1521-0111/92/1/75–87$25.00 https://doi.org/10.1124/mol.117.108621MOLECULAR PHARMACOLOGY Mol Pharmacol 92:75–87, July 2017Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics

Molecular Basis of Metabolism-Mediated Conversion ofPK11195 from an Antagonist to an Agonist of the ConstitutiveAndrostane Receptor s

Bryan Mackowiak, Linhao Li, Matthew A. Welch, Daochuan Li, Jace W. Jones,Scott Heyward, Maureen A. Kane, Peter W. Swaan, and Hongbing WangDepartment of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland (B.M., L.L., M.A.W.,D.L., J.W.J., M.A.K., P.W.S., H.W.); and Bioreclamation In Vitro Technologies, Halethorpe, Maryland (S.H.)

Received February 20, 2017; accepted April 20, 2017

ABSTRACTThe constitutive androstane receptor (CAR) plays an importantrole in xenobiotic metabolism, energy homeostasis, and cellproliferation. Antagonism of the CAR represents a key strat-egy for studying its function and may have potential clinicalapplications. However, specific human CAR (hCAR) antago-nists are limited and conflicting data on the activity of thesecompounds have been reported. 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195),a typical peripheral benzodiazepine receptor ligand, has beenestablished as a potent hCAR deactivator in immortalized cells;whether it inhibits hCAR activity under physiologically relevantconditions remains unclear. Here, we investigated the effects ofPK11195 on hCAR in metabolically competent human primaryhepatocytes (HPH) and HepaRG cells. We show that althoughPK11195 antagonizes hCAR in HepG2 cells, it induces theexpression of CYP2B6 and CYP3A4, targets of hCAR and the

pregnane X receptor (PXR), in HPH, HepaRG, and PXR-knockoutHepaRG cells. Utilizing a HPH-HepG2 coculture model, wedemonstrate that inclusion of HPH converts PK11195 from anantagonist to an agonist of hCAR, and such conversion wasattenuated by potent CYP3A4 inhibitor ketoconazole. Meta-bolically, we show that the N-desmethyl metabolite is re-sponsible for PK11195-mediated hCAR activation by facilitatinghCAR interaction with coactivators and enhancing hCARnuclear translocation in HPHs. Structure-activity analysisrevealed that N-demethylation alters the interaction of PK11195with the binding pocket of hCAR to favor activation. Together,these results indicate that removal of a methyl group switchesPK11195 from a potent antagonist of hCAR to an agonist in HPHand highlights the importance of physiologically relevant metab-olism when attempting to define the biologic action of smallmolecules.

IntroductionThe constitutive androstane receptor (CAR) is a xenobiotic

sensor primarily expressed in the liver that regulates expres-sion of genes associated with drug-metabolizing enzymes andtransporters (Zelko and Negishi, 2000; Qatanani and Moore,2005; Omiecinski et al., 2011). Under physiologic condi-tions, CAR is sequestered in the cytoplasm of hepatocytesand translocates to the nucleus upon activation, in which it

dimerizes with the retinoid X receptor, recruits coactivatorssuch as SRC-1, and induces the transcription of its targetgenes (Kawamoto et al., 1999; Pascussi et al., 2007). Activation ofCAR leads to the coordinated induction of a cellular defensivenetwork in the liver, which enables hepatocytes to metabolizeand excrete foreign compounds. However, induction of importantdrug-metabolizing enzymes through CAR activation can alsosignificantly alter the levels of toxic metabolites and lead tounexpected drug-drug interactions (Tolson and Wang, 2010;Kobayashi et al., 2015). Recently, novel functions of CAR beyondmodulation of drug disposition have come to light, including itsroles in energy metabolism and cell proliferation (Yamamotoet al., 2004; Dong et al., 2009; Gao et al., 2009). Thus, un-derstanding the molecular basis of CAR modulators would notonly benefit early prediction of drug-drug interactions but alsooffer potential therapeutic choices for metabolic disorders andcancers.

This work was supported by the National Institutes of Health NationalInstitute of General Medicine [Grant R01 GM107058]. B.M. and M.A.W. arepartly supported by The University of Maryland’s Center of Excellence inRegulatory Science and Innovation (M-CERSI) Scholars Program funded bythe Food and Drug Administration [Grant 1U01FD005946].

The authors state no conflict of interest and have received no payment inpreparation of this manuscript.

https://doi.org/10.1124/mol.117.108621.s This article has supplemental material available at molpharm.aspetjournals.

org.

ABBREVIATIONS: ACN, acetonitrile; AF2, activation function 2; CAR, constitutive androstane receptor; CINPA1, constitutive androstane receptorinhibitor not pregnane X receptor activator 1; CITCO, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime;COOH-PK, 1-(2-chlorophenyl)isoquinoline-3-carboxylic acid; DMSO, dimethylsulfoxide; EYFP-hCAR, enhanced yellow fluorescent protein-taggedhuman constitutive androstane receptor; hCAR, human constitutive androstane receptor; HPH, human primary hepatocyte; hPXR, human pregnaneX receptor; KET, ketoconazole; KO, knockout; LC, liquid chromatography; MS, mass spectrometry; ND-PK, (R)-N-desmethyl 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide; P450, cytochrome P450; PB, phenobarbital; PCR, polymerase chain reaction; PK11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide; PXR, pregnane X receptor; RIF, rifampicin.

