cancers Article A Targeted-Covalent Inhibitor of 17β-HSD1 Blocks Two Estrogen-Biosynthesis Pathways: In Vitro (Metabolism) and In Vivo (Xenograft) Studies in T-47D Breast Cancer Models Donald Poirier 1,2,* , Jenny Roy 1 and René Maltais 1 1 Laboratory of Medicinal Chemistry, Endocrinology and Nephrology Unit, CHU de Québec—Research Center, Québec, QC G1V 4G2, Canada; [email protected] (J.R.); [email protected] (R.M.) 2 Department of Molecular Medicine, Faculty of Medicine, Université Laval, Québec, QC G1V 0A6, Canada * Correspondence: [email protected]; Tel.: +1-418-654-2296 Simple Summary: 17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) is responsible for the production of estrogens estradiol (E2) and 5-androsten-3β,17β-diol (5-diol). This enzyme is there- fore a target of choice for the treatment of estrogen-dependent diseases such as breast cancer and endometriosis, by blocking estrogen biosynthesis. After we developed the first irreversible and non- estrogenic 17β-HSD1 inhibitor, a molecule named PBRM, our goal was to demonstrate its therapeutic potential. PBRM was able to block the formation of E2 and 5-diol in T-47D human breast cancer cells. When given orally to mice, PBRM was also able to block the tumor growth without any observed toxic effects. Thanks to its irreversible type of inhibition, PBRM retained its anti-tumor growth effect, even after reducing its frequency of administration to only once a week, a clear advantage over Citation: Poirier, D.; Roy, J.; Maltais, reversible inhibitors. These results strongly support the use of PBRM as a new approach in the R. A Targeted-Covalent Inhibitor of treatment of breast cancer. 17β-HSD1 Blocks Two Estrogen-Biosynthesis Pathways: In Abstract: 17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) plays an important role in estrogen- Vitro (Metabolism) and In Vivo dependent breast tumor growth. In addition to being involved in the production of estradiol (E2), (Xenograft) Studies in T-47D Breast the most potent estrogen in women, 17β-HSD1 is also responsible for the production of 5-androsten- Cancer Models. Cancers 2021, 13, 1841. https://doi.org/10.3390/ 3β,17β-diol (5-diol), a weaker estrogen than E2, but whose importance increases after menopause. cancers13081841 17β-HSD1 is therefore a target of choice for the treatment of estrogen-dependent diseases such as breast cancer and endometriosis. After we developed the first targeted-covalent (irreversible) and Academic Editor: Jason Roszik non-estrogenic inhibitor of 17β-HSD1, a molecule named PBRM, our goal was to demonstrate its therapeutic potential. Enzymatic assays demonstrated that estrone (E1) and dehydroepiandrosterone Received: 12 February 2021 (DHEA) were transformed into E2 and 5-diol in T-47D human breast cancer cells, and that PBRM Accepted: 7 April 2021 was able to block these transformations. Thereafter, we tested PBRM in a mouse tumor model (cell- Published: 13 April 2021 derived T-47D xenografts). After treatment of ovariectomized (OVX) mice receiving E1 or DHEA, PBRM given orally was able to reduce the tumor growth at the control (OVX) level without any Publisher’s Note: MDPI stays neutral observed toxic effects. Thanks to its irreversible type of inhibition, PBRM retained its anti-tumor with regard to jurisdictional claims in growth effect, even after reducing its frequency of administration to only once a week, a clear published maps and institutional affil- advantage over reversible inhibitors. iations. Keywords: breast cancer; estrogen; hormone; steroid; inhibitor; DHEA; estrone Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article distributed under the terms and Breast cancer is the most common cancer in women and the second leading cause conditions of the Creative Commons of cancer death [1]. Approximately 70% of breast cancers are estrogen-dependent, which Attribution (CC BY) license (https:// means that they are likely to respond positively to treatment targeting either the action creativecommons.org/licenses/by/ or formation of estrogens. To block the action of estrogens on the estrogen receptor alpha 4.0/). (ERα), several families of antagonists (pure antiestrogens and selective estrogen receptor Cancers 2021, 13, 1841. https://doi.org/10.3390/cancers13081841 https://www.mdpi.com/journal/cancers Cancers 2021, 13, x 2 of 15 Cancers 2021, 13, 1841 2 of 15 (ERα), several families of antagonists (pure antiestrogens and selective estrogen receptor modulators) have been developed [2–5]. Although these molecules are effective and com- monly used in modulators)clinical practice, have some been patients developed develop [2– 5resistance]. Although [6], hence these the molecules interest arein effective and working on othercommonly therapeutic used agents in clinical such practice, as enzyme some inhibitors patients develop that would resistance reduce [6], the hence the interest concentration ofin estrogens. working on other therapeutic agents such as enzyme inhibitors that would reduce the Several