Send Orders of Reprints at [email protected] 1164 Current Topics in Medicinal Chemistry, 2013, 13, 1164-1171 A Challenge for Medicinal Chemistry by the 17-hydroxysteroid Dehydro- genase Superfamily: An Integrated Biological Function and Inhibition Study

S.-X. Lina,b, D. Poiriera and J. Adamskic,d.e aLaboratory of Molecular Endocrinology and Oncology, Centre Hospitalier Universitaire (CHU) de Quebec Research Center (CHUL) and Laval University, Québec City, Québec, G1V4G2, Canada; bWHO Collaborating Center for Re- search in Human Reproductive Health, Shanghai, 200031, China; cHelmholtz Zentrum München, Institute of Experi- mental Genetics, Genome Analysis Center, 85764 Neuherberg, Germany; dLehrstuhl für Experimentelle Genetik, Tech- nische Universität München, 85350 Freising-Weihenstephan, Germany; eGerman Center for Diabetes Research (DZD), 85764 Neuherberg, Germany

Abstract: Members of the 17-hydroxysteroid dehydrogenase (17-HSD) superfamily perform distinct multiple catalyses by the same enzyme, apparently contradictory to the long-held beliefs regarding the high specificity of enzymes. Surpris- ingly, these multi-catalyses can combine synergistically in vitro and in vivo and their dysfunction may result in the stimu- lation of breast or prostate cancer. 17-HSD1 possesses high activation activity, while its inactivation is significant for decreasing the week concentration of dihydrotestosterone (DHT) in breast cancer cells, an important fac- tor for cell proliferation. 17-HSD5 can also carry out multiple catalyses in hormone-dependent cancer cells. In addition to 17-HSDs 1 and 5 some other family members possess such dual-activity as well, and their inhibition decreases hor- mone-dependent cancer proliferation. The multi-specificity of 17-HSD1 is structurally based on the pseudo-symmetric that can accommodate the narrow enzyme substrate tunnel by both normal and alternative binding. The atypical family member 17-HSD5 possesses a spacious binding site, which is accessible to several substrates. Expression of 17- HSD1 can also control other estrogen-responsive elements such as pS2, and can regulate steroid-hormone receptors. The fundamental involvement of 17-HSD1 in catalysis and regulation underlies its close relationship to breast cancer, attributable to its long evolutionary process. These observations stimulated detailed study of steroid-converting enzyme inhibition. The most significant efforts in designing 17-HSD1 inhibitors in decades have progressed through structure ac- tivity relationship studies supported by the availability of both small and protein molecule structures, with the elimination of residual estrogenic activity in the inhibitors. The first non-estrogenic inhibitors of 17-HSD1 to show activity in vivo (breast cancer animal model) are now reported. Keywords: 17-hydroxysteroid dehydrogenase (17-HSD) superfamily, estrogen-dependent breast cancer, pre-receptor modu- lation of steroid hormone action, rational design of inhibitors, structure-activity relationship.

INTRODUCTION [15], can generate many useful data for which it would be.time-consuming and costly to obtain by experiments During the last two decades or so, considerable pro- alone Actually, these data, combined with the information gresses have been made in developing various cheminfor- derived from the structural bioinformatics tools [see, e.g., matics methods or tools for new drug target exploration, reference 16] and structural cheminformatics tools [see, e.g., such as those for identifying predicting the network of sub- references 17-19] can timely provide very useful insights for strate-enzyme-product triads [1], identify recombination both medicinal chemistry research and drug development. spots [2], identify nuclear receptors and their types [3], iden- The use of structure-activity relationship can be considered tifying HIV cleavage sites in proteins [4-6], predicting including both protein and small molecule structures [20, GPCRs and their types [7], predicting proteases and their 21]. The current review is to summarize the progresses in types [8], identifying colorectal cancer related [9], studying the 17-hydroxysteroid dehydrogenase (17-HSD) classifying hepatocellular cirrhosis and carcinoma [10], pre- superfamily characterized by an integrated biological func- dicting secretory proteins of malaria parasite [11], predicting tion and inhibition study to further stimulate the develop- protein subcellular locations [12], QSAR [13, 14], and a se- ment of cheminformatics in this area. ries of powerful web-server predictors listed in Table 3 of

ENZYMOLOGY IN 17-HSD FAMILY *Address correspondence to this author at the Laboratory of Molecular Enzyme-mediated catalysis characterized by kinetic Endocrinology and Oncology, Centre Hospitalier Universitaire (CHU) de Quebec Research Center (CHUL) 2705, boulevard Laurier Québec City, properties, substrate specificity, and varying mechanisms of Québec, G1V4G2, Canada; Tel/Fax: 1-4186542296/6542761; reaction have been studied for more than a century providing E-mail: [email protected] the most precise information regarding biological processes

1568-0266/13 $58.00+.00 © 2013 Bentham Science Publishers A Challenge for Medicinal Chemistry by the 17-hydroxysteroid Dehydrogenase Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 10 1165 to date. This is pivotal for the understanding of cellular ho- are unknown. Steroid conversion capability apparently meostasis, response to environmental challenge, and mecha- evolved as a multi-step process and has its roots in retinoid, nisms of disease. Recent studies of the biological function of fatty acyl, or acetate converting enzymes [31-33]. members of the 17-HSD superfamily revealed that their Our knowledge of enzyme catalysis often comes from activities and specificities can vary extensively by several assays with purified homogeneous enzymes (e.g. 17-HSD1 orders of magnitude toward different steroids, and their and 5)[24-35]. Enzymes may display specificities at varying multi-specificities can be significant. Not only can some levels toward different substrates with accompanying con- critical enzymes affect cancer cell proliferation, but their formational rearrangements in the binding site, thus