title: 17β Hydroxysteroid Dehydrogenase type 14 (17β-HSD14): Development and Synthesis of Potent and Selective Inhibitors for the Structural Characterization and Investigation of the Physiological Role of the Enzyme
title: 17β Hydroxysteroid Dehydrogenase 14 (17β-HSD14): Entwicklung und Synthese potenter und selektiver Inhibitoren zur strukturellen Charakterisierung und Untersuchung der physiologischen Rolle des Enzyms
Creators: name: Braun, Florian
Publisher: name: Philipps-Universität Marburg
Year: 2017
Subjects: Synthese, Inhibitor
Pharmakologie, Therapeutik
Pharmacology &amp; therapeutics, prescription drugs
Steroidhormon Dehydrogenasen, Wirkstoffdesign

description: In dieser Arbeit wird die Familie
      der 17β-Hydroxysteroid Dehydrogenasen (17β-HSDs) vorgestellt,
      die in vitro die Oxidation und/oder Reduktion von
      Steroidhormonen in Position 17 des Steroidgerüstes
      katalysieren. 17β-HSDs besitzen die Fähigkeit lokale
      Konzentrationen von aktiven und inaktiven Hormonderivaten in
      bestimmten Gewebearten nach dem intrakrinen Konzept
      regulieren zu können.1 Demnach können sie als molekulare
      Schalter betrachtet werden, die als Prärezeptorregulatoren
      auf die Funktion von Steroidhormonen wirken, bevor sie an die
      entsprechenden Rezeptoren angreifen.2,3 Die Blockade dieser
      Enzyme durch potente und selektive Inhibitoren kann daher ein
      vielversprechender therapeutischer Ansatz zur Behandlung von
      steroidhormonabhängigen Erkrankungen sein. So konnten
      Inhibitoren für die 17β-HSDs 1, 2 und 3 entwickelt werden,
      mit denen nach der in vivo Verabreichung ein „proof of
      concept“ für die Behandlung von Brustkrebs, Osteoporose und
      Prostatakrebs erbracht wurde.4–10 Vor allem für die Subtypen
      4-14 wurde weiterhin eine weitaus ausgeprägtere
      Multifunktionalität beschrieben, sodass diese ebenfalls
      andere Substrate als Steroidhormone zulassen.11–13 17β-HSD14
      ist das zuletzt identifizierte Mitglied dieser Enzymfamilie,
      das überwiegend im Gehirn, Leber, Plazenta und den Nieren
      exprimiert wird.14,15 Durch die oxidative Deaktivierung von
      E2 durch das Enzym wurde ihm eine steroidogene Funktion
      zugeschrieben, für das JANSSON et al. erste Argumente
      erbrachten.16 Der langsame katalytische Umsatz (kcat-Wert)
      von E2 lässt diese Annahme jedoch fraglich erscheinen.15 Da
      die physiologische Rolle des Enzyms nicht vollständig
      aufgeklärt und die Frage nach dem natürlichen Substrat nicht
      zweifelsfrei beantwortet ist, war es das Ziel dieser Arbeit
      potente und selektive Inhibitoren der 17β-HSD14 zu
      entwickeln. Diese sollten eine ergänzende strukturelle
      Charakterisierung des Enzyms ermöglichen und ferner zu tool
      compounds weiterentwickelt werden, um die physiologische
      Rolle des Enzyms untersuchen zu können.
