Ligands

TNC, EE2, E2 and CC were purchased from Sigma-Aldrich. SR12813 was purchased from Tocris Bioscience. The provenance of compounds used for medium-throughput screening is described in Supplementary Table 1. All compound stock solutions were prepared at 10 mM in DMSO.

Plasmids

The [−7.8/−7.2] XREM-CYP3A4 (XREM-CYP3A4 pGL3b) luciferase plasmid has been described previously36. The 17Mx5-Glob-LUC containing five GAL4 binding sites upstream of the luciferase reporter gene and the VP16 (activation domain)-TIF2 (amino acids 624-869 containing three LXXLL motifs) are gifts from Hinrich Gronemeyer (IGBMC, Illkirch, France). The pM-hPXR expression vector was generated by inserting a PCR fragment corresponding to the full-length hPXR in the pM vector (CLONTECH).

Cell lines

LS174T stable human PXR transfectant (LS-PXR2) and the corresponding control cells (LS-CTRL), HEPG2-PXR and HG5LN GAL4-PXR reporter cell lines were previously described17,37,38. Briefly, LS-PXR2 was obtained after stable transfection of the LS174T cells with the pcDNA3.1-hPXR (residues 1-434)-neomycin expressing plasmid. HEPG2-PXR was obtained after stable transfection of the same PXR expressing plasmid and the XREM-CYP3A4 pGL3b reporter plasmid38. HG5LN GAL4-PXR reporter cell line was established by stable transfection of pSG5-GAL4DBD (residues 1-147)-hPXR LBD (residues 107-434)-puromycin and GAL4-RE5-βGlobin-luciferase-neomycin plasmids17. Finally, LS174T-PXR reporter cell line was established by stable transfection of the CYP3A4-luciferase-hygromycine plasmid in the LS-PXR2 cell line. All cell lines were grown in DMEM medium (Invitrogen) supplemented with 10% fetal calf serum, L-glutamine, and antibiotics (Invitrogen).

Medium-throughput screening

HG5LN GAL4-PXR reporter cell lines (100 μl) were seeded at a density of 25,000 cells per well in 96-well white opaque tissue culture plates (Greiner CellStar). 24 h later, negative (DMSO 0,1%) and positive (SR12813 3 μM) controls, and the 40 tested compounds (50 μl) were added into the wells as indicated in Supplementary Table 1. Then the ligand to be tested in combination with the different ligands was added (50 μl). Cells were incubated at 37 °C for 16 h. At the end of the incubation period, culture medium was replaced with medium containing 3.10−4 M luciferin. Luciferase activity was measured for 2 s in intact living cells using a plate reader (PerkinElmer Luminometer).

Transactivation assays

HG5LN GAL4-PXR, HEPG2- and LS174T-PXR reporter cell lines were seeded at a density of 25,000 cells per well in 96-well white opaque tissue culture plates (Greiner CellStar). Compounds to be tested were added 24 h later, and cells were incubated at 37 °C for 16 h. At the end of the incubation period, culture medium was replaced with medium containing 3.10−4 M luciferin. Luciferase activity was measured for 2 s in intact living cells using a plate reader (PerkinElmer Luminometer). EC 50 values were measured using GraphPad Prism (GraphPad Software Inc.).

Preparation of primary human hepatocytes

Liver samples were obtained from liver resections performed in adult patients for medical reasons unrelated to our research program or from donors when the liver was considered unsuitable for organ transplantation. The use of human specimens for scientific purposes was approved by the French National Ethics Committee. Written or oral informed consent was obtained from each patient or family prior to surgery. The clinical characteristics of the liver donors are presented in Supplementary Table 2. Hepatocytes were isolated by using a two-step perfusion protocol and cultured as described previously39. Briefly, several veins apparent on the cut edge of the lobectomy were used for sequential perfusions with a washing buffer (10 mM HEPES, 136 mM NaCl, 5 mM KCl, 0.5% glucose, pH 7.6), with a calcium chelating buffer (washing buffer complemented with 0.5 mM EGTA), with the washing buffer and then with a collagenase IV solution (washing buffer supplemented with collagenase IV, 200 U ml−1, Sigma) for 20 min. After gentle disruption of the tissue and filtration through a 250 μm mesh, the post-collagenase homogenate was centrifuged at low speed (50 g for 5 min) to pellet the hepatocytes. Hepatocytes were seeded in collagen type-I coated dishes (Becton Dickinson) at 1.7 105 cells per cm2 in a hormonally and chemically defined medium for short term culture consisting of a mixture of William’s E and Ham’s F-12 (1:1 in volume) and additives as previously described40 supplemented with 2% heat inactivated fetal calf serum (FCS). After overnight attachment, the medium and unattached cells were removed and fresh medium without FCS was added. Hepatocytes were treated with the molecules at day 2 post-seeding for 24 or 48 h.

