11β-HSD inhibitors pharmacophore model.

<p>The hydrogen bond acceptor features are represented in green, and the hydrophobic features in blue. The shape of carbenoxolone, as a steric constraint to prevent too large molecules from fitting, is shown as a grey volume.</p

Binding of aldosterone and the silane AB110873 to the MR.

<p><i>A</i>, MR pharmacophore model with the cocrystallized ligand aldosterone. <i>B,</i> the silane AB110873 fitted to the MR ligand binding site with the pharmacophore model. Hydrogen bond acceptors are shown as red arrows, hydrophobic areas as yellow spheres. Exclusion volumes are not shown for clarity. Receptor binding pocket is colored by aggregated lipophilicity (grey)/hydrophilicity (blue).</p

Effect of the silane AB110873 on the activation of mineralocorticoid (MR) and glucocorticoid receptors (GR).

<p>The impact of silane AB110873 on MR and GR transactivation was measured in HEK-293 cells transfected with MMTV-LacZ reporter, MR (<i>A,B</i>) or GR (<i>C</i>) and luciferase transfection control. Cells were incubated with the receptor ligands for 24 h, followed by analysis of reporter activity as given in the Methods section. <i>A</i>, Concentration-dependent activation of the human MR by the silane AB110873. Maximal activation at 30 µM was set as 100%. <i>B</i>, effect of the antagonist spironolactone (1 µM) on MR activation by aldosterone (5 nM) or by the silane AB110873 (20 µM); <i>C</i>, activation of human GR by cortisol (100 nM) and effect of AB110873 (20 µM) and antagonist RU-486 (1 µM) (MR and GR activity of the vehicle control was set as 1, data represent fold activation). Data were obtained from four independent experiments, each performed in triplicates (mean ± SD).</p

AB110873 binds to both 11β-HSD2 and MR.

<p>Proposed binding modes of AB110873 in 11β-HSD2 (<i>A,B</i>) and in the MR (<i>C,D</i>). In the 3D-figures, selected amino acid residues are highlighted with ball and stick, and the receptor binding pockets are colored by aggregated lipophilicity (grey)/hydrophilicity (blue). The 2D figures represent the binding interactions, color-coded as following: red arrow – hydrogen bond acceptor, yellow – hydrophobic interaction.</p

Inhibition of 11β-HSD1 and 11β-HSD2 by compounds predicted by virtual screening.

<p>11β-HSD1-dependent conversion of cortisone (200 nM cortisone, 500 µM NADPH) to cortisol was determined by incubating lysates of HEK-293 cells stably expressing the human enzyme in the presence of vehicle or 20 µM of the respective compound for 10 min at 37°C. Similarly, 11β-HSD2-dependent conversion of cortisol (50 nM cortisol, 500 µM NAD⁺) to cortisone was measured. Data represent mean ± SD from at least three independent experiments.</p><p>T0504 and fenofibrate were used as synthetic positive controls, glycyrrhetinic acid as natural product positive control. N.D., not detectable (complete inhibition).</p

Schematic overview of corticosteroid receptor regulation by 11β-HSD enzymes.

<p>Schematic overview of corticosteroid receptor regulation by 11β-HSD enzymes.</p

Increased mitochondrial superoxide generation and induction of IL-6 production upon MR activation in BV2 cells.

<p>BV2 microglial cells were incubated for 24 h with increasing concentrations of aldosterone (<i>A</i>) or silane AB110873 (<i>B</i>) in the presence or absence of 1 µM spironolactone, followed by determination of mitochondrial superoxide using MitoSox staining as described in the Methods section. Fluorescence was analyzed using a Cellomics ArrayScan high-content screening system. <i>C</i>, BV2 cells were incubated for 24 h with aldosterone (5 nM) or various concentrations of silane AB110873 in the presence or absence of spironolactone, followed by quantification of IL-6 protein levels in the medium of the cultured cells by ELISA. Data (mean ± SD) were obtained from three independent experiments each performed in six replicates.</p

MR with silane AB110873.

<p>The amino acids that form the hydrogen bond network with steroidal agonists are highlighted in ball and stick style. AF-2 (helix 12) is colored in dark blue, helix 5 in green, helix 3 in light blue, and the β-sheet in lilac. The buried hydrophobic pocket additionally occupied by AB110873 is formed by amino acids from helices 3 (Val780) and 5 (Leu809, Ala813), and from the β-sheet (Val750).</p

Inhibition of human 11β-HSD2 by AB110873 and lasalocid.

<p>Concentration-dependent inhibition of 11β-HSD2 by the silane-coupling agent AB110873 (<i>A</i>) and the anti-biotic lasalocid (<i>B</i>) measured in cell lysates of stably transfected HEK-293 cells. Data represent mean ± SD from three independent experiments.</p