enzymesconcentration are involved of estrogens. in the synthesis of estrogens [7], but aromatase (CYP19A1; EC 1.14.14.1),Several steroid enzymes sulfatase are involved (STS; EC in 3.1.6.2) the synthesis and 17β of-hydroxysteroid estrogens [7], but dehy- aromatase (CYP1- drogenase type9A1; 1 (17 ECβ-HSD1; 1.14.14.1), EC 1.1.1.51) steroid sulfatase have been (STS; targeted EC 3.1.6.2) for the and development 17β-hydroxysteroid of in- dehydroge- hibitors for therapeuticnase type purposes 1 (17β-HSD1; (Figure EC 1) 1.1.1.51)[8–12]. Of have these been enzymes, targeted aromatase for the development is the best of inhibitors known, since itfor is involved therapeutic in the purposes aromatization (Figure1 of)[ the8–12 A-ring]. Of these of C19-steroids enzymes, aromatase to obtain the is the best known, C18-steroids estronesince it(E1) is involvedand estradiol in the (E2) aromatization [13]. Until now, of the aromatase A-ring of is C19-steroids the only one to of obtain the C18- these three enzymessteroids whose estrone development (E1) and estradiol of inhibitors (E2) [13has]. led Until to now,marketed aromatase drugs is to the treat only one of these breast cancer, namelythree enzymes anastroz whoseole, letrozole development and exemestane of inhibitors [14]. has The led development to marketed of drugs STS to treat breast inhibitors is morecancer, recent namely but has anastrozole, also given letrozolerise to a great and exemestane deal of work [14 [15–20].]. The developmentThis en- of STS in- zyme is responsiblehibitors for is the more hy recentdrolysis but of has sulfated also given hydroxys rise toteroids a great to deal free of hydroxyster- work [15–20]. This enzyme oids, the form generallyis responsible active for on the a receptor hydrolysis or which of sulfated can serve hydroxysteroids as precursor to for free estrogen hydroxysteroids, the biosynthesis. Onlyform one generally of these active STS inhibitors on a receptor (Irosustat) or which has canreached serve clinical as precursor trials (phase for estrogen biosyn- II) showing clinicalthesis. benefits Only when one of combined these STS wi inhibitorsth an aromatase (Irosustat) inhibitor, has reached but larger clinical stud- trials (phase II) ies are requiredshowing before to clinical be translating benefits this when to a combined marketed with drug an to aromatase treat breast inhibitor, cancer [21– but larger studies 23]. The third enzyme,are required 17β-HSD1, before to also be translatinghas an important this to arole marketed in estrogen drug synthesis to treat breast and cancer [21–23]. β tumor growth [24–28].The third In enzyme,addition 17to being-HSD1, directly also hasinvolved an important in the production role in estrogen of E2, the synthesis and tu- mor growth [24–28]. In addition to being directly involved in the production of E2, the most potent estrogen in women, from E1, this enzyme is also responsible for the produc- most potent estrogen in women, from E1, this enzyme is also responsible for the pro- tion of 5-androstene-3β,17β-diol (5-diol) from dehydroepiandrosterone (DHEA), a less duction of 5-androstene-3β,17β-diol (5-diol) from dehydroepiandrosterone (DHEA), a potent estrogen than E2 [29,30], but a compound whose production increases after meno- less potent estrogen than E2 [29,30], but a compound whose production increases after pause (Figure 1). 17β-HSD1 is therefore a target of choice for the treatment of breast cancer menopause (Figure1 ). 17β-HSD1 is therefore a target of choice for the treatment of breast because its inhibition would make it possible to obtain optimal blocking of estrogen bio- cancer because its inhibition would make it possible to obtain optimal blocking of estrogen synthesis (E2 and 5-diol) and of estrogen-dependent breast cancer cell proliferation. biosynthesis (E2 and 5-diol) and of estrogen-dependent breast cancer cell proliferation. Figure 1. InvolvementFigure 1. Involvement of three enzymes of three (STS, enzymes 17β-HSD1 (STS, 17 andβ-HSD1 aromatase) and aromatase) in the formation in the formation of estrogens of (5-diolestro- and E2) in breast cancergens cells (5-diol and tumors. and E2) Thein breast formation cancer of cells 5-diol and is nottumors. prevented The formation by an aromatase of 5-diol inhibitor is not prevented (AI), unlike by with the use of a 17β-HSD1an aromatase inhibitor suchinhibitor as PBRM. (AI), unlike with the use of a 17β-HSD1 inhibitor such as PBRM. The developmentThe of development 17β-HSD1 inhibitors of 17β-HSD1 has given inhibitors rise to has several given studies rise to several[31–38], studies [31–38], mainly at the beginningmainly at of the the beginning 21st century, of the but 21st this century, research but has this no researcht yet produced has not a yet drug produced a drug to treat breast cancerto treat [39]. breast However, cancer [a39 17].β However,-HSD1 inhibitor a 17β-HSD1 is curre inhibitorntly undergoing is currently phase undergoing phase 1 clinical studies for use in treating endometriosis [40].