expand- expression regulates the transcription and expression of other ing their functional involvement. In vitro study and animal genes, thereby demonstrating pivotal biological roles. This models of several enzymes contributed extensively to under- review will provide a succinct presentation of several func- standing the systemic or physiological effects of the enzymes tional aspects of 17-HSDs to illustrate the scope of the ca- (e.g. yeast complementation assay of 17-HSD7, siRNA- pacity of these enzymes and their inhibition. mediated silencing of 17-HSD1, transgenic 17-HSD1, or At least 14 different mammalian 17-HSDs have been knock-out 17-HSD types 2 and 7) [36-40]. identified [22-25], of which 12 members have been found in On the basis of substrate specificity, 17-HSDs can be humans [22, 26]. Their kinetic parameters (substrate speci- assigned to two groups. The first comprises steroid- ficity, reaction direction, cofactor dependence), tissue distri- converting enzymes and includes 17-HSDs types 1–3. The bution, and sub-cellular localization differ significantly. The second constitutes steroid-converting enzymes, and those 17-HSDs stereo-specifically interconvert ketones and their interacting with acyl-CoAs (17-HSDs 4, 8, 10, and 12), bile corresponding secondary alcohols using NAD(P)H or acids (17-HSDs 4 and 10), and retinoids (17-HSDs 6 and NAD(P)+ as cofactor, with reduction or oxidation as their 9) [41]. Most of the 17-HSDs are members of the function- respective principal reaction under physiological conditions. ally promiscuous SDR (short chain dehydrogenase reduc- The 17-HSD enzymes are molecular switches in pre- tase) group [42], except for 17-HSD5, which is an AKR receptor modulation of steroid hormone action for both ge- (aldo-keto reductase) enzyme [43]. nomic (nuclear mediated) and non-genomic pathways like those of GPCR (G protein-coupled receptor) [26-29]. Oxida- The reaction mechanisms of 17-HSDs are different from tion of the C17 hydroxyl of dihydrotestosterone, testoster- each other, which is consistent with their modest sequence one, or 17-, reduces the potency of these steroids. identity and subdivision into different protein families: SDR Similarly, the reduction of the C17-keto group of 5alpha- and AKR. Nevertheless, they share the common feature of a androstane-3,17-dione, 4-androstene-3,17-dione, or reversible hydride transfer from NADPH to a ketosteroid, or yields the biologically active steroid forms (Fig. 1). Further- hydride transfer from a hydroxysteroid to NAD+ [44, 45]. more, reductive 17-HSDs catalyze the final step in the bio- The 17-HSDs of the SDR-family share conserved amino synthesis of the most active (estradiol and 5- acids that constitute a catalytic triad essential for steroid androstene-3,17-diol) and androgens ( and conversion: Ser142, Tyr155, and Lys159 (numbering for dihydrotestosterone) [30]. 17-HSD1) [46, 47]. Further interactions with a water mole- cule and Asn114 are critical for enzyme activity [48, 49]. 17-HSDs are involved in the etiology of several human diseases. Either loss-of-function (e.g. loss of 17-HSD4 in lethal human D-specific bifunctional protein deficiency), or over-expression (e.g. 17-HSD1 in breast cancer) may result in human disorders [25, 50, 51]. Upregulation in disease makes the corresponding enzymes pharmaceutical targets for therapeutic inhibitor development. Furthermore, enzyme- specific inhibitors are valuable tools for the functional as- sessment of 17-HSDs. The present generation of inhibitors are not only type-specific, but are devoid of cognate receptor binding and include both steroidal and non-steroidal com- pounds [25, 52-56]. Recent inhibitor developments for 17- HSDs illustrated that the best performing compound in en- zymatic assays was also highly ranked by molecular docking scoring [57].

BIOLOGICAL FUNCTION OF 17-HSDs An extensively studied example of 17-HSDs is the type Fig. (1). Sex-hormone activation and inactivation by 17-HSDs (for 1 enzyme, which was among the earliest steroid-converting detailed roles of different 17-HSDs members refer to the Fig. 2 in enzymes to evolve [58]. The homogeneous enzyme demon- reference 56. strates a significant specific activity with a high estrogen turnover [59, 60], while displaying considerable lower Despite the ever-growing wealth of genomic data and specificities for other steroids. The latter “non-cognate” elucidation of 17-HSD activities in different species, the specificities are still clearly significant when compared to the enzyme origins, as well as their functions in early organisms same conversion by other enzymes in the family. This is an 1166 Current Topics in Medicinal Chemistry, 2013, Vo l. 13, No. 10 Lin et al. excellent evolutionary design by nature to meet varying biology, and are likely to be generalized phenomena, at least physiological requirements. A detailed structure-function for steroid-converting enzymes. For instance, 17-HSD7 has study revealed that the enzyme’s multi-specificity toward C- similar estrogen activating and androgen inactivating roles in 19 steroids, including androgens, is due to their pseudo- breast cancer cells, while possessing a stronger DHT inacti- symmetrical small molecular structures, resulting in the co- vation capacity in comparison to 17-HSD1. Interestingly, existence of normal and alternative bindings even in the nar- 17-HSD7 exhibits 3-ketoreductase activity in the choles- row binding tunnel of the enzyme [Fig. 2, reference 61]. terol biosynthesis pathway [70]. Both 17-HSD types 5 and However, the specificity is reduced by several orders of 7 exhibit remarkable dual activities at positions C3 and C17. magnitude for C-19 steroids compared to C-18 steroids, due The importance of basic enzymology and biological func- to steric hindrance of the C-19 with a sandwich structure in tion, as well as their interrelationship should be explored in the enzyme [62]. more depth, with a view to contributing to the development of contemporary biology. Such a combinatorial study will, in Such a configuration permits the following enzyme ca- turn, further advance our understanding of enzymology, the talyses that can also be demonstrated in breast cancer cells: the activation of estrone into the potent estrogen estradiol quantitative biological chemistry of enzymes. and inactivation of the most active androgen, DHT [61-63]. The first demonstration of this was quite surprising, as it had 17-HSD INHIBITORS not been appreciated that such different steroids could be The development of inhibitors is an important aspect of used as substrates for the same enzyme. 