Novel Yeast-based Strategy Unveils Antagonist Binding Regions on the Nuclear Xenobiotic Receptor PXR

The pregnane X receptor (PXR) is a master regulator of xenobiotic metabolism, and its activity is critical toward understanding the pathophysiology of several diseases, including inflammation, cancer, and steatosis. Previous studies have demonstrated that ketoconazole binds to ligand-activated PXR and antagonizes receptor control of gene expression. Structure-function as well as computational docking analysis suggested a putative binding region containing critical charge clamp residues Gln-272, and Phe-264 on the AF-2 surface of PXR. To define the antagonist binding surface(s) of PXR, we developed a novel assay to identify key amino acid residues on PXR based on a yeast two-hybrid screen that examined mutant forms of PXR. This screen identified multiple “gain-of-function” mutants that were “resistant” to the PXR antagonist effects of ketoconazole. We then compared our screen results identifying key PXR residues to those predicted by computational methods. Of 15 potential or putative binding residues based on docking, we identified three residues in the yeast screen that were then systematically verified to functionally interact with ketoconazole using mammalian assays. Among the residues confirmed by our study was Ser-208, which is on the opposite side of the protein from the AF-2 region critical for receptor regulation. The identification of new locations for antagonist binding on the surface or buried in PXR indicates novel aspects to the mechanism of receptor antagonism. These results significantly expand our understanding of antagonist binding sites on the surface of PXR and suggest new avenues to regulate this receptor for clinical applications

In Vivo and In Vitro Characterization of a First-in-Class Novel Azole Analog That Targets Pregnane X Receptor ActivationS⃞

The pregnane X receptor (PXR) is a master regulator of xenobiotic clearance and is implicated in deleterious drug interactions (e.g., acetaminophen hepatotoxicity) and cancer drug resistance. However, small-molecule targeting of this receptor has been difficult; to date, directed synthesis of a relatively specific PXR inhibitor has remained elusive. Here we report the development and characterization of a first-in-class novel azole analog [1-(4-(4-(((2R,4S)-2-(2,4-difluorophenyl)-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)ethanone (FLB-12)] that antagonizes the activated state of PXR with limited effects on other related nuclear receptors (i.e., liver X receptor, farnesoid X receptor, estrogen receptor α, peroxisome proliferator-activated receptor γ, and mouse constitutive androstane receptor). We investigated the toxicity and PXR antagonist effect of FLB-12 in vivo. Compared with ketoconazole, a prototypical PXR antagonist, FLB-12 is significantly less toxic to hepatocytes. FLB-12 significantly inhibits the PXR-activated loss of righting reflex to 2,2,2-tribromoethanol (Avertin) in vivo, abrogates PXR-mediated resistance to 7-ethyl-10-hydroxycamptothecin (SN-38) in colon cancer cells in vitro, and attenuates PXR-mediated acetaminophen hepatotoxicity in vivo. Thus, relatively selective targeting of PXR by antagonists is feasible and warrants further investigation. This class of agents is suitable for development as chemical probes of PXR function as well as potential PXR-directed therapeutics

Alleviation of Gut Inflammation by Cdx2/Pxr Pathway in a Mouse Model of Chemical Colitis

<div><p>Pregnane X Receptor (PXR), a master regulator of drug metabolism and inflammation, is abundantly expressed in the gastrointestinal tract. Baicalein and its <em>O</em>-glucuronide baicalin are potent anti-inflammatory and anti-cancer herbal flavonoids that undergo a complex cycle of interconversion in the liver and gut. We sought to investigate the role these flavonoids play in inhibiting gut inflammation by an axis involving PXR and other potential factors. The consequences of PXR regulation and activation by the herbal flavonoids, baicalein and baicalin were evaluated <em>in vitro</em> in human colon carcinoma cells and <em>in vivo</em> using wild-type, <em>Pxr-</em>null, and humanized (<em>hPXR</em>) PXR mice. Baicalein, but not its glucuronidated metabolite baicalin, activates PXR in a Cdx2-dependent manner <em>in vitro,</em> in human colon carcinoma LS174T cells, and in the murine colon <em>in vivo</em>. While both flavonoids abrogate dextran sodium sulfate (DSS)-mediated colon inflammation <em>in vivo</em>, oral delivery of a potent bacterial β-glucuronidase inhibitor eliminates baicalin’s effect on gastrointestinal inflammation by preventing the microbial conversion of baicalin to baicalien. Finally, reduction of gastrointestinal inflammation requires the binding of Cdx2 to a specific proximal site on the PXR promoter. Pharmacological targeting of intestinal PXR using natural metabolically labile ligands could serve as effective and potent therapeutics for gut inflammation that avert systemic drug interactions.</p> </div

