Electronic Supplementary Material
Identification of approved drugs as potent inhibitors of pregnane X receptoractivationwith differential receptor interaction profiles
Oliver Burk, Maria Kuzikov, Thales Kronenberger, Judith Jeske, Oliver Keminer, Wolfgang E. Thasler, Matthias Schwab, Carsten Wrenger, Björn Windshügel
Correspondence to:
B. Windshügel, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schnackenburgallee 114, 22525 Hamburg, Germany. Phone +49 40 303764 286, Fax +49 40 303764 100, E-mail
O. Burk, Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Auerbachstrasse 112, 70376 Stuttgart, Germany. Phone +49 711 81013753, Fax +49 711 859295, E-mail
Supplementary Methods
Cell viability assays
Potential cell toxicity of the tested compounds was evaluated by ATP quantification using the CellTiter-Glo viability assay kit (Promega). HepG2 cells were seeded at 10,000 cells/well (25µl) in 384-well plates (Greiner) and incubated for 16h.Compounds to be tested (or DMSO) were added at the indicated concentrations aforementioned for an additional incubation of 48h at 37°C. After incubation, 20µl of CellTiter-Glo reagent were added and incubated for 30min at room temperature protected from light. Luminescence was quantified by EnVision plate reader (PerkinElmer, Germany) and compared to DMSO-treated cells.
Mammalian one-hybrid assay
The expression plasmid encoding GAL4-DBD/PXR-LBD(108-434) was constructed by cloning the EcoRI/XbaI insert of the corresponding VP16-AD/PXR-LBD(108-434) expression plasmid (Burk et al., 2005) into vector pM (Takara Clontech, Mountain View, CA).Transient transfections and reporter gene activity measurements for the PXR one-hybrid assay were performed as described in the Methods sectiondose response analysis, using per well a plasmid mixture consisting of 0.3 µg pGL3-G5 reporter gene plasmid, 0.04 µg of the expression plasmid described above, 0.01 µg pRL-CMV and 0.15 µg pUC18.
Supplementary Table S1Overview on evaluated signal window (SW), Z’ value and assay variability ratio (AVR) for the screen.
Assay Plate Barcode / SW / Z' / AVRESP0023198 / 2.84 / 0.44 / 0.56
ESP0023200 / 4.07 / 0.49 / 0.51
ESP0023202 / 3.46 / 0.46 / 0.54
Avg. ±SD / 3.46 ±0.53 / 0.46 ±0.02 / 0.53 ±0.02
Supplementary Table S2Amino acid residues of the PXR LBP(PDB ID: 2O9I) interacting with docked ligands pimecrolimus (PIM) and pazopanib (PAZ). For comparison, also interactions between co-crystallised ligands (T0901317, SR12813, hyperforin) and PXR (PDB ID: 2O9I, 1NRL, 1M13) are listed. An interaction is defined as at least one heavy atom of the ligand and a neighboring amino acid within 4Å distance to each other. Residues involved in hydrogen bonding or salt bridge formation with the ligand are underlined.
PIM / PAZ / T0901317 / SR12813 / Hyperforin1M13 / 2O9I / 2O9I / 1NRL / 1M13
Leu206 / Leu206
Leu209 / Leu209 / Leu209 / Leu209 / Leu209
Val211 / Lys210
Ile236
Phe237
Leu239
Leu240 / Leu240 / Leu240 / Leu240
Met243 / Met243 / Met243 / Met243 / Met243
Ala244
Met246 / Met246 / Met246
Ser247 / Ser247 / Ser247 / Ser247 / Ser247
Phe251
Phe281 / Phe281 / Phe281 / Phe281
Cys284
Gln285 / Gln285 / Gln285 / Gln285 / Gln285
Phe288 / Phe288 / Phe288 / Phe288 / Phe288
Trp299 / Trp299 / Trp299 / Trp299 / Trp299
Tyr306 / Tyr306 / Tyr306 / Tyr306
Met323 / Met323 / Met323 / Met323
Leu324 / Leu324
His327 / His327 / His327
His407 / His407 / His407 / His407
Thr408
Arg410 / Arg410
Leu411 / Leu411 / Leu411 / Leu411
Ile414 / Ile414
Phe420 / Phe420 / Phe420
Met425 / Met425
Phe429 / Phe429
Supplementary Table S3Amino acid residues of the PXR AF-2 site(PDB ID: 2O9I, 5A86) interacting with docked ligands pimecrolimus (PIM), and pazopanib (PAZ). An interaction is defined as at least one heavy atom of the ligand and a neighboring amino acid within 4Å distance to each other. Residues involved in hydrogen bonding or salt bridge formation with the ligand are underlined.