17-HSD1 had been enzyme-related research, and is also true for the 17-HSD known as an estrogenic enzyme for many decades. The two family. The search for inhibitors of 17-HSDs began in the previously mentioned reactions are now demonstrated to be 1970s and gradually gained momentum thereafter before highly synergistic in stimulating breast cancer cell prolifera- culminating in the first decade of the 2000s. The greatest tion [Fig. 3, and reference 63], emphasizing an important number of inhibitors is known for the 17-HSD1 isoform, control of the cancer cell by nM concentration of DHT. The but inhibitors of isoforms 2, 3, 5, 7, 10, and 12 have also multiple levels and sophisticated involvement of this enzyme been reported in the literature. Several review articles re- in biological function and disease states (see below) are ported structure-activity relationship studies, which are cru- likely the results of a long evolutionary process. cial for drug design, and illustrate the huge diversity of 17- With recent developments in the study of the biological HSD inhibitors [25, 54-56, 70-74]. Despite the number of function of 17-HSDs, we found that 17-HSD1 can en- years of research, in particular for isoform 1, no inhibitors of hance the estradiol (E2)-induced expression of the endoge- 17-HSDs are currently in clinical use. This is somewhat nous estrogen-responsive gene pS2 and other genes, demon- surprising since inhibitors of aromatase, 17alpha-lyase, strating its role in the regulation of estrogen-related genes 5alpha-reductase, and other enzymes involved in the synthe- [63]. Moreover, 17-HSD1 expression increases the mRNA sis of estrogens and androgens, are used for the treatment of levels of estrogen receptors (ER) alpha and beta by 171 and breast cancer, prostate cancer and benign prostate hyperpla- 120%, respectively, while decreasing that of the androgen sia [75]. The 17-HSD1 isoform has an established role in receptor by 64% [64]. These results imply an additional role breast cancer, but the presence of undesirable estrogenic for the enzyme as well as its catalytic function, justifying it activity generally associated with first-generation inhibitors as a target for breast cancer therapy. has been a major obstacle to their therapeutic use. This very likely results from the fact that 17-HSD1 has estrogens as ADDITIONAL 17-HSD FAMILY MEMBERS substrates and products, and has a high affinity for estrogens The more significant multi-specificity of 17-HSD5 [60, 76]. The design of inhibitors that are analogues of estro- arises from its spacious binding site [65] that is able to ac- gens makes it difficult to eliminate residual estrogenic activ- commodate several different steroids [66]. The binding site ity. Interesting new developments in vitro describing non- was estimated to be about 960 and 470 Å3 in ternary com- steroidal inhibitors against 17-HSD type 1 have recently plexes with testosterone and 4-dione, respectively, whereas been published [25, 77]. In the following paragraph, we re- the binding site volume of 17-HSD1 is only about 340 Å3 port promising results obtained with four molecules tested on [67]. This more than two-fold difference in size between the tumor xenografts: a non-steroidal derivative and three steroi- two complex binding sites of 17-HSD5 demonstrates the dal derivatives (Fig. 4). The assay measures the ability of an flexibility of the enzyme structure. The enzyme is involved inhibitor of 17-HSD1 to produce a partial or complete in the conversion of 5alpha-androstane-3,17-dione into tes- block of estrone-stimulated human cancer cell growth in tosterone in prostate cancer, in the conversion of progester- nude mice estrone is the precursor of the potent estrogen one, and in the reduction of the anti-proliferative prostaglan- estradiol, and the natural substrate of 17-HSD1. din, thus marking it as a critical target in hormone-dependent The inhibitor I is a non-steroidal derivative with a and hormone-independent cancer therapy [25, 68, 69]. In the pyrimidinone core, which was tested by Solvay Pharmaceu- ternary complex of the 17-HSD5 with testosterone, the ticals [78]. In their animal model, human MCF-7 cells ex- steroid C3-C17 position is quasi-reversed in comparison to pressing 17-HSD1 were inoculated in nude ovariectomized the complex with 4-dione. The enzyme’s multi-specificity (OVX) mice, and tumors generated in presence of estrone contributes significantly to steroid metabolism in peripheral (0.1 μmol/kg/d) were treated for 28 days by subcutaneous tissues, due to the high levels of 17-HSD5 mRNA in both (sc) injection with compound I at a dose of 5 μmol/kg/d (2.8 breast and prostate tissues. mg/kg/d). Since the estrogen-dependent MCF-7 breast can- The multi-catalytic and biological functions of the 17- cer cells express different 17-HSD isoforms [79], the HSD enzymes contribute to the understanding of the systems authors stably transfected the cells with a plasmid expressing A Challenge for Medicinal Chemistry by the 17-hydroxysteroid Dehydrogenase Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 10 1167

Fig. (2). Crystal complex structure of 17-HSD1/DHT. A and B, Electronic density of DHT for 2Fo-Fc map seen at 0.8 sigma cutoff in reverse binding mode (A) and normal binding mode (B). C, Stereo representation showing the H-bond of DHT with the residues His221 in the reverse binding mode (DHT represented in blue), whereas in the normal binding mode, there is no H-bond interaction present (DHT in green). D, Distances between DHT, Tyr155, and the cofactor NADP in 1) reverse mode (the distance between the O3 of DHT with NC4 of NADP as 4.35 Å and between Tyr155 to NC4 of NADP as 5.4 Å) and in 2) normal mode (the distance between the O17 of DHT with NC4 of NADP as 3.75 Å and between Tyr155 to NC4 of NADP as 5.4 Å). Refer to PDB code 3KLM. Adapted from [63] [Fig. 7 in reference 63].