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Although CAR is sequestered in the cytoplasm and musttranslocate to the nucleus to transactivate gene expression inprimary hepatocytes and intact livers, it is spontaneouslylocalized in the nucleus and exhibits constitutive activity inimmortalized cell lines (Kawamoto et al., 1999; Li et al., 2009).This unique feature makes the investigation of chemical-mediatedCARmodulation rather challenging.Wehavepreviouslyidentified 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195), aperipheralbenzodiazepinereceptor ligand, as a potent and selective antagonist of humanCAR (hCAR) with negligible effects on its rodent counterparts(Li et al., 2008). At low micromolar concentrations, PK11195effectively represses hCAR-mediated gene expression in celllines and has been used as a research tool for studying themolecular mechanisms of hCAR activation (Anderson et al.,2011; Lynch et al., 2015). Notably, although a potent antagonistof hCAR,PK11195also activates thehumanpregnaneX receptor(PXR) efficiently (Li et al., 2008; Anderson et al., 2011). Giventhat CAR and PXR share an overlapping array of target genes(Moore et al., 2000) it is challenging to delineate the exact effectsof PK11195 onCARandPXR in cells that express both receptors.To this end, the fact that PK11195 induces the expression ofCYP2B6, the primary target of hCAR, and CYP3A4, the primarytarget of human (hPXR), in human primary hepatocytes (HPHs)has been attributed to its activation of hPXR. Nevertheless,unlike the prototypical hPXR activator rifampicin, whichpreferentially induces the expression of CYP3A4 over CYP2B6,PK11195 favorably enhances the expression of CYP2B6 overCYP3A4, challenging this assumption.It is well established that CAR plays a key role in determining

xenobiotic metabolism and disposition through transactivationof numerous genes in the liver. However, whether and howhepatic metabolism can influence CAR action is not adequatelyunderstood. A previous study showed that although omeprazolerepresents a prototypical activator of the aryl hydrocarbonreceptor, its degradationmetabolite omeprazole-sulfide is a pureantagonist of the aryl hydrocarbon receptor (Gerbal-Chaloinet al., 2006). In screening of antimalarials and their majormetabolites for CAR activation, Burk et al. (2012) found thatartemisinin derivatives and metabolites differentially affectactivities of CAR. Clearly, the metabolic capacity of a cellularsystem can be a key determinant for the biologic function of agiven compound including its role in nuclear receptor activa-tion. Thus, we hypothesize that PK11195 induction of CYP2B6and CYP3A4 in HPHs is not fully mediated by the activation ofPXR, instead, PK11195 is converted from an antagonist to anagonist of hCAR in the metabolically competent HPH.In the current study, we demonstrate that PK11195 is N-

demethylated to a metabolite that robustly activates CAR inHPHs. Using a PXR-knockout (KO) HepaRG cell line, we confirmPK11195 can induce the expression of CYP2B6/CYP3A4 inde-pendent of PXR.A coculture system that addsmetabolism toCARluciferase reporter assayswas used to determine howmetabolisminfluences the agonist/antagonist feature of PK11195. Mamma-lian two-hybrid assays and molecular modeling were used toelucidate the molecular basis and structural features of PK11195and its metabolites in the modulation of CAR activity.

Materials and MethodsChemicals and Biologic Reagents. Phenobarbital (PB), rifampicin

(RIF), ketoconazole (KET), and PK11195 were obtained from Sigma-

Aldrich (St. Louis, MO). 6-(4-Chlorophenyl)imidazo[2,1-b][1,3]-thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) wasobtained from BIOMOL Research Laboratories (Plymouth Meeting,PA). (R)-N-Desmethyl PK11195 (ND-PK) was obtained from ABXAdvanced Biochemical Compounds (Radeberg, Germany). 1-(2-Chlor-ophenyl)isoquinoline-3-carboxylic acid (COOH-PK) was obtained fromBiogene Organics (The Woodlands, TX). Polymerase chain reaction(PCR) primers were synthesized by Integrated DNA Technologies(Coralville, IA). HepaRG wild-type and PXR-KO cells and the cellculture medium were obtained from Sigma-Aldrich. Optima LC/MSGrade water (H2O), acetonitrile (ACN), and formic acid were purchasedfrom Fisher Scientific (Pittsburg, PA). All chemicals and reagents wereused without further purification.

Culture and Treatment of HPHs and HepaRG Cells. HPHswere isolated using a modified two-step perfusion protocol (LeCluyseet al., 2005) from human liver specimens obtained from theUniversityof Maryland Medical Center with prior approval by the InstitutionalReview Board at the University of Maryland at Baltimore or obtainedfrom Bioreclamation In Vitro Technologies (Baltimore, MD). The ageand sex of each liver donor used are detailed in Table 1. Hepatocyteswith viability over 90% were seeded at 0.75 � 106 cells/well in 12-wellcollagen-coated plates as described previously (Faucette et al., 2006).After attachment at 37°C in a humidified atmosphere of 5% CO2,hepatocytes were cultured in serum-free William’s E Medium supple-mented with insulin, transferrin, and selenium, 0.1 mM dexametha-sone, 100 U/ml penicillin, and 100 mg/ml streptomycin, and overlaidwithMatrigel (0.25mg/ml). Thirty-six hours after seeding,HPHsweretreated with vehicle control (0.1% [dimethylsulfoxide (DMSO)],CITCO (1 mM), RIF (10 mM), PB (1 mM), PK11195 (10 mM), ND-PK(10 mM), or COOH-PK (10 mM) for 24 or 72 hours before harvestingcells to collect RNA or protein, respectively. In separate experiments,wild-type and PXR-KO HepaRG cells were plated in 12-well plates(1 � 105 cells/well) and cultured for 21 days following Sigma-Aldrich’sinstructions to induce differentiation before treatment with com-pounds as described previously.

