Supplementary Information for Drug Metabolism and Disposition
Identification of Clinically Utilized Drugs that Activate Pregnane X Receptors
Sunita J. Shukla, Srilatha Sakamuru, Ruili Huang, Timothy A. Moeller, Paul Shinn, Danielle Van Leer, Douglas S. Auld, Christopher P. Austin and Menghang Xia
1 Table S1—Final 1,536-well hPXR assay protocol
Step Parameter Value Description
1 Reagent 5 µL HepG2 (DPX-2) cells 2 Incubation time 5 hr 37°C incubation 3 Library compounds 23 nl Compound dilution series 4 Controls 23 nl Rifampicin
5 Incubation time 24 hr 37°C incubation
6 Reagent 5 µL Detection Reagent 7 Incubation time 30 min Room temperature 8 Detection 30 sec ViewLux luminescent read
Step Notes
1 Medium-binding white solid bottom Greiner plates. 2K cells/well/5 µL plated using an FRD.
3 Pin-tool transfer NPC compound library for a (final) range of 46 µM to 2.8 nM (15 point titration).
Pin-tool transfer, Column 1: two-fold sixteen-point rifampicin titration starting at 46 µM to 1.4 nM 4 (final); Column 2: 23 µM rifampicin (final); Column 3: DMSO; Column 4: 9.2 µM rifampicin (final). Pin-tool transfer tip wash sequence: DMSO, iPA, MeOH, >3 s vacuum dry.
6 ONE-Glo™ Luciferase Assay Reagent. 8 30 s exposure, gain high, and 2x binning luminescent read on Perkin Elmer ViewLux.
2 Table S2—Final 1,536-well rPXR assay protocol
Step Parameter Value Description
1 Reagent 5 µL HepG2 cells 2 Incubation time 5 hr 37°C incubation 3 Library compounds 23 nl Compound dilution series 4 Controls 23 nl Dexamethasone
5 Incubation time 24 hr 37°C incubation
6 Reagent 5 µL Detection Reagent 7 Incubation time 30 min Room temperature 8 Detection 40 sec ViewLux luminescent read
Step Notes
1 Medium-binding white solid bottom Greiner plates. 2K cells/well/5 µL plated using an FRD.
3 Pin-tool transfer NPC compound library for a (final) range of 46 µM to 2.8 nM (15 point titration).
Pin-tool transfer, Column 1: two-fold sixteen-point dexamethasone titration starting at 92 µM to 2.8 4 nM (final); Column 2: 46 µM dexamethasone (final); Column 3: DMSO; Column 4: 23 µM dexamethasone (final). Pin-tool transfer tip wash sequence: DMSO, iPA, MeOH, >3 s vacuum dry.
6 ONE-Glo™ Luciferase Assay Reagent. 8 40 s exposure, gain high, and 3x binning luminescent read on Perkin Elmer ViewLux.
3 Table S3—Final 1,536-well CYP3A4 induction assay protocol
Step Parameter Value Description
1 Reagent 5 µL HepG2 cells (DPX-2) 2 Incubation time 3.5 hr 37°C incubation 3 Library compounds 23 nl Compound dilution series 4 Controls 23 nl Rifampicin
5 Incubation time 24 hr 37°C incubation
6 Reagent 1 µL Assay substrate
7 Incubation time 3 hr 37°C incubation
8 Reagent 5 µL Detection reagent 9 Incubation time 30 min Room temperature 10 Detection 30 sec ViewLux luminescent read
Step Notes
1 Medium-binding white solid bottom Greiner plates. 2K cells/well/5 µL plated using an FRD.
3 Pin-tool transfer NPC compound library for a (final) range of 46 µM to 2.8 nM (15 point titration).
Pin-tool transfer, Column 1: two-fold sixteen-point rifampicin titration starting at 46 µM to 1.4 nM 4 (final); Column 2: 46 µM rifampicin (final); Column 3: 23 µM rifampicin (final); Column 4: DMSO. Pin-tool transfer tip wash sequence: DMSO, iPA, MeOH, >3 s vacuum dry.