description: In vitro 17β-hydroxysteroid
      dehydrogenases (17β HSDs) can catalyze the oxidation and the
      reduction of the hydroxyl or keto group in position 17 of
      steroid hormones. Because of their ability to modulate the
      concentration of active and inactive steroids in the cell,
      these enzymes can be described as prereceptor molecular
      switches. So far, fourteen isoforms have been identified, of
      which twelve can be found in human tissues. In the recent
      years 17β HSD types 1-3 gained major interest as potential
      drug targets for the treatment of sex steroid hormone-related
      diseases for which different “proof of concepts” were
      described, whereas the other 17β HSDs are known to be
      multifunctional. 17β-HSD14 is the latest isoform discovered
      and it belongs to the short-chain dehydrogenase-reductase
      family. In vitro the enzyme can catalyze the oxidation of
      estradiol in the presence of the cofactor NAD+. However, in
      vivo, the natural substrate is still unknown. This enzyme is
      predominantly expressed in the brain, liver, placenta and in
      the kidney and it was revealed to be cytosolic. The goal of
      the project was to further characterize the protein to get a
      deeper insight into the function of 17β-HSD14. Inhibitors are
      useful chemical tools to elucidate the binding site of an
      enzyme and even more to disclose the physiological role of
      the latter upon in vivo administration. As no inhibitor has
      been reported for this enzyme by that time, the aim of this
      work was to identify the first nonsteroidal 17β-HSD14
      inhibitors. From the existing 3D-structure of the target
      enzyme no information about the protein/ligand interactions
      could be gained. Even more, it was not evident whether the
      crystallized enzyme, showing an extensive and open site
      cleft, was present in its active conformation. Therefore, no
      structure-based inhibitor design and no docking studies could
      be applied. Instead, a ligand-based approach was utilized
      starting from a small library of 17β-HSD1 and HSD2
      inhibitors, which were tested for type 14 inhibitory
      activity. The initial hit identified in this preliminary
      screen (Ki around 250 nM) was optimized to result in six
      highly active compounds with Ki &lt; 15 nM, showing good
      physicochemical properties, which should be associated with a
      promising bioavailability profile. Furthermore, these
      compounds, bearing a 2,6-pyridine ketone motife, also possess
      a good selectivity profile with respect to 17β-HSD1 and -2.
      In a close collaboration with NICOLE BERTOLETTI (KLEBE group,
      Philipps-University Marburg) it was possible to obtain
      crystal structures of five inhibitors in ternary complex with
      the target enzyme, which provided together with the
      biological results the basis for the understanding of an
      initial first structure-activity relationship of 17β-HSD14.
      It appears that an inhibitor’s acidic phenol group, being in
      close H-bond distance with Tyr154 and Ser141 of the catalytic
      triad, is essential to achieve high potency. Thereby the
      strong interaction with Tyr is stabilized through an
      extensive H-bonding network. The determined crystal
      structures give important insights to characterize the target
      protein and even more they provide the basis for a
      structure-based inhibitor modification. The scaffold of a
      promising 2,6-pyridine ketone derivative was used as starting
      point to improve the structure of the inhibitor for optimal
      binding to 17β-HSD14. In a first optimization strategy, the
      focus was set on the carbonyl group, which only serves to
      induce a V shape of the inhibitor. As no direct interaction
      of this group was observed in the crystal structure, this
      motif was replaced by different substituents. Even though a
      ten-fold higher binding affinity toward 17β-HSD14 was
      observed for one compound bearing a methylene group, all the
      newly synthesized compounds showed a less satisfactory
      selectivity profile toward the types 1 and 2 compared to the
      initial inhibitor. In a second optimization round the
      pyridine core was extended to address a hydrophobic pocket
      observed in the crystal structures. A quinoline-based
      inhibitor was designed and modeled into the active site of
      the target protein prior to its synthesis. The predicted
      binding mode was verified later on with a co-crystal
      structure. The resulting highly active quinoline (Ki = 12 nM)
      was decorated with different substituents showing a varying
      hydrophobicity profile. Four crystal structures of different
      qunioline-based inhibitors in ternary complex with 17β HSD14
      were obtained, revealing the variation of one inhibitor’s
      binding mode due to a small change in its substituents. In
      the new binding mode the previously mentioned phenol group is
      no longer in H-bond distance to Tyr154, which was described
      to be essential to achieve a good affinity toward the target.
      Among others, Tyr154 is forming instead an H-bond network via
      a water molecule with the carbonyl group of the ligand. This
      different binding mode was not related to a significant
      change in binding affinity for this compound (Ki = 12 nM)
      compared to its starting structure. Including the initial
      pyridine ketone derivative of this study, three
      quinoline-based inhibitors show a good selectivity profile
      toward the 17β-HSD enzymes 1, 2 and 10. Furthermore, they
      display very low cytotoxicity and they have no effect on the
      multi-drug resistance protein P-gp. Together with their
      calculated physicochemical properties, two ligands can be
      highlighted as promising tool compounds to investigate the
      physiological role of 17β HSD14 after in vivo administration.
      In the last part of this thesis it was the goal to discover
      new inhibitor scaffolds. Two fragments showing very low
      inhibitory potency toward 17β-HSD14 were identified in a
      fragment-based lead discovery (FBLD) campaign by Nicole
      Bertoletti. In a fragment-growing approach compounds with a
      higher target affinity could be obtained, highlighting this
      method as a promising strategy for the identification of new
      lead structures.