Real-Time Reverse-Transcription PCR. Total RNA was iso-lated from cells using TRIzol reagent (ThermoFisher, Rockford, IL)and reverse transcribed using a High Capacity cDNA archive kit(Applied Biosystems, Foster, CA) according to the manufacturer’sinstructions. Real-time PCR assay was performed using SYBR GreenPCR Mastermix (Qiagen, Germantown, MD) on an ABI StepOnePlusreal-time PCR system (Applied Biosystems). He primer sequences forCYP2B6, CYP3A4, and glyceraldehyde-3-phosphate dehydrogenaseare as follows: CYP2B6, 59-AGACGCCTTCAATCCTGACC-39 and 59-CCTTCACCAAGACAAATCCGC-39; CYP3A4, 59-GTGGGGCTTTTA-TGATGGTCA-39 and 59-GCCTCAGATTTCTCACCAACACA-39; andglyceraldehyde-3-phosphate dehydrogenase, 59-CCCATCACCATC-TTCCAGGAG-39 and 59-GTTGTCATGGATGACCTTGGC-39. Expres-sion values were quantified using the following equation: fold overcontrol5 2DDCt method, where DCt represents the differences in cyclethreshold numbers between the target gene and glyceraldehyde-3-phosphate dehydrogenase, andDDCt represents the relative change inthese differences between control and treatment groups.

TABLE 1Age and sex of liver donors

Liver Donor Number Sex Age

107 Male 47116a Female 69117a Female 61118a Male 45119a Male 36120a Male 45121 Male 52122 Male 10123 Female 30

aDenotes livers used for coculture experiments in Fig. 3.

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Western Blot Analysis. Protein samples extracted from treatedcellswere electrophoretically separated onSDS-PAGEgels (4%–12%) andtransferred to polyvinylidine fluoride membranes. Subsequently, mem-branes were incubated with primary antibodies against CYP2B6 (1:200;

Santa Cruz Biotechnology, Santa Cruz, CA), CYP3A4 (1:5000; Sigma-Aldrich), orb-actin (1:50,000, Sigma-Aldrich) at 4°C overnight. Blots weredeveloped with West Pico chemiluminescent substrates (ThermoFisher)after incubation with horse-radish peroxidase secondary antibodies.

Fig. 1. PK11195 induces CYP2B6 andCYP3A4 expression inHPHs. Primary hepatocytes from liver donors 107 (A andB) and 122 (C andD)were treatedwith 1mMPB, 1mMCITCO, 10mMRIF, or 10mMPK11195 for 24 or 72 hours to analyzemRNA or protein expression, respectively. Results are expressedas fold over control, mean 6 S.D. (n = 3); ***P , 0.001.

Fig. 2. PK11195 induces CYP2B6 and CYP3A4 expression in HepaRG cells independent of PXR.Wild-type and PXR-KOHepaRG cells were cultured for21 days in accordance with Sigma-Aldrich instructions to induce differentiation. Differentiated HepaRG cells were treated with 1 mM PB, 1 mMCITCO,10 mMRIF, or 10 mMPK11195 for 24 or 72 hours to analyzemRNA (A and C) or protein expression (B and D), respectively. Data represent the mean6 S.D.(n = 3); n.s., not significant; *P , 0.05; **P , 0.01; ***P , 0.001.

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HepG2 Cell Transfection and HepG2/HPH Coculture. HepG2or HepG2-CAR-CYP2B6 stable cells as described previously (Lynchet al., 2015) were cultured in 24-well plates (1 � 105 cells/well) at 37°Cand 5%CO2 for 24hours.HepG2 cellswere cotransfectedwithCYP2B6-2.2k reporter (60 ng/well), hCAR11A expression vector (30 ng/well),and pRL-TK (10 ng/well) by using X-tremeGENE 9 DNA TransfectionReagent (Sigma-Aldrich) following the manufacturer’s instruction.Twenty-four hours after transfection, cells were treated with solvent(0.1% DMSO) or test compounds at indicated concentrations. Sub-sequently, cell lysates were assayed for firefly activities normalizedagainst the activities of Renilla using a luciferase kit (Promega,Madison,WI). In separate experiments, HPHswere plated on collagen-coated cover slips with the corners bent upward in a 24-well plate. Afterpreincubation of vehicle control and test compounds with HPHs for4 hours, both the media and HPH-containing cover slips were trans-ferred to new 24-well plates containing transfected HepG2 cells or theHepG2-CAR-2B6 stable line and incubated for 24 hours before mea-surement of luciferase activities. Data from a representative liverdonor are shown in Fig. 3B-C and represent the mean6 S.D. of threeindividual transfections.

Liquid Chromatography (LC)–Mass Spectrometry (MS)Measurement of PK11195 Metabolism. Complete William’s EMedium containing 10 mM PK11195 was added to HPHs cultured in24-well plates 36 hours after seeding. Media (450 ml) were collectedfrom each well at 0 and 1 hours and frozen immediately at 280°C.Sampleswere thawed on ice before adding 50ml of medium to 450ml ofice-cold methanol/H2O (8:1, v/v). After centrifugation at 16,000g for30 minutes, 200 ml of supernatant was transferred to a separate tubeand dried down before resuspension in 200 ml H2O/ACN (1:1, v/v) with0.1% formic acid. Authentic standards were prepared to 1 mM in H2O/ACN (1:1, v/v) with 0.1% formic acid. LC-MS/MS analysis was

performed on a TSQQuantumUltra Triple Stage Quadrupole MassSpectrometer coupled to an Ultimate 3000 RS Liquid Chromato-gram System (Thermo Scientific, Waltham, MA). The LC separa-tion was performed on a Waters (Milford, MA) BEH C18 column (2.1 �50 mm, 1.7 mm) operated at 30°C. Solvents A and B consisted of 0.1%formic acid in H2O and 0.1% formic acid in ACN, respectively. Thegradient program was 0.0–0.5 minutes, 50% B; 0.5–2.0 minutes,gradient to 95% B; 2.0–3.5 minutes, 95% B; 3.5–4.0 minutes, gradientto 50% B; and 4.0–5.0 minutes, 50% B. The flow rate was 0.5 ml/minand injection volume was 5 ml. Tandem mass spectrometry wasperformed in the positive-ion mode and the electrospray ionizationsource parameters were as follows: spray voltage, 3000; capillarytemperature, 325; sheath gas pressure, 40; auxiliary gas pressure, 15;capillary offset, 35; and tube lens offset, 80. Selected reactionmonitoring was used for mass detection with the following transi-tions: PK11195 (m/z 353.1 → 238.0), ND-PK (m/z 339.1 → 238.0),and COOH-PK (m/z 284.0 → 238.0). Data collection and analysiswere performed using Xcalibur V 2.1 (Thermo Scientific).

