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Center for Pharmacogenetics (Y.M., S.P.S.S., D.T., S.R., W.X.), Department of Pharmaceutical Sciences (Y.M., S.P.S.S., D.T., S.R., B.W.D., W.X.), Department of Chemistry (C.R.J.S., C.K., B.W.D., P.W.), Center for Chemical Methodologies and Library Development (C.R.J.S., C.K., P.W.), Department of Pathology (H.C., S.C.S.), and Department of Pharmacology (W.X.), University of Pittsburgh, Pittsburgh, Pennsylvania
Received March 28, 2005; accepted May 4, 2005
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Abstract |
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; Kliewer et al., 1998; Xie et al., 2000a). Subsequent functional analysis revealed a much broader role of PXR in xenobiotic regulation. PXR can function as a master regulator in regulating phase I and II enzymes (Falkner et al., 2001; Sonoda et al., 2002; Xie et al., 2003), as well as members of the drug transporter family. This broad regulation is due to the presence of PXR-responsive elements in the promoter regions of many xenobiotic enzyme and transporter genes (for review, see Xie et al., 2004). This comprehensive role of PXR in xenobiotic regulation is supported by several DNA microarray-based gene profiling analyses performed in wild-type, transgenic, and knockout mouse models (Ueda et al., 2002; Rosenfeld et al., 2003). Although the initial functional characterization of PXR has focused on the liver and intestine, recent studies have revealed a broader extrahepatic role of PXR, such as the PXR-mediated regulation of P-glycoprotein expression and transport function at the blood-brain barrier (Bauer et al., 2004).
Even though PXR was identified as a "xenobiotic receptor", emerging evidence has suggested PXR as a potential therapeutic target for several human diseases (Xie et al., 2004). This disease relevance is consistent with the notion that many of the PXR target genes are involved in the biotransformation and homeostasis of endogenous and exogenous chemicals that may influence physiological and pathological processes in mammals. For example, activation of PXR in mice has been shown to promote bilirubin clearance and prevent hyperbilirubinemia (Xie et al., 2003). This beneficial effect has been attributed to activation by PXR of bilirubin detoxifying genes, such as the conjugating enzymes UGT1A1 and the excretion transporter MRP2 (Sugatani et al., 2001; Kast et al., 2002; Huang et al., 2003; Xie et al., 2003).
PXR is also important for the prevention of bile acid toxicity. Bile acids are major by-products of cholesterol catabolism in the liver. Despite their beneficial role in solubilizing and absorbing lipids, accumulation of bile acids can cause irreversible liver damage, resulting in cholestasis. Both pharmacological and genetic activation of PXR in mice has been shown to confer resistance to lithocholic acid (LCA) hepatotoxicity (Staudinger et al., 2001; Xie et al., 2001). This protective effect may be due to a PXR-mediated combined induction of CYP3A (Xie et al., 2001) and the hydroxysteroid sulfotransferase (Sonoda et al., 2002). It is important to note that the constitutive androstane receptor (CAR) has also been shown to be important in the clearance of bilirubin and bile acids via overlapping yet distinct mechanisms (Huang et al., 2003; Xie et al., 2003; Saini et al., 2004). The vitamin D receptor-mediated induction of CYP3A is also suggested to promote bile acid detoxification when this receptor is activated by vitamin D receptor agonists or bile acids (Thummel et al., 2001; Makishima et al., 2002).