PIM / PAZ2O9I / 5A86
Lys252 / Lys252
Ile255 / Ile255
Ser256 / Ser256
Lys259 / Lys259
Phe264
Gln272
Ile273 / Ile273
Leu276 / Leu276
Lys277 / Lys277
Leu424
Glu427 / Glu427
Leu428 / Leu428
Supplementary Figure S1 Cell toxicity of novel PXR antagonists.HepG2 cells were treated with increasing concentrations of pimecrolimus (A), or pazopanib (B) or 0.1% DMSO for 48h. Cells then, had their viability measured by quantifying ATP content in comparison with control cells treated with vehicle (DMSO) using the CellTiter Glo kit. Columns represent means ±SEM (N≥3).
Supplementary Figure S2 Novel antagonists inhibit PXR activation by the high affinity agonist T0901317.HepG2 cells were transfected with empty vector pcDNA3 (negCTR) or expression plasmid encoding human PXR and treated with 0.1% DMSO, 10 µM pimecrolimus (PIM) or pazopanib (PAZ), with or without 1 µM T0901317 (T09) for 24 h. Columns show means ± SEM (N=4) of normalized luciferase activity of co-transfected CYP3A4 reporter, relative to the activity of cells transfected with pcDNA3 and treated with DMSO only. Significant differences to respective treatments with DMSO (asterisks, exclusively for single compound treatments) or T0901317 alone(daggers, exclusively for T0901317 co-treatments) were analysed by repeated measures two-way ANOVA with Dunnett’s multiple comparisons test. ***, ††† p<0.001.
Supplementary Figure S3 PXR antagonists dissociate corepressor SMRT from PXR. HepG2 cells were co-transfected with expression plasmids encoding GAL4-DBD/SMRT-RID and VP16-AD/PXR-LBD(108-434) fusion proteinsor empty vector pVP16-AD. Transfected cells were treated with 0.1% DMSO, 10µM each of rifampicin (RIF), pimecrolimus (PIM), pazopanib (PAZ) or sulforaphane (SFN), 1µM camptothecin (CAM), or 25µM ketoconazole (KCZ) for 24h. Columns show means ±SEM (N=3) of normalized luciferase activity of co-transfected pGL3-G5, relative to the activity of cells transfected with pVP16-AD and treated only with DMSO. Significant differences to respective treatments with DMSO were determined by repeated measure two-way ANOVA with Dunnett’s multiple comparisons test.***p<0.001.
Supplementary Figure S4Pimecrolimus activates the isolated PXR-LBD. HepG2 cells were co-transfected with an expression plasmid encoding GAL4-DBD/PXR-LBD(108-434) fusion protein and pGL3-G5 reporter. Transfected cells were treated for 24h with 0.1% DMSO, 10 µM each of rifampicin (RIF) or pimecrolimus (PIM) or a combination of both. Columns show mean fold induction ± SEM(N=4) of normalized luciferase activity by compound treatment. Significant differences to treatment with DMSO were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test. **p<0, ***p<0.001.
Supplementary Figure S5PXR antagonist fluorescence emission upon specific excitation. Fluorescence intensity emission (450 – 610 nm) of compounds was evaluated after excitation with340 nm. Fluorescence intensity of agonist rifampicin (RIF) and antagonists pimecrolimus (PIM), and pazopanib (PAZ) was measured after 6h incubation (each at 50µM) within TR-FRET assay buffer.
Supplementary Figure S6RPLP0 is only weakly modulated by pimecrolimus in primary human hepatocytes. Cultured cells from 6 donors were each treated with 0.1% DMSO, 1µM camptothecin (CAM), 10µM pimecrolimus (PIM) or pazopanib (PAZ), with or without 10µM rifampicin (RIF) for 24h. mRNA expression of RPLP0 was determined by TaqMan RT-qPCR and normalized to the expression of 18S rRNA. The data are shown as fold induction by chemical treatment, as compared to the expression in cells treated with DMSO only, which was designated 1, and are presented as box and whisker plots with boxes representing the 25th to 75th percentiles, medians indicated by horizontal lines and whiskers showing the minimum and maximum values. Significant differences to treatment with DMSO (asterisk, exclusively for single compound treatments), which was designated 1, were analysed by Wilcoxon signed rank test. Significant differences to treatment with rifampicin alone (dagger, exclusively for rifampicin co-treatments) were analysed by Friedman test with Dunn’s multiple comparisons test. *,† p<0.05.