Mammalian Two-Hybrid Assay. COS1 cells seeded in 24-wellplates were transfected with 110 ng of the reporter gene plasmidpG5luc, 80 ng of expression plasmids encoding the respective VP16-AD/hCAR fusions, 40 ng of expression plasmids encoding GAL4-DBD/coregulatory fusions, and 20 ng of reference plasmid pRL-TK, eachwell using X-tremeGENE 9 DNA Transfection Reagent (Sigma-Aldrich).Twenty-four hours after transfection, the cells were treated with solvent(0.1% DMSO), CITCO (1 mM), PK11195 (10 mM), or ND-PK (10 mM) for24hours.Luciferaseactivitiesweremeasured in cell lysatesusing theDual-Luciferase Kit (Promega). Data represent the mean6 S.D. of threeindividual transfections.

Nuclear Translocation of CAR in HPHs. HPHs were plated incollagen-coated 24-well plates and infected with adenovirus-expressing

Fig. 3. PK11195 is metabolized to a CARactivator in HepG2-HPH coculture. HPHswere plated on plastic cover slips with thecorners bent upward and incubated withtest compounds for 4 hours before themedia and the cover slips were transferredto a 24-well plate containing HepG2 cellsand incubated for 24 hours (A). HepG2-CAR-2B6 stable line (B) or transientlytransfected with CAR1+A and CYP2B6-2.2K (C) were treated with PK11195 andCITCO at indicated concentrations withorwithout coculturewithHPHs for 24hours.HepG2-CAR-2B6cellsalone (D) or coculturedwith HPHs (E) were treated with PK11195alone or cotreated with 8 mM KET for24 hours. Luciferase activitywasmeasuredas described in Materials and Methods.Data represent the mean 6 S.D. (n = 3);*P , 0.05; ***P , 0.001.

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enhanced yellow fluorescent protein-tagged hCAR (EYFP-hCAR) asdescribed previously (Li et al., 2009). Twenty-four hours after infection,HPHs were treated with vehicle control (0.1% DMSO), PB (1 mM),

PK11195 (10 mM), ND-PK (10 mM), or COOH-PK (10 mM) for another8 hours. After treatment, cells were fixed with 4% paraformaldehyde,stainedwith 1mg/ml 49,6-diamidino-2-phenylindole (Sigma-Aldrich) for

Fig. 4. PK11195 is metabolized to COOH-PK and ND-PK in HPHs. Proposed PK11195 metabolism pathway (A). An authentic 1 mM standard mix wasrun on LC-MS/MS as described inMaterials andMethods and retention times for PK11195, COOH-PK, andND-PKwere determined to be 1.81, 0.76, and2.14, respectively (B). Media containing 10 mM PK11195 was cultured with HPHs and harvested at 0 hour (C) and 1 hour (D) to determine whetherPK11195 is metabolized by HPHs to the metabolites of interest.

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Fig. 5. The (R)-N-desmethyl (ND) metabolite of PK11195 mediates CAR activation. HepG2-CAR-2B6 cells (A) and HepG2 cells transfected withCAR1+A and CYP2B6-2.2k (B) were treated with vehicle control (CT), ND-PK, COOH PK, or CITCO at indicated concentrations for 24 hours beforeluciferase activities were determined.Mammalian two-hybrid assays were performed in COS1 cells measuring interaction between CAR/SRC-1 (C) or CAR/GRIP1 (D) as detailed in Materials and Methods. Transfected cells were treated with CITCO, PK11195, ND-PK, or ND-PK+PK1195 for 24 hours before

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30minutes, andEYFP-hCAR localization inhepatocyteswas visualizedon a Nikon Eclipse TI fluorescent microscope (Nikon, Melville, NY).Quantitative distribution of EYFP-hCAR was analyzed using GeneralAnalysis in the Nikon Elements AR High Content Analysis softwarepackage (version 4.50.00). Nuclear localization was defined and quanti-fied as the percentage of total enhanced yellow fluorescent protein thatoverlaps with 49,6-diamidino-2-phenylindole. Data from a representativeliver donor are shown in Fig. 7A-B and represent the mean6 S.D. of fiveindividual images for each treatment.

Molecular Modeling. The hCAR/ligand-binding domain proteincrystal structure (Protein Data Bank identification number 1XVP)was retrieved from the RCSB Protein Data Bank (http://www.rcsb.org). The PK11195 and ND-PK molecular structures were generatedand obtained from ChemAxon Chemicalize (http://chemicalize.com)and the CITCO and CAR inhibitor not PXR activator 1 (CINPA1)structures were obtained from National Center for BiotechnologyInformation PubChem (http://pubchem.ncbi.nlm.nih.gov/). DiscoveryStudio (version 4.5.0.15071; Biovia, San Diego, CA) was used toremove water and ligands from the crystallographic data and isolatethe D chain protein that contains the crystal structure of CAR, whichwas subsequently protonated at pH 7.0. A binding site was definedbased on the CITCO binding cavity and defined as an 11.5 Å radiussphere at 24.972 (x), 54.702 (y), and 29.512 (z). The receptor-liganddocking was performed in Discovery Studio using the CDOCKERprotocol (Wu et al., 2003). Briefly, the docking parameters were set togenerate 255 conformations for each ligand and return the top 10 dockswith the lowest CDOCKER energy, which is the sum of the receptor-ligand interaction energy and internal ligand strain. The completeparameters can be found in Supplemental Fig. 1 and SupplementalTable 1. The ligand-receptor interactions of the dockedmoleculeswereanalyzed and visualized in Discovery Studio.

