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, 22 (4), 838-57

The Phytoestrogen Coumestrol Is a Naturally Occurring Antagonist of the Human Pregnane X Receptor

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The Phytoestrogen Coumestrol Is a Naturally Occurring Antagonist of the Human Pregnane X Receptor

Hongwei Wang et al. Mol Endocrinol.

Abstract

Antagonizing the action of the human nuclear xenobiotic receptor pregnane X receptor (PXR) may have important clinical implications in preventing drug-drug interactions and improving therapeutic efficacy. We provide evidence that a naturally occurring phytoestrogen, coumestrol, is an antagonist of the nuclear receptor PXR (NR1I2). In transient transfection assays, coumestrol was able to suppress the agonist effects of SR12813 on human PXR activity. PXR activity was assessed and correlated with effects on the metabolism of the anesthetic tribromoethanol and on gene expression in primary human hepatocytes. We found that coumestrol was able to suppress the effects of PXR agonists on the expression of the known PXR target genes, CYP3A4 and CYP2B6, in primary human hepatocytes as well as inhibit metabolism of tribromoethanol in humanized PXR mice. Coumestrol at concentrations above 1.0 microm competed in scintillation proximity assays with a labeled PXR agonist for binding to the ligand-binding cavity. However, mammalian two-hybrid assays and transient transcription data using ligand-binding-cavity mutant forms of PXR show that coumestrol also antagonizes coregulator recruitment. This effect is likely by binding to a surface outside the ligand-binding pocket. Taken together, these data imply that there are antagonist binding site(s) for coumestrol on the surface of PXR. These studies provide the basis for development of novel small molecule inhibitors of PXR with the ultimate goal of clinical applications toward preventing drug-drug interactions.