Fig. (3). Estrogen responsiveness modulation by 17-HSD1. A, T47D cells were transfected with 17-HSD1 siRNA or control siRNA for 48 h, and then cells were incubated with 0.01 and 0.1 nM E2 for 8 d (left); MCF7 cells and stably transfected MCF7 cells were cultivated in the presence of 1 nM E2 for 1 and 7 d (right). Experiments were carried out in charcoal-treated medium, and cell growth was evaluated by MTT analysis. CTL represents the control cells incubated without any steroid. B, T47D cells were transfected with 17-HSD1-D1-specific siRNA or with control siRNA for 48 h, and then cells were incubated with 1 nM E2 or ethanol vehicle (EtOH) for an additional 48 h. Total RNA was extracted from cells and used for the evaluation of pS2 mRNA expression by quantitative real-time RT-PCR analysis. Error bars represent SD. *P <0.05 by Student’s t test. Adapted from 64 [Fig. 6 in reference 63]. 1168 Current Topics in Medicinal Chemistry, 2013, Vo l. 13, No. 10 Lin et al.

Fig. (4). Representative inhibitors of 17-HSD1, which demonstrated efficacy in reducing estrogen-dependent breast tumors in vivo (animal models). human 17-HSD1. Compared to the non-treated controls (in antiestrogen. When tested on a T47D xenograft tumor model the presence or absence of estrone), inhibitor I reduced tu- in OVX nude mice [84], compound IV (10 mg/kg/d, sc) mor weight by 54%, and tumor area by 75%. The same completely blocked tumor growth stimulated by estrone (0.1 group also tested five steroidal inhibitors (estrone derivatives μg/mouse/d, sc) comparable to that of the control group level B10721325, B10720511, B10720512, B10720440 and (without estrone). B10715817) in the xenograft model at a dose of 5 μmol/kg/d [80, 81]. Compound B10720511 was more potent than the CONCLUSION other analogues and reduced tumor weight by 86%. This compound also showed a dose-dependent effect in this Forty years after the reports describing the first 17- HSD1 structure-activity relationship of inhibitors [85], stud- xenograft study with an estimated IC50 of 1.58 μmol/kg/d (0.7 mg/kg/d). As an example, the representative compound ies related to both small molecule ligands and enzyme struc- II (B10721325) reduced tumor weight by 60%. By measur- tures have been extensively performed by academic groups ing the uterine weight, the authors also observed that such and pharmaceutical companies [54, 71]. We currently have compounds produced an antiestrogenic effect. access to four different inhibitors of 17-HSD1, which effi- ciently reduce estradiol (produced from estrone) -induced Sterix Ltd. used extensive structure-based drug design tumor growth in mice. We conclude that, an integral biologi- with available crystal structures of 17-HSD1, and devel- cal function and inhibition study is critical and inevitable to oped a family of steroidal inhibitors of 17-HSD1 and se- improve the efficiency of drug design, exemplified by the lected compound III (STX1040) as a non-estrogenic candi- elimination of the estrogenic properties of 17-HSD inhibi- date to be tested in a xenograft model [37, 82]. The authors tors. The new results at the level of cell biology and pro- inoculated estrogen-dependent human T47D breast cancer teomics will further facilitate such design [86]. Thus a new cells into nude OVX mice to generate tumors that could be generation of drugs controlling sex-hormone biosynthesis stimulated by estrone. Although T47D cells express addi- will appear to improve the therapy of ER positive BC, after tional 17-HSDs, such as types 7 and 12, it was demon- the selective estrogen receptor modulators and the aromatase strated in vitro that 17-HSD1 is responsible for transform- inhibitors. ing all estrone to estradiol [79]. After treatment with STX1040 (20 mg/kg/day/sc) for 28 days breast tumor growth CONFLICT OF INTEREST stimulated by injected estrone (0.05 or 0.1 μg/mouse/d) or by The authors confirm that this article content has no con- 0.025 mg estrone pellets was decreased. However, the inhi- flicts of interest. bition of tumor growth was not expressed as a percentage and the control data was not shown. STX1040 also decreased the plasma concentration of estradiol in the xenograft ex- ACKNOWLEDGEMENTS periments and the authors determined that it did not work via The authors would like to thank Dr. Muriel Kelly and estrogen-receptor antagonism (antiestrogen). Miss Danqi Lin for excellent editing work of the manuscript. The last steroidal inhibitor of 17-HSD1, compound IV The relative studies were supported by CIHR (Canadian In- (PBRM), differs from the others by its mechanism of action. stitute of Health Research) operating grants (funding refer- By replacing the phenolic -OH of estradiol by a bromoethyl ence number: MOP 89851 and MOP97917 to SXL and DP). group, and adding a characteristic carbamoylbenzyl side This work was supported in part by a grant from the German chain, the authors obtained a non-estrogenic compound that Federal Ministry of Education and Research (BMBF) to the inhibited the enzyme [83]. Furthermore, this compound did German Center for Diabetes Research (DZD e.V.). not bind to the estrogen receptor and did not function as an A Challenge for Medicinal Chemistry by the 17-hydroxysteroid Dehydrogenase Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 10 1169