In this report, using the combination of chemical library development with functional assays, we report the identification of a novel class of PXR agonists from a library of synthetic peptide bond mimetics. S20, a C-cyclopropylalkylamide, was identified as an efficacious PXR agonist. It is interesting that although racemic S20 activated both human PXR (hPXR) and mouse PXR (mPXR), the two enantiomers of S20 exhibited striking species-specific PXR activation. This species-dependent stereoselectivity may provide guidance to avoid or to achieve species-specific xenobiotic receptor activation during pharmaceutical development.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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; Wipf et al., 2001, 2003). 1,4-bis[2-(3,5-Dichloropyridyloxy)] benzene (TCPOBOP) was a gift from Dr. Stephen Safe (Texas A&M, College Station, TX). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
Plasmids and Transient Transfection. The reporter plasmids (tk-USA-Luc, tk-CYP3A4-Luc, tk-CYP3A23-Luc, tk-MRP2-Luc, and tk-EcRE-Luc) and the expression vectors [Gal-hPXR LBD, Gal-mPXR LBD, CAR, and farnesoid X receptor (FXR)] have been described previously (Xie et al., 2000b; Sonoda et al., 2002; Saini et al., 2004). The creation of rat PXR (rPXR) F305L and hPXR L308F mutants (gifts from Dr. Richard Kim) was described previously (Tirona et al., 2004). All reporter genes contain three copies of the corresponding response elements. Transfections were performed on 48-well plates. CV-1 cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as described previously (Xie et al., 2000b). HepG2 cells were transfected using polyethylenimine polymer (kindly provided by Dr. Xiang Gao). Transfected cells were treated with appropriate compounds for 24 h before harvesting and assay for luciferase activity. Luciferase activity was normalized against the cotransfected and 
-galactosidase activity. All transfections were performed in triplicate.
Human and Mouse Primary Hepatocyte Preparation and Treatment. Human livers were obtained through the Liver Tissue Procurement and Distribution System, and hepatocytes were isolated by three-step collagenase perfusion (Strom et al., 1996). Mouse hepatocytes were also prepared by collagenase perfusion. Cells were plated on gelatin-coated T25 flasks and maintained in hepatocyte maintenance medium (Cambrex Bio Science Walkersville, Inc., Walkersville, MD) supplemented with 0.1 mM dexamethasone, 0.1 mM insulin, 50 µg/ml gentamicin, 50 ng/ml amphotericin, and incubated overnight. Cells were treated with appropriate drugs for 48 h before RNA harvest and Northern blot analysis. The drug treatment was reduced to 24 h when cycloheximide was added.
Animals and Drug Treatment. The PXR null mice and "humanized" hPXR transgenic mice have been described previously (Xie et al., 2000a). Mice were maintained with food and water available ad libitum. When necessary, mice were subjected to a single intraperitoneal injection of 50 mg/kg S20 24 h before sacrifice and tissue harvesting. The use of mice in this study complied with all relevant federal guidelines and institutional policies.
Northern Blot Analysis. Total RNA was isolated from mouse tissues or primary hepatocytes using the TRIzol reagent (Invitrogen). Northern blot analysis was performed as described previously (Xie et al., 2000a). The cDNA probe of the human CYP3A4 and UGT1A1 was cloned by reverse transcription-polymerase chain reaction from human liver mRNA. When necessary, quantification was performed with the NIH Image software.
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Results |
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; Wipf et al., 2001, 2003; Janjic et al., 2005). The library was built to examine the scope of diversity-oriented chemical synthesis offered by the three-component aldimine condensation reaction, as well as to explore new scaffolds for biological activity. The effects of the library compounds on PXR were evaluated with a chimeric receptor in which the ligand-binding domain (LBD) of hPXR was fused to the DNA binding domain of the yeast transcription factor Gal4. The activity of PXR was determined using a Gal4-responsive reporter tk-UAS-Luc that contains three copies of the Gal4 binding site. The reporter gene -fold activations elicited by library compounds (at 10 µM) are summarized in Table 1. The chemical and biological information contained in Table 1 allows some general analysis of structure-activity relationship (SAR) for the library compounds as PXR agonists (see Discussion). It is noteworthy that S20, a C-cyclopropylalkylamide exhibited the highest reporter gene -fold activation among the 73 library compounds. S20 has three asymmetric carbons and can be resolved into two enantiomers, (+)-S20 and (-)-S20 (Fig. 1, A and B). The carbon scaffold of S20 is distinct from the chemical structures of known PXR agonists, such as the hPXR-specific RIF (Fig. 1C), and the rodent-specific pregnenolone-16
-carbonitrile (PCN) (Fig. 1D).
