\nEndocrine-Related Cancer (2008) 15 665–692\nwww.endocrinology-journals.org 681\nDownloaded from Bioscientifica.com at 06/06/2026 10:17:57PM\nvia free access\n\n\nPresently, there is little research into the development\nof inhibitors of the remaining, less well characterised,\n17b-HSDs. However, as their number continues to\nincrease, and the substrate specificity, directional\nactivity and expression pattern of each of the enzymes\nare elucidated, the potential of specific inhibitors of\neach of these enzymes for the treatment of various\ndiseases, of both steroidal and non-steroidal origin, will\nbecome clear. Indeed, the inhibition of 17 b-HSD10\nis already being explored for the treatment of\nAlzheimer’s disease, and a potent inhibitor, AG18051\n(1-azepan-1-yl-2-phenyl-2-(4-thioxo-1,4-dihydropyra-\nzolo[3,4-d]pyrimidin-5-yl)-ethanone; Fig. 7b), with an\nIC\n50 of 92 nM, has been identified ( Kissinger et al .\n2004). As this enzyme also converts 5 a-androstanediol\nto DHT ( Yang et al. 2005a,b) and has been seen to be\noverexpressed in primary prostate cancer cultures\n(He et al . 2003 ), it would also be interesting to see\nwhether inhibitors of 17 b-HSD10 may also have a role\nin the treatment of prostate cancer.\nSummary\nDespite the success of inhibitors of steroidogenic\nenzymes in the clinic, such as those of aromatase and\nsteroid sulphatase, the development of inhibitors of\n17b-HSDs is at a relatively early stage. At present, none\nof these inhibitors has reached clinical trials, and only\nrecently has efficacy been demonstrated for the first of the\nenzymes in in vivo disease models. However, great\nadvances in both the understanding of the function of\nthese enzymes, and in techniques for elucidation of\nprotein structure and computer-aided SAR have been\nmade over the last decade. As the precise substrate and\ncofactor specificity, directional activity and expression\npattern of each of the 15 17 b-HSDs in normal and\ndiseased states becomes clearer, it can be seen that the\ndevelopment of specific inhibitors could provide a great\nopportunity to fine-tune pathways in the therapeutic\nintervention of steroidogenic and other metabolic\ndisorders. Presently, the development of inhibitors of\n17b-HSD1 for the treatment of hormone-dependent\nbreast cancer and endometriosis, and 17b-HSD3 for the\ntreatment of hormone-dependent prostate cancer, are\nmost advanced.\nDeclaration of interest\nProf M J Reed and Dr A Purohit are consultants to Ipsen Ltd.\nProf M J Reed is a director of Sterix Ltd.\nFunding\nThis work was supported by Sterix Ltd, a member of the\nIpsen Group.\nAcknowledgements\nThe authors are grateful to Prof. B V L Potter and Dr N\nVicker for critical reading of the manuscript.\nReferences\nAdamski J, Normand T, Leenders F, Monte ´ D, Begue A,\nSte´helin D, Jungblut PW & de Launoit Y 1995 Molecular\ncloning of a novel widely expressed human 80 kDa 17 b-\nhydroxysteroid dehydrogenase IV. Biochemical Journal\n311 437–443.\nAkaza H 2004 Adjuvant goserelin improves clinical disease-\nfree survival and reduces disease-related mortality in\npatients with locally advanced or localized prostate\ncancer. 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