Chemico-Biological Interactions 191 (2011) 60–65 Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint Biochemical and biological evaluation of novel potent coumarin inhibitor of 17-HSD type 1 ˇ S. Starˇ cevi ´ c a , P. Kocbek a , G. Hribar b , T. Laniˇ snik Riˇ zner c,∗ , S. Gobec a,∗∗ a Faculty of Pharmacy, University of Ljubljana, Aˇ skerˇ ceva 7, 1000 Ljubljana, Slovenia b National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia c Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia article info Article history: Available online 11 January 2011 Keywords: 17-HSD type 1 Soluble recombinant enzyme Hormone-dependent breast cancer Coumarin inhibitor abstract Human 17-hydroxysteroid dehydrogenase (17-HSD) type 1 is an enzyme that acts at the pre-receptor level. It catalyzes the NADPH-dependent reduction of the weak estrogen estrone into the most potent estrogen 17-estradiol, which exerts proliferative effects via estrogen receptors. Overexpression of 17- HSD type 1 in estrogen-responsive tissues is related to the development of hormone-dependent diseases, such as breast cancer and endometriosis. 17-HSD type 1 thus represents an attractive target for devel- opment of new drugs. Recently, we discovered that substituted coumarin derivatives potently and selectively inhibit 17-HSD type 1. In the present study, we have performed additional biochemical and biological evaluation of the most promising coumarin derivative. First, we used an efficient method for isolation and purification of the active, soluble recombinant human 17-HSD type 1 from Escherichia coli. This 17-HSD type 1 showed a specific activity of 0.64 ± 0.08 mol min −1 mg −1 for estrone reduction in the presence of NADPH at pH 6.5, and a K m of 62 nM for estrone. Next, we evaluated the best of the coumarin-derivative inhibitors, showing its reversible and competitive inhibition of 17-HSD type 1 reductive activity with a K i of 53 nM. We confirmed that this coumarin inhibitor acts not only in a cell- free assay, but also decreases endogenous 17-HSD type 1 activity in human T-47D breast cancer cells. This inhibitor also reduced estrone dependent growth of T-47D cells after 48 h of incubation. © 2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction The 17-hydroxysteroid dehydrogenases (17-HSDs) catalyze redox reactions at the C17 position of the steroidal nucleus, and can thus regulate the biological potencies of steroid hormones [1]. To date, 14 mammalian 17-HSDs have been identified [2]. With the exception of 17-HSD type 5, which is a member of the aldo-keto reductase superfamily, all of the 17-HSDs belong to the short-chain alcohol dehydrogenase reductase superfamily, although their overall homology is low [3]. Despite their name that implies that they convert steroid substrates at position 17, their substrate specificities and regiospecificities are quite low [2,4]. As well as having different steroid substrates, they are also active towards retinoids, fatty acids and bile acids [4–7]. Only 17-HSD Abbreviations: HSD, hydroxysteroid dehydrogenase; E1, estrone; E2, 17- estradiol; ER, estrogen receptor; NAD(P)(H), nicotine amide dinucleotide (phos- phate) (reduced form); SAR, structure–activity relationship. ∗ Corresponding author. Tel.: +386 1 5437657; fax: +386 1 5437641. ∗∗ Corresponding author. Tel.: +386 1 4769501; fax: +386 1 4258031. E-mail addresses: tea.lanisnik-rizner@mf.uni-lj.si (T. Laniˇ snik Riˇ zner), stanislav.gobec@ffa.uni-lj.si (S. Gobec). types 1, 2 and 3 appear to have physiological roles solely in steroid metabolism [4,5,8]. The most extensively characterized 17-HSD is 17-HSD type 1, which catalyzes NADPH-dependent reduction of the weakly active estrone (E1) into the potent estrogen 17- estradiol (E2) [8] (Fig. 1A). In this manner, 17-HSD type 1 can regulate the occupancy of estrogen receptors (ERs), and thus the proliferative effects of estrogens [9,10]. 17-HSD type 1 is a sol- uble cytoplasmic homodimer with 327 amino-acid residues and a subunit mass of 34.9 kDa [11]. It is expressed mainly in ovaries, placenta and breast tissue, and also in ectopic endometrium, and its overexpression is related to the development and progression of hormone-dependent breast cancer and endometriosis [12–18]. Microsomal 17-HSD type 2 catalyzes the reverse reaction, NAD + - dependent oxidation of E2 into E1. By lowering the intracellular levels of E2, 17-HSD type 2 has a protective role in estrogen- responsive tissues [19–21]. E2 stimulates the growth of breast cancers, and an increased E2/E1 ratio has been reported in ER-positive breast tumors [22]. Interestingly, significantly greater 17-HSD type 1 activity was detected in breast cancer tissue of postmenopausal than pre- menopausal patients [23]. 17-HSD type 1 overexpression has been reported for the more aggressive hormone-dependent breast can- cers that show poor prognosis and short recurrence times [19,24]. 0009-2797/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2011.01.002