Statistical Analysis. All data are expressed as the mean 6 S.D.Statistical comparisons were made using one-way analysis ofvariance with Dunnett’s post-test or two-way analysis of variancewith Bonferroni post-test as needed. Statistical significance was setat *P , 0.05, **P , 0.01, and ***P , 0.001.

ResultsPK11195 Induces the Expression of CYP2B6 and

CYP3A4 in HPHs. We first examined the effects of PK11195,a known hCAR antagonist, on the expression of CYP2B6 andCYP3A4, two prototypical targets for hCAR and hPXR, inHPHs prepared from liver donors 107 and 122. As shown inFig. 1, PB, CITCO, RIF, and PK11195 at selected concentra-tions robustly induced CYP2B6 and CYP3A4 at mRNA andprotein levels in both liver donors. As expected, CITCO (aselective activator of hCAR) and RIF (a selective activator ofhPXR) preferentially induced the expression of CYP2B6 andCYP3A4, respectively. Intriguingly, PK11195 at 10 mM, aconcentration at which it represses the activity of hCAR bymore than 85% in HepG2 cells (Li et al., 2008), markedlyinduced the expression of both CYP2B6 and CYP3A4withouta clear preference, mimicking PB, a dual activator of hCARand hPXR. These findings call into question whetherPK11195 is as an antagonist of hCAR in physiologicallyrelevant cells and challenge the assumption that PK11195induction of cytochrome P450 (P450) in HPHs relies on PXRactivation.

Induction of CYP2B6 and CYP3A4 by PK11195 inPXR-KO HepaRG Cells. HepaRG cells have been validatedas a promising surrogate for HPHs, and importantly, fullydifferentiated HepaRG cells exhibit proper CAR cellularlocalization and maintain physiologically relevant metaboliccapacity, which are not present in most immortalized cellmodels (Jackson et al., 2016). The PXR-KO HepaRG cell lineobtained from Sigma-Aldrich is a newly generated cell linethat does not express functional PXR (Williamson et al., 2016).As expected, PK11195 and other knownCAR/PXRmodulatorsinduced the expression of CYP2B6 and CYP3A4 mRNA andprotein in wild-type HepaRG cells in a trend that mirrorswhat was observed in HPHs (Fig. 2, A and B). Notably, inPXR-KO HepaRG cells PK11195 significantly induced bothCYP2B6 and 3A4 expression at mRNA and protein levels,although induction of CYP2B6 and CYP3A4 by RIF wasfully abrogated (Fig. 2, C and D). These data suggest thatdifferential metabolism of PK11195 in the physiologicallyrelevant HPH/HepaRG cells versus the immortalized HepG2cells may contribute to the observed PXR-independent inductionof CYP2B6 and CYP3A4.PK11195 Is Metabolized to a hCAR Activator in

HPHs. Lack of metabolism is a major limitation of almostall studies using immortalized cell lines, including HepG2cells. AHPH-HepG2 coculturemodelwas established as depictedin Fig. 3A, which introduces the metabolism-competentHPHs into the culture environment shared withHepG2 cells.In agreement with previous reports, PK11195 concentra-tion dependently inhibits the constitutive hCAR activity inHepG2 cells without the presence of HPHs (Fig. 3B, openbars). Results from the coculture with HPHs from a represen-tative liver donor indicate that PK11195 significantly in-creased the luciferase activity of the CYP2B6 reporter inthe presence of hCAR (Fig. 3B, solid bars) or its low-basalalternative hCAR11A (Fig. 3C). These findings suggest thatPK11195 is converted from a CAR antagonist to an agonist inthe presence of HPHs. To further explore the contribution ofmetabolism in this antagonism/agonism conversion, KET, apotent inhibitor of CYP3A4, the most abundant hepatic P450isoform in humans, was cotreated with different concentra-tions of PK11195 in HepG2-CAR-2B6 cells with and withoutHPH coculture. Repression of CAR activity by PK11195 wasnot influenced by the presence of KET in HepG2-CAR-2B6cells alone (Fig. 3D). On the other hand, when HPHs werecocultured with HepG2-CAR-2B6 cells, KET significantlyinhibited the conversion of PK11195 (1 and 10 mM) from aCAR antagonist to a CAR activator (Fig. 3E). Taken together,these results validate the use of the coculture system toinvestigate the mechanistic effects of metabolism on CARactivity and suggest that CYP3A4 contributes to the bio-transformation of PK11195 in HPHs.PK11195 Is Metabolized to ND-PK and COOH-PK in

HPHs. Previous studies postulated that PK11195 is metab-olized into two major metabolites, ND-PK and COOH-PK,which are formed throughN-demethylation and amide hydro-lysis, respectively (Fig. 4A) (Roivainen et al., 2009). To confirm

determining luciferase activity. PXR-KO HepaRG cells were treated with vehicle control, PB (1 mM), CITCO (1 mM), RIF (10 mM), ND-PK (10 mM), orCOOH-PK (10 mM) and harvested for mRNA (E) or protein (F) expression analysis. Data represent the mean6 S.D. (n = 3); n.s., not significant; *P, 0.05;**P , 0.01; ***P , 0.001.