Reverse transcription-PCR and real-time quantitative PCR

Total RNA was extracted from 200,000 cells using the RNeasy mini kit (Qiagen) or Trizol reagent (Invitrogen) and treated with DNAse-1. The first strand cDNA was synthetized using Superscript II (Invitrogen) or MMLV (Invitrogen) and random hexamers. Primer sequences are provided in Supplementary Table 3. Gene expression was normalized with GAPDH and the level of expression was compared with the mean level of the corresponding gene expression in untreated cells and expressed as n-fold ratio. The relative amount of RNA was calculated with the 2ΔΔCT method.

Western-immunoblotting analysis

Total protein extracts were prepared from 500,000 cells with RIPA buffer (Tebu-Bio) in presence of antiproteases (Roche). The protein concentration was determined by the bicinchoninic acid method, according to the manufacturer’s instructions (Pierce Chemical Co.). Bovine serum albumin (Pierce Chemical Co.) was used as standard. Cell lysates were resolved on SDS-PAGE and transferred to a Hybond-ECL membrane (GE Healthcare). Membranes were incubated with rabbit anti-CYP3A4 (5316, 1/10,000, Epitomics) or mouse anti-GAPDH (sc#32233, 1/5,000, Santa Cruz) monoclonal antibodies. Immunocomplexes were detected with horseradish peroxidase-conjugated rabbit (A0545, 1/10,000, Sigma) or mouse (A9044, 1/10,000, Sigma) secondary antibodies followed by enhanced chemiluminescence reaction (Millipore). Chemiluminescence was monitored using a ChemiDoc-XRS+ apparatus (Bio-Rad Laboratories).

Measurement of CYP3A4 activity

Primary human hepatocytes were seeded in 96-well plates and treated with the test compounds or vehicle for 48 h. CYP3A4 activity was detected using the P450-Glo CYP3A4 luciferin-IPA Enzyme Activity Kit (Promega) according to the manufacturer's instructions. Cell number was normalized using CellTiter-Glo Luminescent Cell Viability Assay (Promega).

Statistical analyses

For the analysis of the correlation between parametric data, Stutent’s t-test was used, while the Mann–Whitney U-test was used for nonparametric data. Differences were considered statistically significant when P-values were ***P<0.001 **P<0.01 *P<0.05.