REFERENCES ture with estradiol-adenosine hybrids with high affinity. FASEB J, 2002, 16(13), 1829-31. [1] Chen, L.; Feng, K.Y.; Cai, Y.D.; Chou, K.C.; Li, H.P. Predicting [22] Moeller, G.; Adamski, J. Integrated view on 17beta-hydroxysteroid the network of substrate-enzyme-product triads by combining dehydrogenases. Mol Cell Endocrinol, 2009, 301(1-2), 7-19. compound similarity and functional domain composition. BMC [23] Luu-The, V. Analysis and characteristics of multiple types of hu- Bioinformatics, 2010, 11, 293. man 17beta-hydroxysteroid dehydrogenase. J Steroid Biochem Mol [2] Chen, W.; Feng, P.M.; Lin, H.; Chou, K.C. iRSpot-PseDNC: iden- Biol, 2001 , 76(1-5), 143-51. tify recombination spots with pseudo dinucleotide composition. [24] Meier, M.; Möller, G.; Adamski, J. Perspectives in understanding Nucleic Acids Res, 2013, 41, e68 (open accessible with the role of human 17beta-hydroxysteroid dehydrogenases in health doi:101093/nar/gks1450). and disease. Ann N Y Acad Sci, 2009 , 1155, 15-24. [3] Wang, P.; Xiao, X.; Chou, K.C. NR-2L: A two-level predictor for [25] Marchais-Oberwinkler, S.; Henn, C.; Möller, G.; Klein, T.; Negri, identifying nuclear receptor subfamilies based on sequence-derived M.; Oster, A.; Spadaro, A.; Werth, R.; Wetzel, M.; Xu, K.; features. PLoS ONE, 2011, 6(8), e23505. Frotscher, M.; Hartmann, R.W.; Adamski, J. 17-Hydroxysteroid [4] Chou, K.C. A vectorized sequence-coupling model for predicting dehydrogenases (17-HSDs) as therapeutic targets: protein struc- HIV protease cleavage sites in proteins. J Biol Chem, 1993, tures, functions, and recent progress in inhibitor development. J 268(23), 16938-48. Steroid Biochem Mol Biol, 2011, 125(1-2), 66-82. [5] Chou, K.C. Prediction of human immunodeficiency virus protease [26] Labrie, F.; Luu-The, V.; Lin, S.X.; Simard, J.; Labrie, C.; El-Alfy, cleavage sites in proteins. Anal Biochem, 1996, 233(1), 1-14. M.; Pelletier, G.; Bélanger, A. Intracrinology: role of the family of [6] Shen, H.B; Chou, K.C. HIVcleave: a web-server for predicting 17 beta-hydroxysteroid dehydrogenases in human physiology and HIV protease cleavage sites in proteins. Anal Biochem, 2008, disease. J Mol Endocrinol, 2000, 25(1), 1-16. 375(2), 388-90. [27] Penning, T.M. Hydroxysteroid dehydrogenases and pre-receptor [7] Xiao, X.; Wang, P.; Chou, K.C. GPCR-2L: Predicting G protein- regulation of steroid hormone action. Hum Reprod Update, 2003, coupled receptors and their types by hybridizing two different 9(3), 193-205. modes of pseudo amino acid compositions. Mol Biosyst, 2011, 911- [28] Prossnitz, E.R.; Maggiolini, M. Mechanisms of estrogen signaling 9. and gene expression via GPR30. Mol Cell Endocrinol, 2009, [8] Chou, K.C.; Shen, H.B. ProtIdent: A web server for identifying 308(1-2), 32-8. proteases and their types by fusing functional domain and sequen- [29] Revankar, C.M.; Cimino, D.F.; Sklar, L.A.; Arterburn, J.B.; tial evolution information. Biochem Biophys Res Commun, 2008, Prossnitz, E.R. A transmembrane intracellular estrogen receptor 376(2), 321-5. mediates rapid cell signaling. Science, 2005, 307(5715), 1625-30. [9] Li, B.Q.; Huang, T.; Liu. L.; Cai, Y.D.; Chou, K.C. Identification [30] Prehn, C.; Möller. G.; Adamski, J. Recent advances in 17beta- of colorectal cancer related genes with mRMR and shortest path in hydroxysteroid dehydrogenases. J Steroid Biochem Mol Biol, 2009, protein-protein interaction network. PLoS One, 2012, 7(4), e33393. 114(1-2), 72-7. [10] Huang, T.; Wang, J.; Cai, Y.D.; Yu, H.; Chou, K.C. Hepatitis C [31] Baker, M.E. Evolution of adrenal and sex steroid action in verte- virus network based classification of hepatocellular cirrhosis and brates: a ligand-based mechanism for complexity. Bioessays, 2003, carcinoma. PLoS One, 2012, 7(4), e34460. 25(4), 396-400. [11] Lin, W.Z.; Fang, J.A.; Xiao, X.; Chou, K.C. Predicting secretory [32] Mindnich, R.; Adamski, J. Functional aspects of 17beta- proteins of malaria parasite by incorporating sequence evolution in- hydroxysteroid dehydrogenase 1 determined by comparison to a formation into pseudo amino acid composition via grey system closely related retinol dehydrogenase. J Steroid Biochem Mol Biol, model. PLoS One, 2012, 7(11), e49040. 