17β-Hydroxysteroid dehydrogenase 2, inhibition

Biological state

17β-hydroxysteroid dehydrogenase 2 (HSD17B2) belongs to the superfamily of short-chain dehydrogenase/reductase (SDR). SDRs constitute a large protein family of oxidoreductases, widely present in prokaryotes and eukaryotes. These enzymes typically display a subunit chain-length of about 250–350 amino acid residues and share few distinct sequence motifs comprising conserved nucleotide cofactor binding and active site residues. Despite their low level of conservation (15–30% overall residue identity), enzymes of this family show a conserved α/β sandwich folding pattern, where a central β -sheet is flanked by several helices in a typical motif (Rossmann tertiary fold) binding with NADP as cofactor. These NADP (H) dependent dehydrogenases/reductases act on heterogenous substrates, and a highly variable C-terminal segment creates unique active sites and ligand binding properties (reviewed in Lucacik et al., 2006) (Fig. 1).

The enzymes from the HSD17B family catalyse the NADP(H) dependent oxidation or reduction of hydroxyl/keto groups of estrogens and androgens. The major redox activity at the 17b-position of the steroid, however, several of the HSD17Bs are able to convert multiple substrates at multiple sites (e.g., at the 3rd position on the steroid ring, Day et al., 2008). Being involved in the last step of the biosynthesis of sex steroids from cholesterol, the HSD17B family contributes to regulating intracellular availability of steroid hormone ligands to their nuclear receptors as pre-receptor control mechanism, since the 17β -hydroxylated steroids bind androgen and estrogen receptors with much higher affinity than the 17-oxo steroids.

Figure 1 Human steroidogenic enzymes and peripheral steroid synthesis. Enzymes involved in intracrine metabolism of sex steroid ligands (testosterone (red), 5- dihydrotestosterone (red), estradiol (yellow)); 1: steroid sulfatase, 2: 3-HSD/4,5 isomerase, 3: aromatase (CYP19), 4: 5-reductase, 5: 3-HSD/3-oxo reductase. The numbers of the HSD17Bs involved in reductive and oxidative reactions are marked below and above the arrows respectively (for details c.f. text). Further abbreviations: DHEA-S: dehydroepiandrosterone-sulfate, DHEA: dehydroepiandrosterone, 5A-diol: 5-androstene, 3α,17β diol; 4-Adione: 4-androstene, 3,17-dione; E1: estrone; E2: estradiol, E1-S: estronesulfate; E2-S: estradiolsulfate; androstanediol: 3α,17β-androstanediol; 5α-DHT: 5α-dihydrotestoste (from Lukacik et al., 2006)

At present, 14 mammalian HSD17Bs have been described or annotated, of which 12 orthologs in humans. Out of the 12 human forms, some have been structurally characterised, including HSD17B1, 4, 5, 10, 11 and 14. Each type of HSD17B has a selective substrate affinity, directional (reductive or oxidative) activity in intact cells, and a particular tissue distribution (Poirier 2003). Orthologs to HSD17B1 and HSD17B2 are described in various mammalian species (including rat and mouse) (bgee.org).

At the current status of knowledge, the most relevant HSD17Bs for estrogen metabolism are HSD17B1 and 2. HSD17B1 catalyses the reduction of estrogens (and androgens), converting estrone (E1) into 17beta-estradiol (E2) using preferentially NADH (UniProt KB). Its expression pattern and the preferred reductive reaction direction makes HSD17B1 the major determinant of gonadal and peripheral estradiol synthesis (Peltoketo et al., 1999). HSD17B2 is widely distributed and catalyses the NAD-dependent oxidation of the highly active 17beta-hydroxysteroids, such as estradiol, testosterone, and dihydrotestosterone, to their less active forms (estrone, androstenedione, and 5alpha-androstan-3,17-dione respectively), regulating the biological potency of these steroids (UniProtKB). Other HSD17Bs either catalyse oxidoreduction of substrates other than (female) steroids or are not expressed in uterus, or are not fully characterised, therefore are not described in this context (see a review in Marchais-Oberwinkler et al., 2011).

HSD17Bs can be inhibited by various natural dietary constituents (e.g., phytoestrogens) and endogenous and exogenous compounds such as parabens, steroidal and nonsteroidal inhibitors (Salah et al., 2019). Their inhibition could cause derangements in their interplay at local level, and this can contribute to local imbalance in estrogen “activation” and “deactivation” and proliferative pathological conditions in the uterus (Miyashita et al., 2015).