6 P450-Glo™ Assay substrate [3uM (final)]
8 P450-Glo™ Assay Reagent. 10 30 s exposure, gain high, and 2x binning luminescent read on Perkin Elmer ViewLux.
4 Table S4—Assay statistics
Assay Positive controls EC50 (mean, μM) S/B ratio* Z’ hPXR Rifampicin 3.5 5.7 0.23 hPXR LBD SR12813 0.44 3.1 0.68 rPXR Dexamethasone 2.6 4.9 0.24 CYP3A4 Rifampicin 3.3 2.7 0.36 * S/B = Signal to Background
5 Table S5--Potencies (μM) and efficacies (%) of 72 compounds from cherry-pick studies
Compound Structure hPXR EC50, μM hPXR LBD EC50, rPXR CYP3A4 (Efficacy, %) μM (Efficacy, %) EC50, μM EC50, μM (Efficacy, %) (Efficacy, %) AA-861 28.2 (44.3) 12.6 (119.8) inactive 7.1 (29.3)
Alpha-Lipoic acid inactive 2.8 (107.9) inactive inactive
Ambrisentan 31.6 (46.7) Inactive inactive 31.6 (117.4)
AMI-193 inactive 31.6 (138.7) inactive inactive
Amlodipine Besylate 31.6 (90.5) 28.2 (47.2) inactive 31.6 (257.6)
Aurothioglucose inactive 2.5 (111.7) inactive inactive
Bithionol Inactive 1.0 (50.0) Inactive 12.6 (269.9)
6 Bosentan 14.1 (71.5) 8.9 (82.8) Inactive 12.6 (163.5)
Bumecainum 31.6 (200) 5.0 (80.8) Inactive 7.9 (237.0)
Cephaloridine inactive 3.2 (97.7) inactive inactive
Choroxine OH 39.8 (107.9) inactive inactive inactive Cl N
Cl Ciglitazone 14.1 (96.1) 4.5 (108.0) inactive 10 (209.4)
Clofenvinfosum 12.6 (87.2) 7.9 (108.7) 12.6 (63.4) 10 (198.8)
Colforsin 7.9 (120.6) 14.1 (54.7) 7.9 (76.0) 2.8 (237.0)
7 Demecolcine 12.6 (51.1) 14.1 (103.0) 0.09 (103.0) inactive
Desoximetasone inactive inactive 10 (239.4) 31.6 (101.9)
Dibunate Sodium 25.1 (187.1) 6.3 (98.8) inactive 8.9 (165.6)
N Diclazuril Cl 31.6 (48.8) 7.9 (128.3) inactive 35.5 (96.2)
N N Cl Cl
O N O H Dimercaprol inactive 5.0 (88.0) inactive 50.1 (35.5)
Dinaline 28.2 (52.5) inactive 12.6 (324.2) inactive
Docusate Calcium 25.1 (129.4) 0.89 (104.1) inactive 15.8 (68.8)
Dodecylbenzenesulfonic inactive 15.8 (100.1) inactive inactive acid, sodium salt
8 Dronabinol 31.6 (79.3) 12.6 (60.8) inactive 12.6 (81.5)
Droxidopa inactive 3.2 (84.9) 63.1 (48.4) 0.004 (29.9)
Eburnamonine (-) 14.1 (60.3) 12.6 (49.9) 14.1 (144.3) inactive
Ecopipamum 4.0 (84.7) 6.3 (68.3) Inactive 12.6 (264.0)
Enzacamene 31.6 (60.4) 5.0 (93.2) inactive 31.6 (118.1)
Epothilone B 12.6 (62.5) 31.6 (89.9) 0.01 (102.9) inactive
9 Famprofazone 8.9 (98.3) 1.3 (97.8) 12.6 (41.8) 4.0 (136.2)
Febantel 15.8 (67.6) inactive 31.6 (136.5) 10 (54.2)
Felodipine 12.6 (69.5) 7.1 (79.9) 28.2 (77.3) 2.5 (53.3)