2007, 104(3-5), 334-9. [12] Chou, K.C.; Wu, Z.C.; Xiao, X. iLoc-Hum: Using accumulation- [33] Haller, F.; Moman, E.; Hartmann, R.W.; Adamski, J.; Mindnich, R. label scale to predict subcellular locations of human proteins with Molecular framework of steroid/retinoid discrimination in 17beta- both single and multiple sites. Mol Biosyst, 2012, 8(2), 629-41. hydroxysteroid dehydrogenase type 1 and photoreceptor-associated Chou, K.C. Some Remarks on Predicting Multi-Label Attributes in retinol dehydrogenase. J Mol Biol, 2010, 399(2), 255-67. Molecular Biosystems. Mol Biosyst, 2013, 9, 1092-1100. [34] Zhu, D.W.; Lee, X.; Breton, R.; Ghosh, D.; Pangborn, W.; Daux, [13] Prado-Prado, F.J.; Martinez, V.O.; Uriarte, E.; Ubeira, F.M.; Chou, W.L.; Lin, S.X. Crystallization and preliminary X-ray diffraction K.C.; González-Díaz, H. Unified QSAR approach to antimicrobi- analysis of the complex of human placental 17 beta-hydroxysteroid als. 4. Multi-target QSAR modeling and comparative multi- dehydrogenase with NADP+. J Mol Biol, 1993, 234(1), 242-4. distance study of the giant components of antiviral drug-drug com- [35] Zhou, M.; Qiu, W.; Chang, H.J.; Gangloff, A.; Lin, SX. Purifica- plex networks. Bioorg Med Chem, 2009, 17(2), 569-75. tion, crystallization and preliminary X-ray diffraction results of [14] Du, Q.S.; Huang, R.B.; Wei, Y.T.; Pang, Z.W.; Du, L.Q.; Chou, human 17beta-hydroxysteroid dehydrogenase type 5. Acta Crystal- K.C. Fragment-based quantitative structure-activity relationship logr D Biol Crystallogr, 2002, 1048-50. (FB-QSAR) for fragment-based drug design. J Comput Chem, [36] Marijanovic, Z.; Laubner, D.; Moller, G.; Gege, C.; Husen, B.; 2009, 30(2), 295-304. Adamski, J.; Breitling, R. Closing the gap: identification of human [15] Chou, K.C.; Shen, H.B. Review: recent advances in developing 3-ketosteroid reductase, the last unknown enzyme of mammalian web-servers for predicting protein attributes (doi: 10.4236/ns.2009. cholesterol biosynthesis. Mol Endocrinol, 2003, 17(9), 1715-25. 12011). Natural Science, 2009, 2, 63-92. (openly accessible at [37] Day, J.M.; Foster, P.A.; Tutill, H.J.; Parsons, M.F.; Newman, S.P.; http://www.scirp.org/journal/NS/) Chander, S.K.; Allan, G.M.; Lawrence, H.R.; Vicker, N.; Potter, [16] Chou, K.C. Review: Structural bioinformatics and its impact to B.V.; Reed, M.J.; Purohit, A. 17beta-hydroxysteroid dehydro- biomedical science. Curr Med Chem, 2004, 11(16), 2105-34. genase Type 1, and not Type 12, is a target for endocrine therapy of [17] Ma, Y.; Wang, S.Q.; Xu, W.R.; Wang, R.L.; Chou, K.C. Design hormone-dependent breast cancer. Int J Cancer, 2008, 122, 1931- novel dual agonists for treating type-2 diabetes by targeting perox- 40. isome proliferator-activated receptors with core hopping approach. [38] Saloniemi, T.; Welsh, M.; Lamminen, T.; Saunders, P.; Mäkelä, S.; PLoS One, 2012, 7(6), e38546. Streng, T.; Poutanen, M. Human HSD17B1 expression masculin- [18] Li, X.B.; Wang, S.Q.; Xu, W.R.; Wang, R.L.; Chou, K.C. Novel izes transgenic female mice. Mol Cell Endocrinol, 2009, 301(1-2), inhibitor design for hemagglutinin against H1N1 influenza virus by 163-8. core hopping method. PLoS One, 2011, 6(11), e28111. [39] Rantakari, P.; Strauss, L.; Kiviranta, R.; Lagerbohm, H.; Paviala, [19] Chou, K.C.; Wei, D.Q.; Zhong, W.Z. Binding mechanism of J.; Holopainen, I.; Vainio, S.; Pakarinen, P.; Poutanen, M. Placenta coronavirus main proteinase with ligands and its implication to defects and embryonic lethality resulting from disruption of mouse drug design against SARS. Biochem Biophys Res Commun, 2003, hydroxysteroid (17-beta) dehydrogenase 2 gene. Mol Endocrinol, 308(1), 148-51. 2008, 22(3), 665-75. [20] Poirier, D.; Boivin, R.P.; Bérabé, M.; Lin, S.X. Synthesis of a first [40] Jokela, H.; Rantakari, P.; Lamminen, T.; Strauss, L.; Ola, R.; estradiol- adenosine hybrid compound. Synth Commun, 2003, Mutka, A.L.; Gylling, H.; Miettinen, T.; Pakarinen, P.; Sainio, K.; 33(18), 3183-92. Poutanen, M. Hydroxysteroid (17beta) dehydrogenase 7 activity is [21] Qiu, W.; Campbell, R.L.; Gangloff, A.; Dupuis, P.; Boivin, P.; essential for fetal de novo cholesterol synthesis and for neuroecto- Tremblay, M.R.; Poirier, D.; Lin, S.X. A concerted, rational design of 17beta-hydroxysteroid dehydrogenase inhibitors: complex struc- 1170 Current Topics in Medicinal Chemistry, 2013, Vo l. 13, No. 10 Lin et al.

dermal survival and cardiovascular differentiation in early mouse [60] Jin, J.Z.; Lin, S.X. Human estrogenic 17beta-hydroxysteroid dehy- embryos. Endocrinology, 2010, 151(4), 1884-92. drogenase: predominance of estrone reduction and its induction by [41] Möller, G.; Adamski, J. Multifunctionality of human 17beta- NADPH. Biochem Biophys Res Commun, 1999, 259(2), 489-93. hydroxysteroid dehydrogenases. Mol Cell Endocrinol, 2006, 248(1- [61] Gangloff, A.; Shi, R.; Nahoum, V.; Lin, SX. Pseudo-symmetry of 2), 47-55. C19 steroids, alternative binding orientations, and multispecificity [42] Persson, B.; Kallberg, Y.; Bray, J.E.; Bruford, E.; Dellaporta, S.L.; in human estrogenic 17beta-hydroxysteroid dehydrogenase. FASEB Favia, A.D.; Duarte, R.G.; Jörnvall, H.; Kavanagh, K.L.; J, 