In particular, the inhibition of HSD17B2 has been indicated to potentially increase the bioavailability of E2 in the uterus (Wikoff et al., 2015) and Sanders et al., (2016) reported that TBBPA at 250mg/kg body weight per day for 5 days is associated with downregulation of HSD17B2 in the uterus of the rat.

Selective HSD17B2 inhibitors have been identified, such as C18-C19 scaffold derivatives of E2 (e.g., spirolactones at position C17) and nonsteroidal compounds (e.g., spirodeltalactones, hydroxyphenylnapthole and biphenyl amides. These compounds were identified in silico and tested in vitro cellular systems however they have not reached in vivo phase (preclinical or clinical, Salah et al., 2019)

Biological compartment

Distribution of mRNA varies according to the specific enzyme; HSD17B1 is predominantly expressed in humans in placenta, breast, ovary, as well as in the uterus (endometrium), adipose tissues, brain and gastrointestinal tract (UniProt, Wang et al., 2019); HSD17B2 showing widespread distribution in human, including the gastrointestinal tract (intestine, liver, pancreas), bone, ovary, placenta, endometrium and prostate (see bgee.org).

In a normal cycling endometrium, HSD17B2 protein and mRNA are present at all the stages of the secretory phase, located in the endoplasmic reticulum, not in the endometrial mucosa during the proliferative phase, while HSD17B1 are not detectable in any stage (Utsunomiya et al., 2001).

General role in biology

The HSDs are integral parts of systemic (endocrine) and local (intracrine) mechanisms. In target tissues, they convert inactive steroid hormones to their corresponding active forms and vice versa; their interplay modulates the transactivation of steroid hormone receptors (or other elements of the non-genomic signal transduction pathways), acting as pre-receptor molecular switches (Marchais-Oberwinkler et al., 2011, Salah et al., 2019). In details, the 17β -hydroxysteroid dehydrogenase enzymes (HSD17s) catalyze the NAD(P)(H) dependent oxidoreduction of hydroxyl/keto groups at position C17 of androgens and estrogens; and estrogen receptors transactivate their target genes by binding the 17β -hydroxylated steroids with much higher affinity than the 17-oxo steroids.

In the uterus, estrone is converted into the more potent estradiol by HSD17B1, and the reverse reaction leading to the production of estrone from estradiol is catalysed mainly by HSD17B2. Derangements in this interplay can contribute to local imbalance in estrogen “activation” and “deactivation” and proliferative pathological conditions in the uterus (e.g., Miyashita et al., 2015, Hashimoto et al., 2018). High levels of HSD17B1 mRNA and increased E2/E1 ratio are described in uterine proliferative conditions such as endometriosis, endometrial hyperplasia and uterine leiomyoma and inhibitors of this enzyme are investigated as potential therapeutics for these conditions (Marchais-Oberwinkler et al., 2011, Salah et al., 2019).

The role of HSD17B2 in pathologies of estrogen-sensitive tissues is emerging, and its inhibition could potentially increase the bioavailability of E2 in the uterus (Wikoff et al., 2015). Down-regulation of HSD17B2 gene was noted in the distal uterus of TBBPA-treated rats (Sanders et al., 2014). Utsunomiya et al., 2001 demonstrated 17b-HSD type 2 immunoreactivity in the cytoplasm of endometrial endometrioid adenocarcinomas cell; Hashimoto et al (2018)  demonstrated that androgen-mediated increase in HSD17B2 mRNA levels in HEC-1B cells is associated with significantly reduced E2 induced cell proliferation; in addition, the author investigated the HSD17B2 status of endometroid endometrial carcinoma cells and 19/53 sample immunoreactive for the enzyme, positively correlated with the intratumorally DHT concentration; this status was inversely associated with the histological grade, proliferation index and clinical stage and patients tended to have better prognosis than those negative for HSD17B2 immunoreactivity.

High levels of HSD17B2 mRNA have been demonstrated in the epithelial cell component of whole endometrial tissue upon exposure to progesterone both in vivo and in vitro Bulun et al., 2010.

Expression:

• qRT-PCR, Northern blot:  HSD17B2 mRNA expression
• Western blotting:  HSD17B2 protein levels
• Immunohistochemistry: HSD17B2 protein (Miyashita et al., 2015, 2004, Hashimoto et al., 2018).

Activity:

• Radiometric assays (Singh and Reed 1991) after extraction and separation by thin layer chromatography (Kitawaki et al., 2000) or HPLC in whole cells and lysates
• Homogeneous proximity (Kokko et al.,2006) and fluorescence resonance energy transfer (FRET)-based assays (Kokko et al., 2007) (high throughput in vitro screening)
• Sensitive HPLC-based methods can be used for tissue sample analysis (Delvoux et al., 2007).