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), the S20 enantiomers were prepared separately and evaluated for PXR activation using the Gal-PXR transfection systems. The potential species preference was evaluated by comparing the reporter activity elicited by Gal-hPXR with that elicited by Gal-mPXR. Although the racemic S20 activated both hPXR and mPXR, the activation of PXR by the two enantiomers was remarkably species-dependent (Fig. 2, A and B). As shown in Fig. 2A, when Gal-hPXR was used, enantiomer (+)-S20 showed a modestly higher activity than the racemic S20. In sharp contrast, enantiomer (-)-S20 was a weak hPXR activator, inducing the reporter only 2-fold. The opposite enantiomorphic preference was observed when Gal-mPXR was used. The activity of (+)-S20 on mPXR was less than half of that of racemic S20, whereas (-)-S20 was fully active (Fig. 2B). The stereoselectivity was further evaluated in primary hepatocyte cultures. In human hepatocytes, (+)-S20 was a potent CYP3A4 mRNA inducer, whereas (-)-S20 had little effect (Fig. 2C). When wild-type mouse hepatocytes were used (-)-S20, but not (+)-S20, induced CYP3A11 expression (Fig. 2D). RIF and PCN were included to verify the hepatocyte responsiveness. We conclude that the activation of hPXR and mPXR by S20 shows opposing enantioselectivity. Note that the respective induction of CYP3A4 and CYP3A11 in the human and mouse hepatocytes was sustained in the presence of 1 µg/ml of the protein synthesis inhibitor cycloheximide (Fig. 2, E and F). This concentration of cycloheximide has been shown to inhibit protein synthesis in primary hepatocytes (Kotokorpi et al., 2004), suggesting that the S20-mediated CYP3A mRNA induction does not require new protein synthesis.
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S20 Is an Efficacious PXR-Specific Agonist that Induces Coactivator Recruitment. The efficacy of racemic S20 and its enantiomers as hPXR agonists was measured and compared with RIF using the Gal-hPXR transfection system. As shown in Fig. 3A, both S20 and RIF activated hPXR in a concentration-dependent manner. Compared with RIF, racemic S20 was more potent but had a similar EC50 of 2 µM. The enantiomer (+)-S20, on the other hand, was not only more potent but also more efficacious than RIF, with an estimated EC50 of 400 nM. The superior potency and efficacy of (+)-S20 compared with RIF was also confirmed when the wild-type hPXR and a PXR-responsive reporter gene (tk-CYP3A4-Luc) were used in the transfection (Fig. 3B). The ability of (-)-S20 to activate mPXR was compared with that of PCN, a prototypical mPXR-specific agonist. Although (-)-S20 is preferred by mPXR (Fig. 2B), this enantiomer had a similar potency and efficacy in activating the wild-type mPXR and inducing tk-CYP3A23-Luc, another PXR-responsive reporter gene (Fig. 3C). The approximate equivalency of (-)-S20 in mPXR activation (compared with PCN) was also confirmed in an independent Gal-mPXR transfection assay (data not shown).
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A hallmark of ligand-dependent activation of nuclear receptors is the recruitment of p160 nuclear receptor coactivators, such as the steroid receptor coactivator 1 (SRC-1). The Gal-hSRC-1 receptor interaction domain transfection system was used to examine whether treatment with S20 caused interaction between hSRC-1 and PXR. As shown in Fig. 3D, cotransfection of VP-mPXR or VP-hPXR, in the absence of agonists, induced the reporter activity by 2- to 3-fold, consistent with our recent report (Saini et al., 2005). The PXR-hSRC-1 interaction was enhanced by the treatment with racemic S20. RIF and PCN were also analyzed to verify the responsiveness and specificity of this coactivator recruitment assay.