2003, 17(2), 274-6. Kedishvili, N.; Kisiela, M.; Maser, E.; Mindnich, R.; Orchard, S.; [62] Nahoum, V.; Gangloff, A.; Shi, R.; Lin, S.X. How estrogen- Penning, T.M.; Thornton, J.M.; Adamski, J.; Oppermann, U. The specific proteins discriminate estrogens from androgens: a common SDR (short-chain dehydrogenase/reductase and related enzymes) steroid binding site architecture. FASEB J, 2003, 17(10), 1334-6. nomenclature initiative. Chem Biol Interact, 2009, 178(1-3), 94-8. [63] Aka, J.A.; Mazumdar, M.; Chen, C.Q.; Poirier, D.; Lin, SX. [43] Jin, Y.; Penning, T.M. Aldo-keto reductases and bioactiva- 17beta-hydroxysteroid dehydrogenase type 1 stimulates breast can- tion/detoxication. Annu Rev Pharmacol Toxicol, 2007, 47, 263-92. cer by dihydrotestosterone inactivation in addition to estradiol pro- [44] Penning, T.M. Molecular endocrinology of hydroxysteroid dehy- duction. Mol Endocrinol, 2010, 24(4), 832-45. drogenases. Endocr Rev, 1997, 18(3), 281-305. [64] Aka, J.A.; Zerradi, M.; Houle, F.; Huot, J.; Lin, S.X. 17beta- [45] Penning, T.M. Molecular determinants of steroid recognition and hydroxysteroid dehydrogenase type 1 modulates breast cancer pro- catalysis in aldo-keto reductases. Lessons from 3alpha-hydroxy tein profile and impacts cell migration. Breast Cancer Res, 2012, steroid dehydrogenase. J Steroid Biochem Mol Biol, 1999, 69(1-6), 14(3), R92. 211-25. [65] Qiu, W.; Zhou, M.; Labrie, F.; Lin, S.X. Crystal structures of the [46] Puranen, T.J.; Poutanen, M.H.; Peltoketo, H.E.; Vihko, P.T.; multispecific 17beta-hydroxysteroid dehydrogenase type 5: critical Vihko, R.K. Site-directed mutagenesis of the putative active site of androgen regulation in human peripheral tissues. Mol Endocrinol, human 17 beta-hydroxysteroid dehydrogenase type 1. Biochem J, 2004, 18(7), 1798-807. 1994, 304( Pt 1), 289-93. [66] Byrns, M.C.; Jin, Y.; Penning, T.M. Inhibitors of type 5 17- [47] Ghosh, D.; Pletnev, V.Z.; Zhu, D.W.; Wawrzak, Z.; Duax, W.L.; hydroxysteroid dehydrogenase (AKR1C3): overview and structural Pangborn, W.; Labrie, F.; Lin, S.X. Structure of human estrogenic insights. J Steroid Biochem Mol Biol, 2011, 125(1-2), 95-104. 17 beta-hydroxysteroid dehydrogenase at 2.20 A resolution. [67] Lin, S.X.; Shi, R.; Qiu, W.; Azzi, A.; Zhu, D.W.; Dabbagh, H.A.; Structure, 1995, 3(5), 503-13. Zhou, M. Structural basis of the multispecificity demonstrated by [48] Hwang, C.C.; Chang, Y.H.; Hsu, C.N.; Hsu, H.H.; Li, C.W.; Pon. 17beta-hydroxysteroid dehydrogenase types 1 and 5. Mol Cell En- HI. Mechanistic roles of Ser-114, Tyr-155, and Lys-159 in 3alpha- docrinol, 2006, 248(1-2), 38-46. hydroxysteroid dehydrogenase/carbonyl reductase from Coma- [68] Qiu, W.; Zhou, M.; Mazumdar, M.; Azzi, A.; Ghanmi, D.; Luu- monas testosteroni. J Biol Chem, 2005, 280(5), 3522-8. The, V.; Labrie, F.; Lin, S.X. Structure-based inhibitor design for [49] Filling, C.; Berndt, K.D.; Benach, J.; Knapp, S.; Prozorovski, T.; an enzyme that binds different steroids: a potent inhibitor for hu- Nordling, E.; Ladenstein, R.; Jörnvall, H.; Oppermann, U. Critical man type 5 17beta-hydroxysteroid dehydrogenase. J Biol Chem, residues for structure and catalysis in short-chain dehydro- 2007, 282(11), 8368-79. genases/reductases. J Biol Chem, 2002, 277(28), 25677-84. [69] Aka, J.A.; Mazumdar, M.; Lin, S.X. Reductive 17beta- [50] van Grunsven, E.G.; van Berkel, E.; Ijlst, L.; Vreken, P.; de Klerk, hydroxysteroid dehydrogenases in the sulfatase pathway: critical in J.B.; Adamski, J.; Lemonde, H.; Clayton, P.T.; Cuebas, D.A.; the cell proliferation of breast cancer. Mol Cell Endocrinol, 2009, Wanders, R.J. Peroxisomal D-hydroxyacyl-CoA dehydrogenase de- 301(1-2), 183-90. ficiency: resolution of the enzyme defect and its molecular basis in [70] Penning, T.M. 17-hydroxysteroid dehydrogenase: inhibitors and bifunctional protein deficiency. Proc Natl Acad Sci U S A, 1998, inhibitors design. Endocr. Relat Cancer, 1996, 3, 41-56. 95(5), 2128-33. [71] Poirier, D. Inhibitors of 17 beta-hydroxysteroid dehydrogenases. [51] Jansson, A. 17Beta-hydroxysteroid dehydrogenase enzymes and Curr Med Chem, 2003, 10(6), 453-77. breast cancer. J Steroid Biochem Mol Biol, 2009, 114(1-2), 64-7. [72] Brozic, P.; Lanisnik Risner, T.; Gobec, S. Inhibitors of 17beta- [52] Schuster, D.; Kowalik, D.; Kirchmair, J.; Laggner, C.; Markt, P.; hydroxysteroid dehydrogenase type 1. Curr Med Chem, 2008, Aebischer-Gumy, C.; Ströhle, F.; Möller, G.; Wolber, G.; 15(2), 137-50. Wilckens, T.; Langer, T.; Odermatt, A.; Adamski, J. Identification [73] Poirier, D. Contribution to the development of inhibitors of 17- of chemically diverse, novel inhibitors of 17-hydroxysteroid de- hydroxysteroid dehydrogenase types 1 and 7: key tools for studying hydrogenase type 3 and 5 by pharmacophore-based virtual screen- and treating estrogen-dependent diseases. J Steroid Biochem Mol ing. J Steroid Biochem Mol