Biological domains of applicability

1. Taxonomic applicability
   • Mammalians, birds. Invertebrates were not investigated.
2. Life stage applicability
   • All life stages, mainly adulthood (UniProt)
3. Sex applicability
   • Males, females.
4. Evidence for the biological domain of applicability
   • HSD17B2 enzyme catalyses the oxidation of E2 to the less potent E1 (estrone), participating in the modulation of the estrogenic signalling in the uterus.
   • Inhibition of HSD17B2 could increase E2 available to estrogenic activation pathways in estrogenic sensitive tissues (uterus).  

Bgee, online. Available online: https://bgee.org/

Bulun SE, Cheng Y-H, Pavone ME, Yin P, Imir G, Utsunomiya H, Thung S, Xue Q, Marsh EE, Tokunaga H, Ishikawa H, Kurita T and Su EJ, 2010. 17β-Hydroxysteroid Dehydrogenase-2 Deficiency and Progesterone Resistance in Endometriosis. Semin Reprod Med, 28:044-050

Day JM, Tutill HJ, Purohit A and Reed MJ, 2008. Design and validation of specific inhibitors of 17beta-hydroxysteroid dehydrogenases for therapeutic application in breast and prostate cancer, and in endometriosis. Endocr Relat Cancer, 15:665-692. doi: 10.1677/erc-08-0042

Delvoux B, Husen B, Aldenhoff Y, Koole L, Dunselman G, Thole H and Groothuis P, 2007. A sensitive HPLC method for the assessment of metabolic conversion of estrogens. J Steroid Biochem Mol Biol, 104:246-251. doi: 10.1016/j.jsbmb.2007.03.006

Hashimoto C, Miki Y, Tanaka S, Takagi K, Fue M, Doe Z, Li B, Yaegashi N, Suzuki T and Ito K, 2018. 17β-Hydroxysteroid Dehydrogenase Type 2 Expression Is Induced by Androgen Signaling in Endometrial Cancer. Int J Mol Sci, 19:1139

Kitawaki J, Koshiba H, Ishihara H, Kusuki I, Tsukamoto K and Honjo H, 2000. Progesterone Induction of 17β-Hydroxysteroid Dehydrogenase Type 2 during the Secretory Phase Occurs in the Endometrium of Estrogen-Dependent Benign Diseases But Not in Normal Endometrium. The Journal of Clinical Endocrinology & Metabolism, 85:3292-3296. doi: 10.1210/jcem.85.9.6829

Kokko L, Jaakohuhta S, Lindroos P and Soukka T, 2006. Improved homogeneous proximity-based screening assay of potential inhibitors of 17beta-hydroxysteroid dehydrogenases. Assay Drug Dev Technol, 4:671-678. doi: 10.1089/adt.2006.4.671

Lukacik P, Kavanagh KL and Oppermann U, 2006. Structure and function of human 17beta-hydroxysteroid dehydrogenases. Mol Cell Endocrinol, 248:61-71. doi: 10.1016/j.mce.2005.12.007

Marchais-Oberwinkler S, Henn C, Möller G, Klein T, Negri M, Oster A, Spadaro A, Werth R, Wetzel M, Xu K, Frotscher M, Hartmann RW and Adamski J, 2011. 17β-Hydroxysteroid dehydrogenases (HSD17Bs) as therapeutic targets: protein structures, functions, and recent progress in inhibitor development. J Steroid Biochem Mol Biol, 125:66-82. doi: 10.1016/j.jsbmb.2010.12.013

Miyashita M, Koga K, Takamura M, Izumi G, Nagai M, Harada M, Hirata T, Hirota Y, Fujii T and Osuga Y, 2014. Dienogest reduces proliferation, aromatase expression and angiogenesis, and increases apoptosis in human endometriosis. Gynecological endocrinology : the official journal of the International Society of Gynecological Endocrinology, 30:644-648. doi: 10.3109/09513590.2014.911279

Poirier D, 2003. Inhibitors of 17 beta-hydroxysteroid dehydrogenases. Curr Med Chem, 10:453-477. doi: 10.2174/0929867033368222

Salah M, Abdelsamie AS and Frotscher M, 2019. Inhibitors of 17β-hydroxysteroid dehydrogenase type 1, 2 and 14: Structures, biological activities and future challenges. Mol Cell Endocrinol, 489:66-81. doi: 10.1016/j.mce.2018.10.001