Figures

Fig. 1.
Fig. 1.
Effects of Coumestrol on Nuclear Receptor Activity A, Structure of the phytoestrogen coumestrol; B, coumestrol activity was assessed across a broad panel of nuclear receptors. Coumestrol was tested at 25 μm and activity plotted as either fold induction relative to basal activity (left axis) or as percentage decrease in basal activity (right axis).
Fig. 2.
Fig. 2.
Effects of Coumestrol Analogs on PXR Activity CV-1 cells were transfected with mouse PXR (pSG5 plasmid) (A) or human PXR (B) and XREM-tk-luciferase reporter vector. Cell extracts were assayed for luciferase activity after drug treatment for 43 h. Drugs included PCN (10 μm), coumestrol (I), coumestrol diacetate (CD) (II), and coumestrol ester (CE) (III) (25 μm each) alone or in combination with PCN. For the human PXR transfection studies, the drugs included rifampicin (10 μm), coumestrol (25 μm), coumestrol diacetate, or coumestrol ester (25 μm each) alone or in combination with rifampicin. Data for each column represent the mean of three independent assays performed in triplicate ± se. *, P > 0.08; **, P > 0.1.
Fig. 3.
Fig. 3.
Effect of Coumestrol on PXR and CAR Activity CV-1 cells were transfected with expression plasmids for human PXR (A) or human CAR (B) and the XREM-tk-Luciferase reporter vector. Cell extracts were assayed for luciferase activity after compound treatment for 24 h. In the PXR antagonist assay, 300 nm SR12813 was added to increase the basal activity of the reporter to approximately 70% of maximal activity. A full dose-response curve is shown for coumestrol in this assay. Data at each point represent the means of assays performed in triplicate and are plotted relative to the maximal response relative to SR12813 (the ±se was less than 0.05% for all data points plotted). The mean IC50 of coumestrol was 11.6 μm for PXR in these assays. In the CAR inverse agonist assay (did not require addition of agonist to increase basal activity), coumestrol demonstrated an EC50 of 29.6 μm.
Fig. 4.
Fig. 4.
Binding Assays A, Purified human PXR bound to SPA beads was incubated in the presence of 10 nm PXR agonist [3H]NMTB in the presence of increasing concentrations of unlabeled SR12813 (⋄) or coumestrol (▪). Displacement of [3H]NMTB was measured in a Wallac Microbead counter. Data are expressed as a percentage of bound [3H]NMTB in the absence of competitor. A Ki of 37 nm was observed for SR12813 and a Ki of 13 μm for coumestrol. B, Purified human CAR LBD bound to SPA beads was incubated with 10 nm [3H]clotrimazole in the presence of increasing concentrations of unlabeled clotrimazole (•) or coumestrol (▴). A Ki of 250 nm was observed for clotrimazole, and a Ki of 54 μm was observed for coumestrol.
Fig. 5.
Fig. 5.
Effect of Coumestrol on PXR Mutant Activity A and B, CV-1 cells were transfected with expression plasmids for wild-type human PXR or PXR mutant (S247W) and the XREM-tk-Luciferase (A) or tk-MRP2-luc (B) reporter plasmid. Cell extracts were assayed for luciferase activity after compound treatment for 24 h. Drugs included rifampicin (10 μm) or coumestrol (25 μm) alone or in combination with rifampicin and/or T1317 (1 μm) or 17β-estradiol (20 μm). Data for each column represent the mean of two independent assays each performed in quadruplicate ± se (*, P > 0.8; **, P < 0.003; ***, P < 0.001). LBP, Ligand-binding pocket. C and D, CV-1 cells were transfected with expression plasmids for Gal4-PXR or Gal4-PXR mutant (S247W) (C) or Gal4-VP16 fusion construct or Gal4-PXR double mutant (S247W/C287W) (D) and treated with drug(s) as shown. E, The identical experiments as shown in D were performed in HEK293T cells. C, Top panel, Data for each column represent the mean of two independent assays each performed in triplicate ± se (*, P < 0.001; **, P < 0.0001); bottom panel, data for each column represent the mean of three independent assays each performed in triplicate ± se (***, P > 0.2). F, Same experiment as described in D. The PXR expression plasmid is Gal4-PXR (I) double mutant (S247W/S208W) or (II, III) triple mutant (S247W/S208W/C284W). D–F, Data for each column represent the mean of three independent assays each performed in triplicate ± se (*, P > 0.1;**, P < 0.0001; #, P < 0.002; ***, P < 0.01; ****, P < 0.003; ##, P < 0.002; @, P > 0.3). Illustration in F, S247W is a mutation that is known to lead to ligand-independent activation of PXR (30 ). It also fills a significant portion of the ligand-binding pocket of the receptor. Combining that mutation with either S208W or C284W will effectively fill the ligand-binding pocket of PXR, leaving room insufficient to allow even the smallest established ligands (like SR12813) from binding to the receptor.
Fig. 6.
Fig. 6.
