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

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

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- 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 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 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 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 Cl 31.6 (48.8) 7.9 (128.3) inactive 35.5 (96.2)

N N Cl Cl

O N O H inactive 5.0 (88.0) inactive 50.1 (35.5)

Dinaline 28.2 (52.5) inactive 12.6 (324.2) inactive

Docusate 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 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 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 Sodium + inactive 5.6 (93.6) inactive 17.8 (140.7) - Na O OH O

O O

HO 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- 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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