Conicasterol E, a Small Heterodimer Partner Sparing Farnesoid X Receptor Modulator Endowed with a Pregnane X Receptor Agonistic Activity, from the Marine Sponge <i>Theonella swinhoei</i>

We report the isolation and pharmacological characterization of conicasterol E isolated from the marine sponge <i>Theonella swinhoei.</i> Pharmacological characterization of this steroid in comparison to CDCA, a natural FXR ligand, and 6-ECDCA, a synthetic FXR agonist generated by an improved synthetic strategy, and rifaximin, a potent PXR agonist, demonstrated that conicasterol E is an FXR modulator endowed with PXR agonistic activity. Conicasterol E induces the expression of genes involved in bile acids detoxification without effect on the expression of small heterodimer partner (SHP), thus sparing the expression of genes involved in bile acids biosynthesis. The relative positioning in the ligand binding domain of FXR, explored through docking calculations, demonstrated a different spatial arrangement for conicasterol E and pointed to the presence of simultaneous and efficient interactions with the receptor. In summary, conicasterol E represents a FXR modulator and PXR agonist that might hold utility in treatment of liver disorders

Theonellasterol is a selective FXR antagonist.

<p>(A) (C) and (D) HepG2 cells were co-transfected with the Gal4 luciferase reporter and a series of chimeras in which the Gal4 DNA binding domain is fused to the LBD of the indicated nuclear receptors. Cells were treated with the appropriate agonists or specific agonists in combination with <i>theonellasterol</i>. (B) HepG2 cells were co-transfected with pSG5-PXR, pSG5-RXR and with the reporter pCYP3A4promoter-TKLuc and then stimulated with rifaximin, a PXR agonist, alone or in combination with <i>theonellasterol</i>. Data are the mean ± S.E. of three experiments. *P<0.05 versus not treated cells. (E) Microarray analysis showing the relative mRNA expression of various nuclear receptors and nuclear receptors co-activators following stimulation of HepG2 cells with CDCA, 10 µM, alone or in combination with <i>theonellasterol</i>, 50 µM. Data are the mean ± S.E. of three experiments.</p

Theonellasterol from <i>Theonella swinhoei</i>.

<p>(A) Chemical structure of theonellasterol isolated from <i>Theonella swinhoei</i>. (B) Superimposition of the different docking poses of theonellasterol in the rat FXR (theonellasterol and 1OSV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030443#pone.0030443-Mi1" target="_blank">[20]</a> yellow), and human FXRs (theonellasterol and 3BEJ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030443#pone.0030443-Soisson1" target="_blank">[15]</a> orange; theonellasterol and 1OSH <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030443#pone.0030443-Downes1" target="_blank">[17]</a> pink; theonellasterol and 3DCT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030443#pone.0030443-AkwabiAmeyaw1" target="_blank">[18]</a> purple; theonellasterol and 3RUU <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030443#pone.0030443-AkwabiAmeyaw2" target="_blank">[19]</a> light blue). (C) Superimposition of theonellasterol (yellow) with 6-ECDCA (sky blue), and Z (pink)/ E (light pink) gugglusterone in the binding pocket of FXR (1OSV). Amino acids interacting with theonellasterol (yellow) are depicted in green, amino acids interacting with 6-ECDCA and theonellasterol are depicted in sky blue, amino acids interacting with Z/E gugglusterone and theonellasterol are depicted light pink, amino acids interacting with 6-ECDCA and Z/E gugglusterone in red, and amino acids interacting with all molecules are depicted in blue.</p

<i>Theonellasterol</i> alters the liver expression of genes involved in bile acid metabolism in BDL mice.

<p>(A) Real-Time PCR analysis of OSTα, (B) BSEP, (C) SHP and (D) MRP4. Data are the mean ± S.E. of 4 mice per group. *P<0.05 versus sham. **P<0.05 versus BDL. #P<0.05 versus BDL plus <i>theonellasterol</i>.</p

Theonellasterol is an FXR antagonist.

<p>Luciferase reporter assay performed in HepG2 transiently transfected with pSG5-PXR, pSG5-RXR, pCMV-βgal, pCYP3A4promoter-TKLuc vectors and stimulated 18 h with (A) 10 µM of CDCA or <i>theonellasterol</i> and (B) 10 µM of CDCA alone or in combination with <i>theonellasterol</i> 50 µM. Data are the mean ± S.E. of three experiments. *P<0.05 versus cells left untreated. #P<0.05 versus CDCA. (C) CHiP assay of NCoR binding to the OSTα promoter. CDCA displaces NCoR from OSTα and this effect is reversed by theonellasterol. RT-PCR analysis of proteins immune-precipitated with a control IgG are shown as control. Data are the mean ± S.E. of three experiments. *P<0.05 versus anti IgG immunoprecipitates. #P<0.05 CDCA versus cells left untreated; P<0.05 <i>thenollasterol</i> versus CDCA alone.</p

Inhibition of the ATPase activity of Hsp90 by different concentration of compounds 1–4.

<p>Radicicol and 17-AAG were used as positive controls. Data are the mean of two independent experiments performed in triplicate and were analyzed by t test (Hsp90 vs Hsp90+ testing compound): *P<0.05, **P<0.005.</p

Schematic representation of the results obtained from limited proteolysis experiments.

<p>The preferential cleavage sites detected on recombinant Hsp90, and on the Hsp90/<b>1</b> complex are in black. The Hsp90 N-terminal domain is highlighted in light grey, the middle domain is boxed and the C-terminal domain is highlighted in grey.</p

Theonellasterol reverses the effect of CDCA on the expression of canonical FXR target genes.

<p>Relative mRNA expression of (A) OSTα, (B) BSEP, (C) SHP and (D) MRP4 in HepG2 cells treated with 10 µM CDCA alone or with the combination of CDCA plus <i>theonellasterol</i> 50 µM. (E) CHiP assay performed in HepG2 cells not stimulated or primed with CDCA, 10 µM, alone or in combination with <i>theonellasterol</i>, 50 µM. Theonellasterol antagonizes the recruitment of FXR on the MRP4 promoter. Data are the mean ± S.E. of three experiments. *P<0.05 versus cells left untreated. #P<0.05 versus CDCA alone.</p

BDL causes liver cell injury and theonellasterol attenuates liver necrosis.

<p>(A and B) Liver necrosis was robustly attenuated by <i>theonellasterol</i> as confirmed by assessment of ALT and histopathology analysis. Data are the mean ± S.E. of 6 mice per group. *P<0.05 versus sham. **P<0.05 versus BDL. #P<0.05 versus BDL plus <i>theonellasterol</i>. (C) Representative liver histology from an individual mice per group. Liver sections were stained with H&E, original magnification 10×.</p

SPR analysis results.

<p>Sensorgrams obtained by injecting different concentrations (from 0.020 to 1 <i>µ</i>M) of <b>1</b> (A), <b>2</b> (B), <b>3</b> (C), <b>4</b> (D), <b>5</b> (E), <b>6</b> (F) <b>7</b> (G) and radiciol (H) on immobilized Hsp90.</p