Biol, 2011, 125(1-2), 148-61. Biol, 2011, 125(1-2), 83-94. [53] Saloniemi, T.; Järvensivu, P.; Koskimies, P.; Jokela, H.; [74] Day, J.M.; Tutill, H.J.; Purohit, A.; Reed, M.J. Design and valida- Lamminen, T.; Ghaem-Maghami, S.; Dina, R.; Damdimopoulou, tion of specific inhibitors of 17beta-hydroxysteroid dehydrogenases P.; Mäkelä, S.; Perheentupa, A.; Kujari ,H.; Brosens, J.; Poutanen, for therapeutic application in breast and prostate cancer, and in en- M. Novel hydroxysteroid (17beta) dehydrogenase 1 inhibitors re- dometriosis. Endocr Relat Cancer, 2008, 15(3), 665-92. verse estrogen-induced endometrial hyperplasia in transgenic mice. [75] Poirier, D. New cancer drugs targeting the biosynthesis of estro- Am J Pathol, 2010, 176(3), 1443-51. gens and androgens, Drug Devel Res, 2008, 69, 304-318 [54] Day, J.M.; Tutill, H.J.; Purohit, A. 17ß-hydroxysteroid dehydro- [76] Huang, Y.W.; Pineau, I.; Chang, H.J.; Azzi, A.; Bellemare, V.; genase inhibitors. Minerva Endocrinol, 2010, 35(2), 87-108. Laberge, S.; Lin, S.X. Critical residues for the specificity of cofac- [55] Poirier, D. Advances in development of inhibitors of 17beta hy- tors and substrates in human estrogenic 17beta-hydroxysteroid de- droxysteroid dehydrogenases. Anticancer Agents Med Chem, 2009, hydrogenase 1: variants designed from the three-dimensional struc- 9(6), 642-60. ture of the enzyme. Mol Endocrinol, 2001, 15(11), 2010-20. [56] Poirier, D. 17beta-Hydroxysteroid dehydrogenase inhibitors: a [77] Marchais-Oberwinkler, S.; Frotscher, M.; Ziegler, E.; Werth, R.; patent review. Expert Opin Ther Pat, 2010, 20(9), 1123-45. Kruchten, P.; Messinger, J.; Thole, H.; Hartmann, R.W. Structure- [57] Möller, G.; Husen, B.; Kowalik, D.; Hirvelä, L.; Plewczynski, D.; activity study in the class of 6-(3'-hydroxyphenyl)naphthalenes Rychlewski, L.; Messinger, J.; Thole, H.; Adamski, J. Species used leading to an optimization of a pharmacophore model for 17beta- for drug testing reveal different inhibition susceptibility for 17beta- hydroxysteroid dehydrogenase type 1 (17beta-HSD1) inhibitors. hydroxysteroid dehydrogenase type 1. PLoS One, 2010, 5(6), Mol Cell Endocrinol, 2009, 301(1-2), 205-11. e10969. [78] Messinger, J.; Hirvelä, L.; Husen, B.; Kangas, L.; Koskimies, P.; [58] Mindnich, R.; Adamski, J. Zebrafish 17beta-hydroxysteroid dehy- Pentikäinen, O.; Saarenketo, P.; Thole, H. New inhibitors of drogenases: an evolutionary perspective. Mol Cell Endocrinol, 17beta-hydroxysteroid dehydrogenase type 1. Mol Cell Endocrinol, 2009, 301(1-2), 20-6. 2006, 248(1-2), 192-8. [59] Lin, S.X.; Yang, F.; Jin, J.Z.; Breton, R.; Zhu, D.W.; Luu-The, V.; [79] Laplante, Y.; Rancourt, C.; Poirier, D. Relative involvement of Labrie, F. Subunit identity of the dimeric 17 beta-hydroxysteroid three 17beta-hydroxysteroid dehydrogenases (types 1, 7 and 12) in dehydrogenase from human placenta. J Biol Chem, 1992, 267(23), the formation of estradiol in various breast cancer cell lines using 16182-7. selective inhibitors. Mol Cell Endocrinol, 2009, 301(1-2), 146-53. [80] Husen, B.; Huhtinen, K.; Poutanen, M.; Kangas, L.; Messinger, J.; Thole, H. Evaluation of inhibitors for 17beta-hydroxysteroid dehy- A Challenge for Medicinal Chemistry by the 17-hydroxysteroid Dehydrogenase Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 10 1171

drogenase type 1 in vivo in immunodeficient mice inoculated with carbamoylbenzyl)estradiol. ACS Med Chem Lett, 2011, 2(9),678- MCF-7 cells stably expressing the recombinant human enzyme. 681. Mol Cell Endocrinol, 2006, 248(1-2), 109-13. [84] Ayan, D.; Maltais, R.; Roy, J.; Poirier, D. A new nonestrogenic [81] Husen, B.; Huhtinen, K.; Saloniemi, T.; Messinger, J.; Thole, H.H.; steroidal inhibitor of 17-hydroxysteroid dehydrogenase type I Poutanen, M. Human hydroxysteroid (17-beta) dehydrogenase 1 blocks the estrogen-dependent breast cancer tumor growth induced expression enhances estrogen sensitivity of MCF-7 breast cancer by estrone. Mol Cancer Ther, 2012, 11(10), 2096-104. cell xenografts. Endocrinology, 2006,147(11), 5333-9. [85] Jarabak, J.; Sack GH, J.r. A soluble 17beta-hydroxysteroid dehy- [82] Lawrence, H.R.; Vicker, N.; Allan, G.M.; Smith, A.; Mahon, M.F.; drognease from human placenta. The binding of pyrimidine nucleo- Tutill, H,J.; Purohit, A.; Reed, M.J.; Potter, B.V. Novel and potent tides and steroids. Biochemistry, 1969, 8(5), 2203-12. 17beta-hydroxysteroid dehydrogenase type 1 inhibitors. J Med [86] Lin, S.X.; Chen, J.; Mazumdar, M.; Poirier, D.; Wang, C.; Azzi, A.; Chem, 2005, 48(8), 2759-62. Zhou, M. Molecular therapy of breast cancer: extensive endocrine [83] Maltais, R.; Ayan, D.; Poirier, D. Crucial Role of 3-bromoethyl in research and clinic trials for decades. Nat Rev Endocrinol, 2010, removing the estrogenic activity of 17-HSD1 inhibitor 16-(m- 6(9), 485-93.

Received: January 06, 2013 Revised: March 08, 2013 Accepted: April 01, 2013