## Endpoint to Predict
- Endpoint scope: assay
- Measurement type: EC50
- Label type: continuous
- Binary semantics: pchembl
- Assay description: transient human FXR/Gal4-luciferase reporter assay: A Gal4-hFXR fusion construct reporter system was used as the primary assay to characterize compound activity. A construct including 5 copies of the Gal4 promoter response element upstream of a firefly luciferase reporter cDNA was stably expressed in HEK293 cells. This reporter cell line was maintained in Dulbecco's Modified Eagle's medium (DMEM; Gibco) supplemented with 1% penicillin-streptomycin (P/S) solution, 500 μg/ml Zeocin and 10% charcoal/dextran-treated fetal bovine serum (cs-FBS) at 37° C. in a humidified 5% CO2 atmosphere. Another plasmid was constructed in which the human cytomegalovirus promoter in the pcDNA3.1 vector directs the expression of the cDNA encoding a fusion protein comprised of the DNA binding domain from the Gal4 transcription factor fused to the ligand binding domain from human FXR.The day prior to transfection, the reporter cells in culture are detached from the plate with trypsin and plated into a T75 flask at a sufficient density to achieve approximately 90% confluence the next morning. The transfection reagents are prepared by separately diluting 25 μg of the pcDNA3.1-Gal4-FXR plasmid into 1.87 mL of Opti-MEM (Thermo-Fisher), and 40 μL of Lipofectamine 2000 (Thermo-Fisher) into 1.87 mL of Opti-MEM, and then adding the diluted DNA solution into the diluted Lipofectamine 2000 solution and incubating at room temperature for 15-20 minutes. The mixture is further diluted with 10 mL of a solution comprised of DMEM, 10% cs-FBS, and 1% P/S immediately prior to transferring to the cells. The maintenance culture media is aspirated from the cells and the final transfection mixture is added before the cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. This protocol can be scaled up, and the transiently transfected cells can be cryopreserved in an assay-ready format.For compound testing, 100 nL of the compounds (serial dilutions in DMSO) are dispensed with an Echo acoustic dispenser (Labcyte) into the wells of a Corning/Costar clear bottom 384-well white plate. The transfected cells are harvested, counted, and diluted such that 10-25,000 cells in 25 μL are plated into each well of the 384-well compound assay plate. The compound-treated cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. The next morning 25 μL of Steady-Glo (Promega) are added to each well of the plate, the mixture is incubated for 15 min. with shaking, and luminescence is measured on an Envision (Perkin Elmer) plate reader. Background counts from cells treated with DMSO alone are subtracted from all raw counts, and the corrected values are converted to a percentage of the control response attained with 8 μM GW-4064. These data are fit to a 4-parameter log agonist-response equation to calculate an EC50 value.
- Assay organism: Homo sapiens
- Target name: Bile acid receptor
- Target type: SINGLE PROTEIN
- Target organism: Homo sapiens

## Reference Molecule A
- Canonical SMILES: O=C(O)c1ccc2nc(C34CCC(OCc5c(-c6c(Cl)cncc6Cl)noc5C5CC5)(CC3)CC4)ccc2n1
- Properties:
- ChEMBL ID: CHEMBL5998796
- Molecular weight: 565.46
- Heavy atoms: 39
- Hydrogen bond donors: 1
- Hydrogen bond acceptors: 7
- TPSA: 111.23
- LogP: 7.12
- Rotatable bonds: 7
- Ring count: 8
- Aromatic rings: 4
- Formal charge: 0
- Fraction Csp3: 0.41
- QED: 0.25

## Known Reference Result on This Endpoint
- Molecule A clean result: median pChEMBL = 7.28
- This result is provided as context for predicting Molecule B on the same endpoint.

## Reference Molecule A Other ChEMBL Evidence
- type EC50; relation =; value 53; units nM; pChEMBL 7.28; comment 933008

## Query Molecule B
- Canonical SMILES: O=C(O)c1ccc(-c2noc(C34CCC(OCc5c(-c6ccccc6OC(F)(F)F)noc5C5CC5)(CC3)CC4)n2)nc1
- Properties:
- ChEMBL ID: CHEMBL5796472
- Molecular weight: 596.56
- Heavy atoms: 43
- Hydrogen bond donors: 1
- Hydrogen bond acceptors: 9
- TPSA: 133.6
- LogP: 6.82
- Rotatable bonds: 9
- Ring count: 8
- Aromatic rings: 4
- Formal charge: 0
- Fraction Csp3: 0.43
- QED: 0.22

## Molecule Similarity
- Weighted Tanimoto: 0.44

Answer: 5.82