Fenbendazole 10 (33.7) inactive 4.0 (317.2) inactive
Fentanyl 31.6 (93.9) 31.6 (102.9) inactive 31.6 (205.6)
Fluorometholone 12.6 (31.0) 1.3 (88.2) 1.6 (221.0) 3.2 (182.4)
10 Haloprogin Inactive 10 (123.8) inactive inactive
Hetacillin Potassium Inactive 6.3 (84.0) inactive inactive
Hydralazine Hydrochloride inactive Inactive 0.45 (48.1) inactive
Idinavir Sulfate 14.1 (79.2) inactive inactive 15.8 (221.6)
OH O Lasalocid Sodium + inactive 5.6 (93.6) inactive 17.8 (140.7) - Na O OH O
O O
HO Levothyroxine Inactive 11.2 (105.2) Inactive 100 (87.7)
Linolenic Acid inactive 2.5 (110.6) inactive 0.002 (16.3)
11 LOE 908 Hydrochloride 3.2 (35.8) 15.8 (72.8) 12.6 (56.7) inactive
Loratadine Cl 10.0 (60.0) 10 (97.4) inactive 12.6 (297.1)
N
N
O O
Methacycline Inactive 3.2 (79.5) inactive 12.6 (50.6) Hydrochloride
6-α-Methylprednisolone inactive inactive 4.0 (84.7) 12.6 (112.2)
Nilvadipine 10.0 (133.4) 0.89 (102.4) 14.1 (61.2) 4.0 (273.7)
12 Nisoldipine 10.0 (46.1) 7.9 (77.5) 31.6 (75.4) 12.6 (223.5)
Norelgestromin 25.1 (64.5) inactive inactive inactive
Orlistat Inactive 1.6 (52.0) inactive 28.2 (49.6)
Oxatomide 7.1 (67.9) 7.9 (71.5) 25.1 (52.2) 4.0 (129.4)
Plicamycin inactive 15.8 (36.7) 0.32 (116.5) inactive
Proadifen Hydrochloride 25.1 (97.2) 14.1 (70.2) 35.5 (61.9) 12.6 (259.7)
Rifampicin 7.9 (147.0) 0.20 (43.9) Inactive 3.2 (459.2)
13 Rifaximin 14.1 (65.3) inactive inactive 14.1 (265.6)
Rimexolone 4.5 (82.1) 2.5 (118.5) 1.4 (165.4) 1.6 (140.8)
Riodipine 11.2 (96.7) 14.1 (82.3) 22.4 (150.6) 1.6 (122.3)
Sirolimus inactive 0.89 (80.7) Inactive 10 (93.2)
Spiroxatrine 20.0 (49.3) 25.1 (71.0) inactive 35.5 (95.1)
Suberoylanilide 35.5 (55.1) 25.1 (82.7) 2.5 (307.6) inactive
Suramin Inactive 1.3 (66.0) inactive inactive Tacrolimus inactive 5.0 (109.3) inactive inactive
14 Tenilidona 11.2 (119.0) 10 (57.2) 4.5 (70.7) inactive
Terconazole 8.9 (32.3) inactive Inactive 14.1 (46.1)
Testosterone Isocaproate 31.6 (21.4) 11.2 (75.5) Inactive 28.2 (50.9)
Thiamylal Sodium 25.1 (115.7) 14.1 (62.3) Inactive 8.9 (228.1)
Thonzonium Bromide inactive 11.2 (134.3) Inactive 11.2 (36.0) Tianeptinum 22.4 (66.1) 39.8 (80.6) Inactive 14.1 (49.6)
Triiodothyronine, Reverse inactive 12.6 (103.5) Inactive inactive
Troglitazone 31.6 (79.5) 8.9 (90.1) Inactive 31.6 (136.8)
Tropanyl 3,5- 11.2 (62.1) 14.1 (35.8) Inactive 28.2 (244.0) Dimethulbenzoate