Preparation of PXR/RXR for fluorescence anisotropy assays

The human PXR-LBD (130-434; pDB-His-MBP vector) and the human RXR-LBD (223-462; pET-3a vector) were overproduced in Escherichia coli BL21(DE3). Cells were grown at 37 °C in LB medium supplemented with the appropriate antibiotic until OD 600 reached 0.6. Expression of T7 polymerase was induced by addition of isopropyl-β-D-thiogalactoside to a final concentration of 0.5 mM. After 16 h of incubation at 20 °C, cell cultures were harvested by centrifugation at 6,000g for 15 min. Cell pellets from 3 L of His 6 -MBP-PXR-LBD culture and from 1 l of non-tagged RXR-LBD culture were resuspended in 150 ml buffer A (20 mM HEPES pH 7.5, 250 mM NaCl, 15 mM imidazole, 5% (v/v) glycerol) supplemented with a protease inhibitor cocktail tablet (cOmplete, EDTA-free, Roche). The suspension was then lysed by sonication and centrifuged at 18,000g at 4 °C for 30 min. The supernatant was loaded onto a Ni2+-affinity column (HisTrap 5 ml; GE Healthcare) equilibrated with buffer A, using the ÄKTA pure system (GE Healthcare). The column was washed with 20 volumes of buffer A and 20 volumes of buffer A containing 50 mM imidazole. The His 6 -MBP-PXR/RXR heterodimer was eluted with buffer A containing 100 mM imidazole. The fractions containing the heterodimer were pooled and incubated for 48 h at 4 °C with the tobacco etch virus protease to cleave the His 6 -MBP tag, and then further reloaded onto the Ni2+-affinity column to eliminate the tag. The flow-through containing the PXR/RXR heterodimer was then purified using a gel filtration column (Superdex 200 16/60; GE Healthcare) equilibrated with buffer B (10 mM Tris-HCl pH 7.5, 250 mM NaCl, 1 mM DTT and 5% (v/v) glycerol). The fluorescein-labelled SRC-1 fragment (residues 570–780) was prepared using the protocol previously described41. Briefly, the SRC-1fragment was expressed in E. coli BL21(DE3) as a fusion with an inducible self-splicing intein (Sce VMA) and a chitin-binding domain using the vector pTYB1 (New England Biolabs). The cells were grown at 37 °C to 0.6 at OD 600 and then at 17 °C overnight. The fusion protein was purified using chitin resin (New England Biolabs). Intein cleavage was induced using 2 mM cys-fluor with 50 mM MESNA, releasing C-terminally labelled SRC-1. Excess cys-fluor was removed using a phenyl sepharose resin (Amersham). SRC-1 was further purified using size-exclusion chromatography. Thin-layer chromatography was used to confirm that there was no free fluorescein label in the purified samples.

Steady-state fluorescence anisotropy

Measure of the binding affinities of the coactivator fragment for the PXR/RXR heterodimer in the absence and presence of various ligands was performed using a Safire2 microplate reader (TECAN). The excitation wavelength was set at 470 nm and emission measured at 530 nm for the fluorescein-tagged fragment. Assays were carried out in the following buffer solution 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM TCEP and 5% (v/v) glycerol. We initiated the measurements at the highest concentration of protein (20 μM) and diluted the protein sample twofold successively with the buffer solution. For each point of the titration curve, the protein sample was mixed with 5 nM of fluorescent fragment and a 3 molar excess of ligand (60 μM final concentrations). Binding data were fitted using a sigmoidal dose–response model using GraphPad Prism (GraphPad Software Inc.).

Mammalian two-hybrid experiments

Gal4-hPXR and VP16-TIF2 interaction was monitored on 17Mx5-Glob-LUC reporter construct. Transient transfections assays were performed in U2OS cells using Jet-PEI (Ozyme) according to manufacturer’s instructions. Luciferase assays were performed with the Promega dual-reporter kit, according to the manufacturer’s instructions. Renilla luciferase encoded by the normalization vector phRLTK (Promega) was used as internal control for firefly luciferase normalization.

Mass spectrometry

Mass spectrometry experiments were carried out on an electrospray time-of-flight mass spectrometer (LCT, Waters) equipped with an automated chip-based nanoESI device (Triversa Nanomate, Advion Biosciences). External calibration was done in the positive ion mode over the mass range m/z 500–5,000 using the multiply charged ions produced by 0.5 μM horse heart myoglobin solution diluted in water/acetonitrile 50/50 mixture acidified with 0.5% (v/v) formic acid. Purified PXR(130-434)-SRC1 was buffer exchanged against 50 mM ammonium acetate (NH4Ac), pH 8.0 using NAP5 desalting columns (illustra NAP-5 Columns, GE Healthcare Life Sciences). Protein concentration was determined spectrophotometrically (ɛ 280 nm =26,210 l mol−1 cm−1). Analysis of EE2 and TNC binding to PXR(130-434)-SRC1 was achieved in 50 mM NH 4 Ac pH 8.0 keeping a constant 5% amount of isopropanol (v/v). Protein concentration was set to 10 μM and different compound concentrations ranging from 20 to 80 μM were tested. Incubations lasted 5 min at 18 °C. Mass spectra were recorded using low cone voltage (V c , 20 V) and elevated interface pressure (Pi, 5 mbar).