S20 was a PXR-specific agonist, in that it had no effect on the activity of CAR and FXR. When mCAR was cotransfected with the CAR-responsive tk-MRP2-Luc reporter, racemic S20 did not alter the constitutive activity of CAR (Fig. 3E). In the same transfection, CAR activity was inhibited by androstenol and increased by TCPOBOP as expected (Fig. 3E). Neither the racemic nor the enantiomerically pure S20 activated hCAR in a similar transfection assay (data not shown). Likewise, FXR was activated by chenodeoxycholic acid (CDCA), as expected, but not by racemic S20, when the FXR-responsive tk-EcRE-Luc reporter was cotransfected (Fig. 3F).
S20 Induces the Expression of Drug-Metabolizing Enzymes and Transporters in Reporter Gene Assays, in Human Hepatocytes, and in "Humanized" Mice. PXR is known to regulate CYP3A genes (Blumberg et al., 1998; Kliewer et al., 1998). To examine whether or not the potential PXR ligands can bind and activate PXR to induce CYP3A gene expression, we performed an independent ligand activation assay using the full-length mPXR or hPXR and the CYP3A reporter gene tk-CYP3A4-Luc. This reporter contains three copies of the ER-6 type PXR response element derived from the CYP3A4 gene (Blumberg et al., 1998). In both mPXR- and hPXR-transfected cells, the tk-CYP3A4 reporter was activated by racemic S20 (Fig. 4A; hPXR data not shown). Activation of PXR by racemic S20 also induced the expression of tk-MRP2-Luc (Fig. 4B), a PXR-responsive reporter that contains three copies of the ER-8 type element derived from the drug transporter MRP2 gene.
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The capability of S20 to induce PXR target phase I and II enzymes was also evaluated in human hepatocytes. As shown in Fig. 4C, compared with vehicle-treated cells, treatment of hepatocytes (from donor HH1052) with 5 µM racemic S20 induced the mRNA expression of both CYP3A4 and UGT1A1 as revealed by Northern blot analysis. The potency of induction was comparable with that induced by 10 µM RIF. As expected, PCN did not induce the expression of either enzyme. Concentration-dependent induction of CYP3A4 and UGT1A1 by S20 was also observed in hepatocytes prepared from another donor (HH1119) (Fig. 4D). The lower basal expression of CYP3A4 in HH1119 compared with that in HH1052 is consistent with the known individual variation in the expression of this enzyme (Strom et al., 1996).
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). The "humanized" mice were generated by introducing a liver-specific hPXR transgene into the mPXR null background (Xie et al., 2000a). The creation of these mice allowed for the evaluation of the need for PXR in the induction of drug-metabolizing enzymes by S20 in vivo. As shown in Fig. 4E, treatment of wild-type mice with a single dose of S20 (50 mg/kg body weight) induced the expression of CYP3A11 and this induction was abolished in PXR-null mice. In contrast, the CYP3A11 inducibility was restored in the "humanized" hPXR transgenic mice. Therefore, PXR is necessary for CYP3A11 induction by S20, and hPXR alone was sufficient to restore the regulation.
Structural Determinants of PXR in the S20 Enantioselectivity. The LBD of rPXR exhibits 98% amino acid homology with that of mPXR (Zhang et al., 1999). These two receptors share ligand profiles (data not shown). Tirona et al. (2004) recently reported the identification of amino acids in rPXR that determine species-specific rifampicin activation. The Phe305 of rPXR (conserved in mPXR) and its human counterpart Leu308, residues that are located within or are neighboring the flexible loop that forms part of the pore to the ligand-binding cavity, were found to be critical for the species-dependent ligand specificity (Tirona et al., 2004). We then used rPXR F305L and hPXR L308F mutants (Tirona et al., 2004) to examine whether the same residues are also important to determine the S20 enantiospecificity. As shown in Fig. 5, the preference of rPXR to (-)-S20 was completely abolished in the rPXR F305L mutant (Fig. 5A). In contrast, the L308F mutation had little effect on the hPXR's preference to (+)-S20 (Fig. 5B).