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that these metabolites were generated in our experimentalsystem, PK11195 (10 mM) was incubated with HPHs andmedia was collected at 0 and 1 hours. These samples wereprepared and analyzed by LC-MS/MS as detailed inMaterialsand Methods to determine whether the proposed ND-PK andCOOH-PKmetabolites of PK11195 were formed in HPHs. The1 mM standard mix exhibited chromatographic separationand reproducible retention times of 1.81, 0.76, and 2.14 forPK11195, COOH-PK, and ND-PK, respectively (Fig. 4B).As expected, PK11195 was abundantly present in HPHculture medium at 0 hour, while the amounts of COOH-PKand ND-PK were negligible (Fig. 4C). However, the peaksfor COOH-PK and ND-PK significantly increased in in-tensity after 1 hour in HPH culture, indicating that PK11195ismetabolized byHPHs to COOH-PK andND-PK (Fig. 4D). Toour knowledge, this is the first report confirming that the

COOH-PK and ND-PK metabolites of PK11195 are generatedby HPHs, and these metabolites require further study todetermine whether they mediate CAR activation.The (R)-N-Desmethyl Metabolite of PK11195 Medi-

ates hCAR Activation. After confirming that COOH-PKand ND-PK were generated in our experimental system, wetested these metabolites for hCAR activation in HepG2-basedluciferase assays. As shown in Fig. 5A, ND-PK activatedhCAR in a concentration-dependent manner while COOH-PKhad no effect on CAR activity. ND-PK activation of hCAR wasfurther confirmed in the CAR11A/CYP2B6-2.2k reporter assay,where ND-PK robustly activates hCAR to a level similar to thepositive control (CITCO), while COOH-PK exhibits negligibleCAR activation (Fig. 5B). In separate experiments, the mamma-lian two-hybrid assay revealed that PK11195 could significantlyrepress the interaction between hCAR and SRC-1 or GRIP1,

Fig. 6. ND-PK induces CAR target genes in HPHs.Primary hepatocytes from liver donors 120 (A and B) or121 (C and D) were treated with PB (1 mM), CITCO(1 mM), RIF (10 mM), or increasing concentrations (0.1, 1,and 10 mM) of PK11195, ND-PK, or COOH-PK for 24 or72 hours to analyze mRNA (A and C) or protein (B andD) expression, respectively. Data represent the mean6S.D.(n = 3).

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while ND-PK moderately enhanced hCAR recruitment ofthese coactivators in a manner similar to that by CITCO(Fig. 5, C and D). Notably, ND-PKmarkedly rescued PK11195-mediated repression of SRC-1/CAR interaction, whileonly minimally affecting PK11195-repressed binding ofGRIP1 to hCAR. Furthermore, ND-PK significantly in-duced CYP2B6 and CYP3A4 mRNA and protein expression

in the PXR-KO HepaRG cells, indicating that ND-PK caninduce P450 expression independent of PXR (Fig. 5, Eand F). Overall, these results identify ND-PK as themetabolite of PK11195 that is responsible for PK11195-mediated CAR activation in metabolically competenthepatic cells.ND-PK Induces CYP2B6, CYP3A4 Expression, and

Nuclear Translocation of hCAR in HPHs. To fully char-acterize the effect of ND-PK on hCAR activation and targetgene induction, HPHs from two liver donors (120 and 121)were treated withmultiple concentrations of PK11195, ND-PK,and COOH-PK. Our results indicate that ND-PK concentrationdependently and potently induced CYP2B6 and CYP3A4at both mRNA and protein levels, while COOH-PK onlyexhibited negligible effects on the expression of thesegenes (Fig. 6, A–D). Nuclear translocation of CAR in HPHshas been regarded as the essential step in its activation. Totest the nuclear translocation of CAR by PK11195 metab-olites, HPHs were infected with adenovirus-expressingEYFP-hCAR overnight before treatment with compoundsfor 8 hours. Without activation, EYFP-hCAR was pre-dominantly expressed in the cytoplasm of HPHs, and asexpected the prototypical CAR activator PB as well asPK11195 induced significant hCAR nuclear accumulation(Fig. 7, A and B). Importantly, ND-PK at 10 mM robustlyincreased nuclear translocation of hCAR, while COOH-PK(10 mM) caused negligible CAR nuclear accumulation,which correlate well with their capacity for P450 induction.These data further support that ND-PK is the metabolite ofPK11195 that activates hCAR in physiologically relevantsystems.Computational Modeling of PK11195 and ND-PK. The

metabolism of a potent CAR antagonist (PK11195) and itsconversion into a potent CAR activator (ND-PK) with a differ-ence of only one methyl group provided a unique opportunity toprobe the structure-activity relationship for CAR throughmolecular modeling. Docking studies used the 1XVP (http://www.rcsb.org) crystal structure of CITCO bound to the CARligand-binding domain to identify PK11195 and ND-PK inter-actions with different residues in the binding pocket (Xu et al.,2004). Upon validation of ourmodel by docking CITCO into theligand-binding domain, both PK11195 andND-PKwere dockedand found to interact with many of the same residues in thebinding pocket, suggesting that they bind in similar conforma-tions (Fig. 8A). However, PK11195 interacts with residuesimportant to CAR activity such as V199, Y224, and Y326,including the N-methyl group interacting with H203, whereasthe demethylated form of PK11195 (ND-PK) does not (Fig.8, B and C); notably, PK11195 and the CAR antagonistCINPA1 (Fig. 8D) share these interactions, which maycontribute to their antagonism of hCAR (Cherian et al.,2016). The demethylation of PK11195 allows it move awayand not interact with these important residues in the CARbinding pocket, which may explain how the difference of onemethyl group between PK11195 andND-PK can have such asubstantial effect on CAR activity. These docking studiesdetermined the structure-activity relationship of PK11195and ND-PK with CAR and demonstrated that even smallalterations in ligand structure, such as the demethylation ofPK11195, have the potential to alter its interactions withamino acids in the CAR binding pocket and lead to largedifferences in CAR activity.

