Physiological and Biochemical Aspects of 17β-Hydroxysteroid Dehydrogenase Type 2 and 3 Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Roger Thomas Engeli aus Sulgen (TG), Schweiz Basel, 2017 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung-Nicht kommerziell 4.0 International Lizenz. Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Alex Odermatt und Prof. Dr. Rik Eggen Basel, den 20.06.2017 ________________________ Dekan Prof. Dr. Martin Spiess 2 Table of Contents Table of Contents ............................................................................................................................... 3 Abbreviations ..................................................................................................................................... 4 1. Summary ........................................................................................................................................ 6 2. Introduction ................................................................................................................................... 8 2.1 Steroid Hormones ............................................................................................................................... 8 2.2 Human Steroidogenesis..................................................................................................................... 11 2.3 Hydroxysteroid Dehydrogenases ...................................................................................................... 12 2.3.1 17β-Hydroxysteroid Dehydrogenases ........................................................................................ 13 3. Project 1: 17β-Hydroxysteroid Dehydrogenase Type 2 ................................................................... 14 3.1 Introduction ....................................................................................................................................... 14 3.2 Paper 1 (Vuorinen et al., 2014) ......................................................................................................... 17 3.3 Paper 2 (Vuorinen/Engeli et al., 2017a) ............................................................................................ 31 3.4 Paper 3 (Vourinen/Engeli et al., 2017b) ............................................................................................ 36 3.5 Paper 4 (Engeli et al., in preparation) ................................................................................................ 47 3.7 Conclusion ......................................................................................................................................... 62 4. Project 2: 17β-Hydroxysteroid Dehydrogenase Type 3 ................................................................... 64 4.1 Introduction ....................................................................................................................................... 64 4.2 Paper 5 (Engeli et al., 2016) ............................................................................................................... 66 4.3 Paper 6 (Ben Rhouma et al., 2017) .................................................................................................... 75 4.4 Paper 7 (Engeli et al, submitted) ....................................................................................................... 85 4.5 Conclusion ....................................................................................................................................... 106 5. Project 3: Murine Leydig Cell Lines .............................................................................................. 109 5.1 Introduction ..................................................................................................................................... 109 5.2 Paper 8 (Odermatt et al, 2016) ....................................................................................................... 111 5.3 Paper 9 (Engeli et al, in preparation)............................................................................................... 125 5.4 Conclusion ....................................................................................................................................... 152 6. Acknowledgments ...................................................................................................................... 155 7. Appendix .................................................................................................................................... 155 8. References ................................................................................................................................. 157 3 Abbreviations 17β-HSD1: 17β-Hydroxysteroid Dehydrogenase type 1 17β-HSD2: 17β-Hydroxysteroid Dehydrogenase type 2 17β-HSD3: 17β-Hydroxysteroid Dehydrogenase type 3 17β-HSD5: 17β-Hydroxysteroid Dehydrogenase type 5 3β-HSD: 3β-Hydroxysteroid Dehydrogenase AKR: Aldo-keto Reductase AR: Androgen Receptor CAIS: Complete Androgen Insensitivity Syndrome CYP: Cytochrome P450 DHEA: Dehydroepiandrosterone DHT: Dihydrotestosterone DNA: Deoxyribonucleic Acid DSD: Disorder of Sexual Development EDC: Endocrine Disrupting Chemical ELISA: Enzyme-linked Immunosorbent Assay ER: Endoplasmic Reticulum ERα: Estrogen Receptor Alpha ERβ: Estrogen Receptor Beta GC-MS: Gas Chromatography-mass Spectrometry GR: Glucocorticoid Receptor hCG: Human Chorionic Gonadotropin HSD: Hydroxysteroid Dehydrogenase HPA: Hypothalamic-pituitary-adrenal axis LC-MS: Liquid Chromatography-mass Spectrometry 4 LH: Luteinizing Hormone MR: Mineralocorticoid Receptor mRNA: Messenger Ribonucleic Acid NAD+: Nicotinamide Adenine Dinulceotide NADPH: Nicotinamide Adenine Dinucleotide Phosphate P450c17: 17α-hydroxylase, 17,20 lyase P450scc: Cholesterol Side-Chain Cleavage Enzyme PAIS: Partial Androgen Insensitivity Syndrome SAR: Structure-activity Relationship SDR: Short-chain Dehydrogenase/Reductase StAR: Steroidogenic Acute Regulatory Protein TBT: Tributyltin TPT: Triphenyltin 5 1. Summary Steroid hormones regulate a broad variety of physiological functions through the transcriptional regulation of target genes. The active steroid hormones discussed in this thesis largely elicit their physiological effects through the activation of nuclear receptors. Many of these receptors reside in their inactive form in the cytoplasm, translocate to the nucleus upon ligand binding and drive the transcription of target genes. The local interconversion of active and inactive steroid hormones is regulated by members of 17β-hydroxysteroid dehydrogenases/reductases (17β- HSDs). The enzyme 17β-HSD2 converts active estrogen estradiol and the potent androgen testosterone to its inactive keto-forms, whereas 17β-HSD3 mainly converts androstenedione into testosterone. The present thesis is split into three major projects that focus mainly on potential toxicological and therapeutic effects of inhibiting the enzyme 17β-HSD2 and biochemical aspects of the enzymes 17β-HSD3. The first project was designed to develop a 17β-HSD2 pharmacophore model and subsequently use this model as a virtual screening tool to identify novel nonsteroidal 17β-HSD2 inhibitors. It has been hypothesized that pharmacological inhibition of the enzyme 17β-HSD2 expressed in osteoclasts could be a useful strategy to treat osteoporosis through increasing local concentrations of active sex hormones. Our approach was initiated through the development of a pharmacophore model that underwent several rounds of experimental validation and improvement. In silico screening of internal and external compound databases using the optimized pharmacophore model resulted in the identification of several novel nonsteroidal compounds that inhibit 17β-HSD2 at nanomolar concentrations in vitro. Furthermore, the virtual screening of a cosmetic ingredients database revealed several paraben compounds as potential 17β-HSD2 inhibitors. In vitro examinations revealed that all tested paraben compounds were found to significantly inhibit 17β-HSD2 at a concentration of 20 µM. However, parabens are rapidly metabolized to p-hydroxybenzoic acid, which does not influence 17β-HSD2 activity. We reported a novel potential estrogenic effect of paraben compounds by inhibiting 17β-HSD2 although their estrogenic potential is unlikely to be of toxicological concern due to their rapid metabolism by esterases. In the second project we biochemically analyzed six mutations in the HSD17B3 gene that were associated to cause 17β-HSD3 deficiency in Egyptian and Tunisian patients with 46, XY disorder 6 of sexual development (DSD). Patients with 17β-HSD3 deficiency are unable to synthesize sufficient amounts of testosterone during embryogenesis which leads to severe undervirilization of internal and external genitals. All tested mutations (G133R, C206X, T54A, M164T, and L194T) were confirmed to be nearly inactive in vitro and therefore unable to sufficiently convert androstenedione into testosterone. Further analyses showed that the G289S polymorphism exhibited a similar rate of testosterone formation as the wild type 17β-HSD3 enzyme and consequently cannot be causing the pathogenesis of 46, XY DSD. All HSD17B3 mutations associated with 46, XY DSD in this project were predicted by an in silico 17β-HSD3 homology model (based