• Computational investigation of the binding mode of bis(hydroxylphenyl)arenes in 17β-HSD1: molecular dynamics simulations, MM-PBSA free energy calculations, and molecular electrostatic potential maps.

      Negri, Matthias; Recanatini, Maurizio; Hartmann, Rolf W; Pharmaceutical and Medicinal Chemistry, Saarland University and Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus C2.3, 66123 Saarbrücken, Germany. (2011-09)
      17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the last step of the estrogen biosynthesis, namely the reduction of estrone to the biologically potent estradiol. As such it is a potentially attractive drug target for the treatment of estrogen-dependent diseases like breast cancer and endometriosis. 17β-HSD1 belongs to the bisubstrate enzymes and exists as an ensemble of conformations. These principally differ in the region of the βFαG'-loop, suggesting a prominent role in substrate and inhibitor binding. Although several classes of potent non-steroidal 17β-HSD1 inhibitors currently exist, their binding mode is still unclear. We aimed to elucidate the binding mode of bis(hydroxyphenyl)arenes, a highly potent class of 17β-HSD1 inhibitors, and to rank these compounds correctly with respect to their inhibitory potency, two essential aspects in drug design. Ensemble docking experiments resulted in a steroidal binding mode for the closed enzyme conformations and in an alternative mode for the opened and occluded conformers with the inhibitors placed below the NADPH interacting with it synergically via π-π stacking and H-bond formation. Both binding modes were investigated by MD simulations and MM-PBSA binding free energy estimations using as representative member for this class compound 1 (50 nM). Notably, only the alternative binding mode proved stable and was energetically more favorable, while when simulated in the steroidal binding mode compound 1 was displaced from the active site. In parallel, ab initio studies of small NADPH-inhibitor complexes were performed, which supported the importance of the synergistic interaction between inhibitors and cofactor.
    • Structural basis for species specific inhibition of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1): computational study and biological validation.

      Klein, Tobias; Henn, Claudia; Negri, Matthias; Frotscher, Martin; Pharmaceutical and Medicinal Chemistry, Saarland University, Saarbrücken, Germany. (2011)
      17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the reduction of estrone to estradiol, which is the most potent estrogen in humans. Inhibition of 17β-HSD1 and thereby reducing the intracellular estradiol concentration is thus a promising approach for the treatment of estrogen dependent diseases. In the past, several steroidal and non-steroidal inhibitors of 17β-HSD1 have been described but so far there is no cocrystal structure of the latter in complex with 17β-HSD1. However, a distinct knowledge of active site topologies and protein-ligand interactions is a prerequisite for structure-based drug design and optimization. An elegant strategy to enhance this knowledge is to compare inhibition values obtained for one compound toward ortholog proteins from various species, which are highly conserved in sequence and differ only in few residues. In this study the inhibitory potencies of selected members of different non-steroidal inhibitor classes toward marmoset 17β-HSD1 were determined and the data were compared with the values obtained for the human enzyme. A species specific inhibition profile was observed in the class of the (hydroxyphenyl)naphthols. Using a combination of computational methods, including homology modelling, molecular docking, MD simulation, and binding energy calculation, a reasonable model of the three-dimensional structure of marmoset 17β-HSD1 was developed and inhibition data were rationalized on the structural basis. In marmoset 17β-HSD1, residues 190 to 196 form a small α-helix, which induces conformational changes compared to the human enzyme. The docking poses suggest these conformational changes as determinants for species specificity and energy decomposition analysis highlighted the outstanding role of Asn152 as interaction partner for inhibitor binding. In summary, this strategy of comparing the biological activities of inhibitors toward highly conserved ortholog proteins might be an alternative to laborious x-ray or site-directed mutagenesis experiments in certain cases. Additionally, it facilitates inhibitor design and optimization by offering new information on protein-ligand interactions.