Fig. 7. ND-PK induces CAR nuclear translocation in HPHs. Primaryhepatocytes from liver donor 123 were infected with adenovirus-expressing EYFP-hCAR for 24 hours and treated with 1 mM PB,10 mM PK11195, 10 mM ND-PK, or 10 mM COOH-PK for 8 hours beforebeing fixed with 4% paraformaldehyde, stained with 1 mg/ml 49,6-diamidino-2-phenylindole (DAPI), and visualized on a Nikon Eclipse TIfluorescent microscope. Quantitation of nuclear localization was de-termined using General Analysis in the Nikon Elements AR HighContent Analysis software package and defined as the percentage ofenhanced yellow fluorescent protein (YFP) that overlaps with DAPI.Representative images (A) and quantitative analysis (B) of nuclearlocalization are shown. Data represent the mean 6 S.D. of fiveindividual images; **P , 0.01; ***P , 0.001.

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Fig. 8. N-Demethylation of PK11195 leads to reduced interaction with residues important to CAR activity. The hCAR/ligand-binding domain crystalstructure (1XVP) was prepared in Biovia Discovery Studio and docking was carried out using the CDOCKER protocol as detailed in Materials andMethods. After model validation, PK11195 (yellow) and ND-PK (light blue) were docked into the CAR ligand-binding pocket (A). Interactions of PK11195(B), ND-PK (C), CINPA1 (D), and CITCO (E) with residues in the ligand-binding pocket of hCAR. Residues are color-coded as follows: dark blue, interactwith all structures; light blue, PK11195 and ND-PK; orange, PK11195, CINPA1, and CITCO; red, PK11195 and CINPA1; green, CINPA1 and CITCO;yellow, CITCO only; and purple, ND-PK only.

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DiscussionThe biologic function of CAR is regulated by the interplay

between specific cellular factors and small molecular modu-lators. PK11195, a well-known peripheral benzodiazepinereceptor ligand, has been used as a potent hCAR deactivatorin cell-based luciferase reporter assays. In contrast to CARantagonism exhibited in immortalized cell lines, PK11195robustly induces the expression of both CYP2B6 and CYP3A4in HPHs, which are prototypical targets for hCAR and hPXR,respectively. The mechanistic basis for this observed discrep-ancy is largely unknown, although it was presumed thatPK11195-mediated P450 induction in HPHs was attributed toits activation of hPXR. Here, we show that PK11195 signifi-cantly induces the expression of CYP2B6 and CYP3A4 inPXR-KO HepaRG cells, which demonstrates that PK11195can stimulate P450 expression independent of PXR. Utilizinga HPH-HepG2 coculture model, we show that introduction ofmetabolically competentHPHs is sufficient to convert PK11195from an antagonist to an agonist of hCAR in HepG2 cells. InHPHs, PK11195 is biotransformed to ND-PK and COOH-PK.Further studies demonstrated that ND-PK is the activemetabolite that potently activates hCAR and induces theexpression of CYP2B6 and CYP3A4. Moreover, structure-activity analysis reveals that N-demethylation of PK11195allows its side chain to rotate away from residues in the CARligand-binding pocket toward a conformation in favor of CARactivation.CAR and PXR regulate an overlapping array of target genes

and share many common chemical modulators (Hernandezet al., 2009; Mackowiak and Wang, 2016). Although suchcrosstalk betweenCARandPXR can be beneficial by forming adefensive network against xenobiotics, it makes the delinea-tion of specific function of each individual receptor extremelychallenging, particularly in cells such as HPHs, where bothCAR and PXR are abundant and functionally intact. Recently,the HepaRG cell line has emerged as a useful alternative forHPHs; differentiated HepaRG cells exhibit prototypical HPHmorphology, inductive expression of major drug-metabolizingenzymes and transporters, and have been used for in vitrodrug metabolism and toxicology studies (Jossé et al., 2008;Andersson et al., 2012). The PXR-KOHepaRG cell line providesan excellent model to determine the contribution of CAR/PXRto PK11195-mediated P450 induction. Our results uncoveran unexpected induction of both CYP2B6 and CYP3A4 byPK11195 in the PXR-KO HepaRG cells, although inductionby selective hPXR activator RIF was fully abrogated. Thesefindings provide conclusive evidence that PK11195 can induceCYP2B6/CYP3A4 expression in physiologically relevant he-patic cells independent of hPXR. Indeed, previous and currentstudies in HPHs have shown that PK11195 induces bothCYP2B6 and CYP3A4 in a pattern that mimics that of PB, adual activator of hCAR and hPXR (Li et al., 2008; Andersonet al., 2011). Together, these results indicate that PK11195modulates CAR differently in HPHs and HepaRG cells versusin immortalized cell lines, and is most likely metabolized froman antagonist to an agonist in cells exhibiting physiologicallyrelevant metabolism.Lack of metabolism capacity is a significant drawback

associated with the use of immortalized cell lines in toxicityassessment and drug development. Cell-based luciferase re-porter assays using immortalized cell lines in particular have

been extensively used to investigate nuclear receptor activityand predict target gene expression. However, proper inter-pretation of such data has become a heightened concern inboth academia and the pharmaceutical industry. Several linesof evidence indicate that introduction of metabolic capacity tocell cultures appears to be an attractive solution to overcomethis issue. In this regard, we have previously used an HPH-leukemia/lymphoma cell coculture model to show that thepresence of HPHs markedly increases the biotransforma-tion of cyclophosphamide, a chemotherapeutic prodrug, to itspharmacologically active metabolite and leads to enhancedanticancer activity in cocultured HL-60 and SU-DHL-4 cells(Wang et al., 2013; Hedrich et al., 2016). Using a HPH-HepG2coculture system in the current study, we observed thatPK11195 concentration dependently increased CAR activa-tion in contrast to decreasing CAR activity when exposed toHepG2 cells only. More importantly, such agonistic effects ofPK11195 in the coculture can be reversed by KET, suggestingthat CYP3A4 plays a key role in the metabolism-basedconversion of PK11195 in HPHs. It is not uncommon thatmetabolism can influence the pharmacological action of drugs.For instance, chrysin, a dietary flavonoid, markedly inducesthe expression and activity of UDP-glucuronosyltransferase1A1 in HepG2 and Caco-2 cells, but not in HPHs (Smith et al.,2005). Buprenorphine, a potent activator of PXR in HepG2cells, is not a physiologically relevant activator of PXR or aninducer of associated P450s in HPHs (Li et al., 2010). On theother hand, phenytoin, an antiepileptic agent, is a potent inducerof CYP2B6 and CYP3A4 in HPHs but does not activate CAR orPXR in cell-based reporter assays (Wang et al., 2004). Collec-tively, these studies demonstrate that themetabolic capacity of acellular systemcanbe akeydeterminant for the biologic functionof a given compound, including its role in nuclear receptoractivation.Previous reports have postulated that PK11195 is rapidly