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Discussion |
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; Tirona et al., 2004). Mutagenesis studies in the context of the highly conserved rPXR revealed that Phe305, a residue that is important for rifampicin specificity (Tirona et al., 2004), was also critical for the preference of rPXR for the (-)-S20 enantiomer (Fig. 5A). Phe305 is located in the flexible loop that forms part of the pore to the ligand-binding cavity, based on the published crystal structure of hPXR LBD (Watkins et al., 2001). To our surprise, the corresponding L308F mutation had little effect on the preference of hPXR for the (+)-S20 enantiomer (Fig. 5B). A direct S20 enantiomer-PXR LBD docking modeling was not successful because of the flexible structures of the two enantiomers (M. Redinbo, personal communication). Nevertheless, the current findings represent, to our knowledge, the first example that an enantiomeric pair of compounds is capable of activating an orphan nuclear receptor in a species-specific manner. Our findings are potentially implicated in drug development. From the perspective of pharmaceutical development, the conceivable benefits of this knowledge of stereoselectivity are: 1) for pharmaceutical agents whose therapeutic effects do not rely on PXR, the choice of PXR-neutral but therapeutically effective enantiomers may help to avoid untoward drug-drug interactions. The drug-induced enzyme production is the primary mechanism for the side effect of drug-drug interactions; and 2) for drugs whose therapeutic target is PXR (see below for discussion), a hPXR-specific enantiomer will be necessary to achieve intended therapeutic effects in humans. Thus, the identification of species- and enantiomer-specific ligands for PXR can be both desirable and valuable. This work takes a first step in this direction by demonstrating exquisite species-dependent enantiomer-based selectivity. Future studies are necessary to determine whether the stereoselectivity is applicable to other xenobiotic nuclear receptors, such as CAR, or orphan nuclear receptors in general.
The analysis of biological effects summarized in Table 1 and Fig. 6 allows some generalized SAR analysis for PXR agonists with the allylic amide and C-cyclopropylalkylamide scaffolds. Although both scaffolds contain some high-potency agonists (-fold induction >4; i.e., S2, S4, S7, S14, S15, S20, S27, S33, S47, S50, S51, and S66), the percentage of low potency agents (fold induction <2) is considerably higher for the C-cyclopropylalkylamides (i.e., S10, S12, S16, S22, S23, S24, S25, S30, S45, S68, S70, and S73) than for the allylic amides (i.e., S30), thereby making the C-cyclopropylalkylamide scaffold more responsive to SAR fine-tuning. It is interesting that S20, the most potent and, so far, the most intriguing PXR agonist, has a cyclopropylalkylamide scaffold. For structurally closely related allylic amides and C-cyclopropylalkylamides, consistent levels of agonism are observed (S2 and S7). Homoallylic amides also show a relatively flat SAR; only S66 qualifies as a high-potency agonist. Among the C-cyclopropylalkylamides, ester or extended chain amide substitution patterns at the cyclopropane ring are not well tolerated (for example, S16, S22, S24, S25, and S45), and neither are halogen substituents at the cyclopropane methylene group (S68, S70, and S73). In contrast, a broad range of N-substituents are among the most active compounds. Examples include P(O)Ph2 in S14 and S47, CO2Bn in S20, and p-toluenesulfonyl (p-CH3C6H4SO2) in S50. The relative configuration of the cyclopropane ring and the amide methine carbon seems to play a minor role (for example, S50 versus S51), which is not overly surprising because the C(1) terminus of the C-cyclopropylalkylamide scaffold is freely rotating and substituted with large aromatic groups that are likely to fold onto the target in a fashion to optimize hydrophobic interactions.