15 Zafirlukast 14.1 (76.4) 0.71 (101.8) Inactive 12.6 (188.2)
Zearalanol 31.6 (77.5) 4.0 (73.7) Inactive 25.1 (123.2)
16 Table S6--Potencies (μM) and efficacies (%) of 26 re-acquired compounds from confirmation studies
Compound Structure hPXR hPXR LBD rPXR CYP3A4 CYP3A4 EC50, μM EC50, μM EC50, μM EC50, μM EC50, µM (Efficacy, %) (Efficacy, %) (Efficacy, %) (Efficacy, %) (Hepatocytes) Bumecainum 11 (158) inactive 38 (117.2) 7 (102) 1.1
Ciglitazone Chiral 23.6 (170.3) 41 (110.9) inactive 10.8 (84.1) N/A O
O S HN O Colforsin O Chiral 9 (283.4) inactive 7.1 (158.1) 2.7 (93.2) 0.7 HO O H O
HO OHO N Diclazuril Cl 38.7(45.2) inactive 9 (50.9) inactive N/A
N N Cl Cl
ONO H O Famprofazone Chiral 5.1 (83.1) inactive 9.8 (102.7) 3.1 (78.2) 1.4 N N N
17 Felodipine 3.9 (86) 17.1 (81.1) 23.4 (188) 1.9 (60) N/A
Fenbendazole 29.8 (180.2) inactive 5.1 (183.6) inactive N/A
Fluorometholone Chiral 26.9 (63.5) inactive 1.7 (219.3) inactive N/A
O F OHO HO Hetacillin Potassium inactive 19.5 (72) inactive inactive N/A
Hydralazine inactive inactive 1.6 (87.5) inactive N/A Hydrochloride
Methacycline inactive 30 (61.5) inactive inactive N/A Hydrochloride
6-α- inactive inactive 10 (146.5) inactive N/A Methylprednisolone
18 Nilvadipine 2 (92.8) inactive 10.5 (175.2) 1.9 (99.7) 1.4
Oxatomide 9.7 (174.5) 36.4 (76.3) 27.7 (86.8) 3.5 (56.1) 0.4
H H HH HO H H H H H H H O Plicamycin O H H inactive inactive 0.4 (172.8) inactive N/A O H H O H HO O H O O H H H H H H H H H HH H H O H H OH H H H O H H H O OOH H H H H O H H H H O H H H HH H H H O H H HO O H O H H H H O H O H H H HH O H H H H Rimexolone 4 (104.7) inactive 0.68 (114.8) 2.1 (63.3) 0.4
Riodipine 4 (115.8) inactive 12.6 (155.5) 1.8 (84.6) N/A OO
F OO NH
F O
19 H H Chiral H H Sirolimus O HO inactive 22 (83) inactive inactive N/A H H H H H H H H H H H H H H H H H H H H H H H O O H H H H H H H OO H H H H H O H HO H H H H H H H H N H H H H H HHO H O O H H O H H H O HH H H H H H H H H H H H H H H Suramin inactive 1.8 (67) inactive inactive N/A
Tacrolimus inactive 15.4 (60.9) inactive inactive N/A
Terconazole 14.3 (80.2) inactive 9.1 (33.1) inactive N/A
20 Thiamylal Sodium 4.1 (83.6) inactive 45.9 (76.2) 6.4 (118.6) N/A
Thonzonium Bromide inactive 20.7 (94) inactive inactive N/A Troglitazone 38.4 (89.5) 24.4 (56.2) inactive 6.9 (42.6) N/A
Zafirlukast 30.6 (147.8) 2.1 (104) 58.4 (62.8) 9.5 (55.3) N/A
Zearalanol 23.7 (52.1) 15.1 (91.8) 38.4 (46.8) 25.8 (51.5) N/A
21