biotransformed into two major metabolites: the N-desmethylmetabolite, ND-PK, and the amide hydrolysis product,COOH-PK (Roivainen et al., 2009). In the current study,we have evaluated the activation of hCAR and induction ofrelated P450s by both metabolites. Notably, ND-PK signifi-cantly increased the luciferase activity of theCYP2B6 reporterby activating hCAR or hCAR11A in HepG2 cells and inducedthe expression of CYP2B6 and CYP3A4 in HPHs, HepaRG,and PXR-KO HepaRG cells. In contrast, COOH-PK failed toactivate hCAR in HepG2 cells and only marginally inducedCYP2B6/CYP3A4 expression in HPHs. Consistent with theseobservations fluorescent microscopy analysis of adenovirus-expressing EYFP-hCAR-infected HPHs further confirmedthat ND-PK but not COOH-PK efficiently translocates CARfrom the cytoplasm to the nucleus of HPH, the first step inCAR activation. This discovery may also provide a mecha-nism for the previously observed PK11195-mediated CARnuclear translocation in HPHs (Li et al., 2009). Together, thesefindings suggest that removing onemethyl group fromPK11195changes it from an antagonist to an agonist of CAR andcontributes to P450 induction in HPHs.Mechanistically, ligand binding changes the secondary

structure of a nuclear receptor, influences the recruitment ofcoregulators, and alters the target gene expression thereaf-ter. Previous reports have indicated that nuclear localizedCAR can interact with coactivators such as SRC-1 andGRIP1 without the presence of agonists (Muangmoonchai

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et al., 2001; Min et al., 2002). Our mammalian two-hybridresults demonstrate that PK11195-repressed CAR/SRC-1interaction can be efficiently rescued by ND-PK. However,this recovery is moderate in the CAR/GRIP1 interaction,reflecting the differential capacity between PK11195 andND-PK in influencing the recruitment of SRC-1 versusGRIP1 by CAR.Computational modeling studies have shown that the

constitutive activity of CAR is mediated by residues in thebinding pocket interacting with and stabilizing the activationfunction 2 (AF2) domain (Andersin et al., 2003; Xu et al., 2004;Windshügel et al., 2005). Agonists tend to bind and furtherstabilize the AF2 domain in the active conformation, whileantagonists disrupt its stability (Jyrkkärinne et al., 2008). Toexplore how the N-demethylation of PK11195 has such adrastic effect on CAR activity, we used docking studies toprobe the structure-activity relationship of PK11195 andND-PKwith CAR. Although both compounds bound in similarconformations, PK11195 interacted with residues importantto CAR activation, such as V199, H203, Y224, and Y326, whileND-PK does not. Residues V199 and Y326 are thought tostabilize the CAR AF2 domain in the active conformation byinteracting with H12, while Y224 may be involved in localprotein folding. Mutating any of these residues abrogates thebasal activity of CAR while mutating H203 reduces CARactivity by 50%, demonstrating the importance of theseresidues to CAR activity (Jyrkkärinne et al., 2005). Therefore,PK11195 interactions with these residues may destabilize theAF2 domain, while loss of theN-methyl group in ND-PK couldrestabilize H12. Indeed, the potent CAR antagonist CINPA1also interacts with these residues, suggesting such interac-tions are important in CAR antagonism (Cherian et al., 2016).CITCO interacts with V199, Y224, and Y326, but the interac-tion distances are generally greater than those of PK11195 orCINPA1 and thus may not displace these residues enough toinhibit stabilization of H12 (Supplemental Table 1). Althoughdocking studies provide a possible mechanism for this drasticchange in CAR activity, future studies will use moleculardynamics and mutagenesis to further probe the detailedstructure-activity relationship between PK11195 and ND-PKwith CAR.In conclusion, this study demonstrates that PK11195 is

metabolically converted from an antagonist to an agonist ofhCAR and this conversion contributes significantly to theobserved induction of CYP2B6 and CYP3A4 in HPHs andHepaRG cells. We show that ND-PK is the metaboliteresponsible for PK11195-mediated CAR activation byfacilitating CAR interactions with SRC-1 and GRIP1and enhancing CAR nuclear translocation in HPHs. Thedemethylation of PK11195 also disrupts its interactionwith residues critical to CAR activity, providing a possiblemechanism for the activity shift. Additionally, this reporthighlights the importance of metabolic competence whenattempting to identify modulators of nuclear receptors andprovides a possible solution to this problem with a novel HPHcoculture system.

Acknowledgments

The authors thank Michael Mitchell from Sigma Life Sciences forkindly providing the PXR-KO-HepaRG cell line. The LC-MS analysisof PK11195 metabolism was performed in the Mass SpectrometryCenter at the University of Maryland School of Pharmacy.

Authorship Contributions

Participated in research design: Mackowiak, L. Li, Welch, D. Li,Jones, Kane, Swaan, Wang.

Conducted experiments:Mackowiak, L. Li, Welch, Jones, Heyward.Contributed new reagents or analytic tools: Heyward.Performed data analysis: Mackowiak, L. Li, Welch, Jones, Kane,

Swaan, Wang.Wrote or contributed to the writing of the manuscript: Mackowiak,

L. Li, Welch, Jones, Kane, Swaan, Wang.

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