Effect of Coumestrol on PXR-Mediated Transcription and Binding of Coactivator SRC-1 with PXR CV-1 cells were cotransfected with expression plasmids for human PXR and the XREM-tk-Luciferase reporter vector (A) or human FL PXR-VP16 plasmid (B) and Gal4-SRC-1 receptor interacting domain (RID), and UASg-Tk-luc reporter. Cell extracts were assayed for luciferase activity after compound treatment for 24 h. Cell extracts were assayed for luciferase activity after drug treatment for 48 h. Drugs included rifampicin (10–30 μm) and coumestrol (25 μm). Data for each column represent the mean of three independent assays performed in triplicate ± se. *, P > 0.1; **, P > 0.3.
Fig. 7.
Fig. 7.
Effect of Coumestrol on Binding of Coactivator SRC-1 with PXR Mammalian two-hybrid assays were performed in HEK293T cells to study the interaction of human PXR with SRC-1 in the presence or absence of drug(s). The cells were transfected with human FL PXR-VP16 plasmid (A) or human LBD PXR-VP16 plasmid (B) and Gal4-SRC-1 receptor interacting domain (RID), and UASg-Tk-luc reporter. Cell extracts were assayed for luciferase activity after drug treatment for 48 h. Drugs included rifampicin (10 μm), coumestrol, and coumestrol diacetate (25 μm each). Data for each column represent the mean of two independent assays performed in triplicate ± se. *, P < 0.05.
Fig. 8.
Fig. 8.
Effect of Coumestrol on Subnuclear Localization of PXR in Wild-Type Mouse Liver The photomicrographs show the effect of drug(s) on mouse liver tissue expressing PXR. Three mice from each treatment group were killed after completing LORR studies (see Materials and Methods and Fig. 12). A, Liver tissue was immunoassayed for PXR. The remaining panels show controls: B, no primary antibody; C, PXR protein; D, H&E stain. Fifteen sections of each liver were made and stained. The picture is the best representative section of all stained slides.
Fig. 9.
Fig. 9.
Effect of Coumestrol on Subnuclear Localization of PXR in Humanized Mouse Liver The photomicrographs show the effect of drug(s) on mouse liver tissue expressing human PXR. The same numbers of mice as in Fig. 8 were killed. A, Liver tissue was immunoassayed for human PXR. The remaining panels show controls: B, no primary antibody; C, PXR protein; D, H&E stain. Fifteen sections of each liver were made and stained. The picture is the best representative section of all stained slides.
Fig. 10.
Fig. 10.
Effect of Coumestrol on Subnuclear Localization of PXR The photomicrographs show the effect of drug(s) on HEK293T cells expressing GFP (A) or GFP full-length human PXR (B) and/or SRC-1 (D and E). Twenty-four hours after transfection, 293T cells were treated with vehicle (0.2% DMSO), rifampicin (R) 10 μm, coumestrol (C) 25 μm, or both drugs for 4 h in serum-free media. The cells were fixed with 10% paraformaldehyde for 20 min and then mounted using mounting medium containing 4′,6-diamidino-2-phenylindole stain. The cells were imaged by confocal microscopy at ×40 magnification using the appropriate fluorescent channels. For quantification of granular or bright speckled nuclei (B and E, respectively), 60 cells (about four to five cells per high-power field scanning a total of 15 fields) were randomly counted for each experiment, and a representative picture from three independent experiments is shown. *, P > 0.2; **, P > 0.1; ***, P < 0.001.
Fig. 11.
Fig. 11.
Effects of Coumestrol on CYP3A4 and CYP2B6 Expression Total mRNA was isolated from two donors of primary human hepatocytes and treated with vehicle (V, DMSO), coumestrol (C, 25 μm), rifampicin (R10 μm), coumestrol plus rifampicin (R+C, 25 and 10 μm, respectively), SR12813 (S, 1 μm), and coumestrol plus SR12813 (S+C, 25 and 1 μm, respectively). After Northern blotting, the RNAs from were probed with a fragment of the human CYP3A4 coding region (donors 1 and 2) or CYP2B6 (donor 2) and visualized by autoradiography. Relative mRNA abundance (normalized for β-actin mRNA levels) is plotted below each autoradiogram.
Fig. 12.
Fig. 12.
Effect of Coumestrol and Its Analogs on Anesthetic (Tribromoethanolamine) Metabolism in Vivo A and B, LORR duration assessments to determine the effect of coumestrol and its analogs, diacetate and dimethyl ether, in PXR (+/+) (A) and PXR (−/−) (B) mice. Six- to 8-wk-old C57BL/6 female mice (n = 5 per treatment group) were treated with vehicle or PCN or coumestrol, coumestrol diacetate, or the combination of PCN plus coumestrol or its analog (see Materials and Methods for details). On d 6, all mice underwent LORR duration assessments as described previously (23 ). Columns represent the mean and error bars ± se. *, P < 0.001; **, P > 0.3; ***, P > 0.1.

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