Ekins S , Reschly EJ , Hagey LR , Krasowski MD .

K08-GM074238 NIGMS NIH HHS

	Figure 1. Chemical structures of PXR activators. Chemical structures of the PXR activators 5β-pregnane-3,20-dione, 5α-androstan-3α-ol, 5β-lithocholic acid, 5α-cyprinol 27-sulfate, 3-aminoethylbenzoate, and 6-formylindolo-[3,2-b]-carbozole. The key bond positions are numbered for the steroids and bile salts, and the lettering of the steroidal rings is indicated for pregnanedione and lithocholic acid. The structure to the right of lithocholic acid illustrates the most stable orientation of the A, B, and C steroid rings for 5β-bile salts (like lithocholic acid) with the A/B cis configuration (referring to the relative orientation of the hydrogen atom substituents on carbon atoms 5 and 10). The structure to the right of 5α-cyprinol sulfate shows the most stable orientation of 5α-bile salts (like 5α-cyprinol sulfate) that prefentially adopt the A/B trans configuration.
	Figure 2. PXR activation and steroid pathways. Steroid pathways typical of vertebrates are indicated. (A) Human PXR is activated by a large number of steroid hormones, although typically at micromolar concentrations. The coloring indicates at which concentrations the various steroids activate human PXR (see key in bottom right of panel). (B) Zebrafish PXR is activated by a smaller number of steroid hormones than human PXR, although there is much overlap between the selectivity of the two PXRs. Zebrafish PXR tends to be more sensitive to steroid hormone activation, at least for the functional assay used in this study. The coloring indicates at which concentrations the various steroids activate zebrafish PXR using the same key as in (A). Abbreviations: dehydroepiandrosterone, DHEA; DHEA sulfate; DHEA SO4; dihydrotesterone, DHT.
	Figure 3. Pharmacophore models of PXR activators. Pharmacophore models of PXR activators of (A) human PXR, (B) zebrafish PXR, (C) mouse PXR, (D) rat PXR, (E) rabbit PXR, and (F) chicken PXR. The pharmacophores were generated from the same 16 molecules using Catalyst. The molecules mapped to each pharmacophore are TCDD (green) and 5β-pregnane-3,20-dione (grey). It should be noted that TCDD is inactive in rabbit PXR and only maps to the hydrophobic features. The pharmacophore features are hydrophobic (cyan), hydrogen bond acceptor and vector (green), and excluded volume (grey).
	Figure 4. Maximum likelihood phylogeny of VDRs, PXRs, and CARs. Maximum likelihood phylogeny of 49 amino acid sequences of VDRs, PXRs, and CARs (see Methods for details of analysis). Numbered branch labels indicate bootstrap percentages. Node labels 'AncR1', 'AncR2', and 'AncR3' indicate ancestral nodes that were reconstructed (see Additional files 7 and 8).
	Figure 5. Conservation of ligand-binding residues. From published X-ray crystallographic structures of human VDR, rat VDR, zebrafish VDR, human PXR, human CAR, and mouse CAR (see Methods for references), amino acid residues that interact with ligands ('ligand-binding residues') were identified. At these amino acid residue positions, the sequences of Ciona intestinalis VDR/PXR, AncR1, AncR2, and AncR3 were compared with the corresponding sequence for human PXR, mouse PXR, rat PXR, rabbit PXR, chicken PXR, Xenopus laevis PXRα, Xenopus laevis PXRβ, zebrafish PXR, human CAR, human VDR, and sea lamprey VDR. The ordinate represents the percent identity of Ciona intestinalis VDR/PXR, AncR1, AncR2, and AncR3 for the corresponding sequences of PXRs, VDRs, or CAR at these ligand-binding residue positions.

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