Although PXR was isolated as a "xenobiotic receptor" that regulates drug metabolism, accumulating evidence has implicated the role of PXR in the treatment and prevention of human diseases, such as hyperbilirubinemia and bile acid-associated cholestasis. Genetic activation of PXR in transgenic mice prevents hyperbilirubinemia and LCA-induced hepatotoxicity (Xie et al., 2001, 2003). Similar genetic and pharmacological studies suggest that activation of CAR is also protective against hyperbilirubinemia and LCA hepatotoxicity (Huang et al., 2003; Saini et al., 2004). The notion that activation of PXR and CAR may be medically beneficial has also been supported by clinical observations. For example, RIF has been shown to relieve pruritus in cholestatic liver disease by stimulating 6-hydroxylation and elimination of bile acids (Wietholtz et al., 1996). This therapeutic effect of RIF is in agreement with the identification of RIF as a human-specific PXR activator and CYP3A4 inducer. Likewise, phenobarbital and the Chinese herbal remedy "Ying Zhi Huang" have been clinically used to treat neonatal hyperbilirubinemia (Valaes et al., 1980; Huang et al., 2004). This therapeutic effect has recently been attributed to the activation of CAR and subsequent induction of bilirubin detoxifying enzymes and transporters (Huang et al., 2003, 2004; Xie et al., 2003). It is our opinion that accumulating evidence, including animal studies and anecdotal clinical observation, is supporting the role of xenobiotic receptors as potential drug targets, and potent and selective PXR and CAR agonists may gain therapeutic value. However, it remains to be determined whether the S20 enantiomers have the appropriate properties, such as pharmacokinetics and bioavailability, to serve as pharmacological agents in humans. The in vivo induction of CYP3A11 by S20 in the "humanized" hPXR transgenic mice, although modest, was promising, in our opinion, in that it represents our first efforts with a compound from this series in animals, and S20 retained activity. The dose and scheduling of treatment were those used previously for some of the known PXR agonists, such as PCN and RIF. The in vivo pharmacokinetic and pharmacodynamic properties of S20 are still unknown; it is likely that the regimen of treatment we chose is not optimal for target gene regulation.
The majority of the published reports on PXR ligands have focused on either endogenous chemicals, such as bile acids and their intermediates and vitamin K (Staudinger et al., 2001; Xie et al., 2001; Makishima et al., 2002; Dussault et al., 2003; Goodwin et al., 2003; Tabb et al., 2003) or existing xenobiotics, including drugs (e.g., paclitaxel, ritonavir) (Dussault et al., 2001; Synold et al., 2001), herbal medicines (e.g., St. John's wort) (Moore et al., 2000) and environmental xenobiotics (e.g., polychlorinated biphenyls, 1,1-dichloro-2,2-bis (p-chlorophenyl)ethylene) (Schuetz et al., 1998; Wyde et al., 2003; Tabb et al., 2004), and pesticides (e.g., transnonachlor and chlordane) (Schuetz et al., 1998). The current study represents a strategy for PXR ligand identification by combining chemical library synthesis with functional evaluation using both cell cultures and whole animals. Although the results of the functional ligand assays are convincing, we have yet to perform ligand binding studies to demonstrate the direct binding of S20 to PXR. We therefore cannot exclude the possibility that S20 may activate PXR via indirect pathways. Future work will involve the development of a focused library of analogs to obtain sufficient structure-activity relationship data to better understand and increase the potency of PXR activation by this new ligand scaffold. Moreover, a crystal structure analysis of S20-bound PXR LBD would extend our understanding of the molecular basis for ligand recognition and enantiomer preference by PXR.
Although the enantiomer-specific metabolic profiles have been reported for many drugs, the current study represents the first example that enantiomers could have opposite species preference in activating PXR, an important regulator of drug-metabolizing enzymes. It is conceivable that the knowledge of stereoselectivity may be used to guide the development of safer drugs to avoid drug-drug interactions or to achieve human-specific drug effects when a xenobiotic receptor is being used as a therapeutic target.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: PXR, pregnane X receptor; LCA, lithocholic acid; CAR, constitutive androstane receptor; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene; FXR, farnesoid X receptor; LBD, ligand-binding domain; SAR, structure-activity relationship; hPXR, human PXR; PCN, pregnenolone-16-carbonitrile; RIF, rifampicin; mPXR, mouse PXR; SRC, steroid receptor coactivator; rPXR, rat PXR; MRP2, multidrug resistance associated protein 2.
Address correspondence to: Dr. Wen Xie, Center for Pharmacogenetics, Salk Hall 656, University of Pittsburgh, Pittsburgh, PA 15261. E-mail: wex6{at}pitt.edu
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