Baicalein, in contrast to baicalin, docks within the PXR ligand binding domain and induces PXR and Cdx2 in LS174T colon carcinoma cells.

<p>(A) Molecular docking of baicalein and baicalin to PXR ligand binding domain (LBD). Figure (i) represent molecular docking of baicalein to PXR LBD. The 2D schematic diagrams shown in (ii, baicalein) and (iii, baicalin) were generated using the LIGX module of MOE program. The binding site residues are colored by their nature, with hydrophobic residues in green, polar residues in purple and charged residues highlighted with bold contours. Blue spheres and contours indicate matching regions between ligand and receptors. Hydrogen bond interactions are shown by green and blue arrows for side chain and main chain interactions, respectively. (B) LS174T cells were exposed to 0.1% DMSO (vehicle) and baicalein (25 µM) or baicalin (25 µM) for 48 hours and total RNA was isolated for PXR mRNA expression analysis by real-time quantitative (RT-qPCR). (C & D) LS174T cells were exposed to different concentrations of baicalein or baicalin as illustrated, for 48 hours and total RNA isolated for Cdx2 mRNA expression analysis by (C) semi-quantitative polymerase chain reaction and by (D) RT-qPCR. β-actin was used as internal control. (E, top panel) Representative western blot of Cdx2 from the same experiment as in (C). (E, bottom panel) Absolute band intensity was quantified for each lanes of the western blot as in figure (E, top panel), using Image J software. Histogram, mean ± SEM. *<i>P</i><.01; ** <i>P</i><.001; ns, not significant.</p

Flavonoids protect against DSS-induced bloody diarrhea, colitis score and inflammatory cytokine expressions.

<p>(A) Mice exposed to DSS, as represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036075#pone-0036075-g003" target="_blank">Figure 3</a> according to the dosing scheme illustrated in upper right panel of (A), were evaluated for the presence of bloody diarrhea after flavonoids treatment. Data plotted as percentage (%) of total mice that had bloody diarrhea on different days of DSS treatment. (B) The histologic score was determined on day 9, when mice were sacrificed. (C & D) Total RNA was isolated from colons of DSS treated mice as in figure (A & B) and analyzed for (C) TNFα and (D) IL-6 mRNA expressions by RT-qPCR. Histogram and data points, mean ± SEM. *<i>P</i><.05; **<i>P</i><.01; ***<i>P</i><.001; ns, not significant.</p

Baicalein, in contrast to baicalin, induces PXR and Cdx2 mRNA.

<p>(A) Scrambled or Cdx2 siRNA (si-Cdx2) transfected LS174T colon cancer cells were exposed to 0.1% DMSO (vehicle), baicalein (25 µM) or baicalin (25 µM) for 48 hours. mRNA levels of PXR were quantified by RT-qPCR. (B, top panel) Representative western blot of PXR from the same experiment as in (A). (B, bottom panel) Absolute band intensity was quantified for each lanes of the western blot as in figure (B, top panel), using Image J software. (C) Scrambled or PXR shRNA (shPXR) transduced LS174T cells, were exposed to 0.1% DMSO (vehicle), baicalein (25 µM) or baicalin (25 µM) for 48 hours. Cdx2 mRNA levels were quantified by RT-qPCR. (D, top panel) Representative western blot of Cdx2 from the same experiment as in (C). (D, bottom panel) Absolute band intensity was quantified for each lanes of the western blot as in figure (D, top panel), using Image J software. Histogram, mean ± SEM. *<i>P</i><.05; ** <i>P</i><.01;*** <i>P</i><.001; ns, not significant.</p