Lanthascreen TR-FRET PXR competitive binding assay

GST-hPXR-LBD (10 nM) was incubated with different concentrations (10–30 μM) of TNC, EE2, TNC and EE2, and SR12813 in the presence of Fluormone PXR ligand (40 nM) and Lanthascreen terbium-anti-GST antibody (10 nM). To read a LanthaScreen TR-FRET assay, the fluorimeter (PHERAstar FS; BMG LABTECH) is configured to excite the terbium donor around 340 nm, and to separately read the terbium emission peak that is centred at ∼490 nm, and the fluorescein emission that is centred at ∼520 nm. Results are expressed as the signal from the fluorescein emission divided by the terbium signal to provide a TR-FRET emission ratio. Fluorescence ratio data were fitted using a sigmoidal dose–response model using GraphPad Prism (GraphPad Software Inc.).

Isothermal titration calorimetry

Purified PXR(130-434)-SRC-1 was dialysed overnight against Tris-HCl 20 mM, pH 8.5, NaCl 200 mM, TCEP 1 mM using 10 kDa molecular weight cut-off dialysis cassettes (Slide-A-Lyzer 0.5 ml 10 K MWCO, Thermo Scientific). Protein concentration was determined spectrophotometrically (ɛ 280 nm =26,210 l mol−1 cm−1). Duplicate experiments were performed on Microcal ITC200 (Malvern) operating at 25 °C. Titrations were carried out in Tris-HCl 20 mM, pH 8.5, NaCl 200 mM, TCEP 1 mM supplemented with 0.05% Tween 20 and 5% DMSO (syringe, sample and reference cells). PXR (5 μM) was disposed in 200 μl cell and compounds were delivered from 40 μl syringe. Compound solutions were set to 300 μM when tested individually (Fig. 5b,c), 50 μM each when used simultaneously (Fig. 5f) and 50 μM (EE2, Fig. 5d) or 200 μM (TNC, Fig. 5e) when tested after pre-incubation of PXR with 50 μM TNC or EE2, respectively. Heat exchanges were monitored throughout titrations consisting of 19 injections (one time 0.5 μl followed by 18 times 2 μl) of compound solutions into the cell containing PXR solution. Data analysis and thermodynamic parameter fitting used Microcal Origin software (Malvern).

Protein production and purification for structural studies

The human PXR-LBD (130-434) was co-produced with a fragment of the steroid receptor coactivator-1 (SRC-1, 623-710) to enhance PXR stability. The PXR-LBD gene was cloned into pRSET-A with a His 6 tag at the N-terminus (gift from Matthew Redinbo, University of North Carolina at Chapel Hill, USA), and the SRC-1 fragment gene has been inserted into the pACYC184 vector. Proteins were overproduced in E. coli BL21(DE3) cells overnight at 20 °C in LB medium without any ligand. After culture cells were harvested by centrifugation and the pellets resuspended in lysis buffer (20 mM Tris pH 7.5, 250 mM NaCl, 5% (v/v) glycerol) supplemented with lysozyme (1 μg ml−1) and a protease inhibitor cocktail tablet (cOmplete, EDTA-free, Roche), and then subjected to sonication. The clarified cell lysate was applied onto a Ni2+-affinity column (HisTrap 5 ml; GE Healthcare) equilibrated with lysis buffer supplemented with 10 mM imidazole. The eluted PXR-LBD was then applied onto a gel filtration column (Superdex 75 26/60; GE Healthcare) equilibrated with a buffer containing 20 mM Tris pH 7.8, 250 mM NaCl, 5% (v/v) glycerol, 5 mM DTT, 1 mM EDTA. The PXR-LBD was concentrated and stored at −40 °C.

Crystallization

Prior to crystallization assays the purified PXR-LBD (2.4 mg ml−1) was mixed with EE2 (2.5 molar equivalents), TNC (2.5 molar equivalents), or EE2+TNC (2.5 molar equivalents each). Co-crystals with EE2 were obtained in 100 mM NaCl, 100 mM imidazole pH 7.1, 10% (v/v) isopropanol, in 100 mM imidazole pH 7.1, 10% (v/v) isopropanol with TNC alone, and in 50 mM NaCl, 50 mM LiCl, 100 mM imidazole pH 7.1, 10% (v/v) isopropanol with EE2+TNC mixture.

Data collection and structure determination