External ID: CHEMBL4236714

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Journal: J Med Chem
Year: 2018
Volume: 61
Issue: 18
First Page: 8255
Last Page: 8281
DOI: 10.1021/acs.jmedchem.8b00734

Target ChEMBL ID: CHEMBL2863
ChEMBL Target Name: Transient receptor potential cation channel subfamily V member 2
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL683425

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem. Lett.
Year: 2000
Volume: 10
Issue: 23
First Page: 2613
Last Page: 2616
DOI: 10.1016/s0960-894x(00)00528-x

Target ChEMBL ID: CHEMBL3229
ChEMBL Target Name: Anandamide amidohydrolase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: H - Homologous protein target assigned
Confidence: Homologous single protein target assigned

External ID: FXRN10

Protocol: NCGC Assay Protocol Summary

Using a beta-lactamase reporter-gene under control of Farnesoid X receptor (FXR) response element, a cell-based assay [GeneBLAzer FXR-UAS-bla assay developed by Invitrogen] was used to measure FXR ligands signaling. GeneBLAzer FXR-UAS-bla HEK293T cells were used with assay medium containing 10% dialyzed FBS. The assay was performed in clear bottom black Greiner 1536-well plates. Guggulsterone were used in the screen as the positive control. Library compounds were measured for their ability to inhibit the reporter gene activity. Compounds were screened in a titration series in 1536-well format. The fluorescence intensity (405 nm excitation, 460/530 nm emission) was measured using an EnVision plate reader. Data was normalized relative to Chenodeoxycholic acid (50 uM, 100%), and DMSO only wells (basal, 0%). Concentration-response titration points for each compound were fitted to the Hill equation yielding concentrations of half-maximal inhibition (IC50) and maximal response (efficacy) values.

qHTS protocol for FXR beta-lactamase assay in antagonist mode

[Step] [Parameter] [Value] [Description]
1. Reagent; 5 uL; 5000 cells/well
2. Incubation; 37 C for 5-7 hrs
3. Compounds; 23 nL; 0.59 nM to 92 uM
4. Controls (Guggulsterone, FXR antagonist); 23 nL,
- One column of dose response, start 200 uM, 1:2 dilutions in duplicates
5. Agonist (Stimulator); 1uL; Chenodeoxycholic acid at 100 uM and 50 uM final concentrations
6. Incubation; 37 C for 16 hrs
7. Detection reagent; 1 uL of CCF4-dye
8. Incubation; RT for 2 hrs
9. Detection; Fluorescence; Envision plate reader

Comment: Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, active compounds are expected to have the inhibitor phenotype and apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

External ID: CHEMBL4236713

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Journal: J Med Chem
Year: 2018
Volume: 61
Issue: 18
First Page: 8255
Last Page: 8281
DOI: 10.1021/acs.jmedchem.8b00734

Target ChEMBL ID: CHEMBL2863
ChEMBL Target Name: Transient receptor potential cation channel subfamily V member 2
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: FXRA10

Protocol: NCGC Assay Protocol Summary

Using a beta-lactamase reporter-gene under control of Farnesoid X receptor (FXR) response element, a cell-based assay [GeneBLAzer FXR-UAS-bla assay developed by Invitrogen] was used to measure FXR ligands signaling. GeneBLAzer FXR-UAS-bla HEK293T cells were used with assay medium containing 10% dialyzed FBS. The assay was performed in clear bottom black Greiner 1536-well plates. Chenodeoxycholic acid and Guggulsterone were used in the screen as agonist and antagonist positive controls respectively. Library compounds were measured for their ability to stimulate the reporter gene activity. Compounds were screened in a titration series in 1536-well format. The fluorescence intensity (405 nm excitation, 460/530 nm emission) was measured using an EnVision plate reader. Data was normalized relative to Chenodeoxycholic acid control (100 uM, 100%), and DMSO only wells (basal, 0%). Concentration-response titration points for each compound were fitted to the Hill equation yielding concentrations of half-maximal stimulation (EC50) and maximal response (efficacy) values.

qHTS protocol for FXR beta-lactamase assay in agonist mode

[Step] [Parameter] [Value] [Description]
1. Reagent; 6 uL; 5000 cells/well
2. Incubation; 37 C for 5-7 hrs
3. Compounds; 23 nL; 0.59 nM to 92 uM
4. Controls (Chenodeoxycholic acid); 23 nL,
- One column with dose response, start 200 uM, 1:2 dilutions in duplicates
- Final concentrations of 100 uM and 60 uM in two columns
5. Incubation; 37 C for 16 hrs
6. Detection reagent; 1 uL of CCF4-dye
7. Incubation; RT for 2 hrs
8. Detection; Fluorescence; Envision plate reader

Comment: Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, active compounds are expected to have the activator phenotype and apparent activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

External ID: ARE853

Protocol: Assay Protocol Summary:

ARE-bla HepG2 cells were dispensed at 2,000 cells/5 ul/well in 1536-well black wall/clear bottom plates using a Multidrop Combi (ThermoFisher Scientific, Waltham, MA) dispenser. After the assay plates were incubated at 37C/5% CO2 for 5 hrs, 23 nL of compounds dissolved in DMSO, positive controls or DMSO only was transferred to the assay plate by a pintool work station (Kalypsys, San Diego, CA). The plates were incubated at 37C/5% CO2 for 16 hrs. After 1 uL of LiveBLAzer B/G FRET substrate was added using a Flying Reagent Dispenser (FRD) (Aurora Discovery, San Diego, CA), the assay plates were incubated at room temperature for 2 hrs. The fluorescence intensity was measured by an Envision (PerkinElmer, Shelton, CT) plate reader. Cell viability using CellTiter Glo viability assay (Promega, Madison, WI) was also measured by adding 4 ul/well of CellTiter-Glo reagent into the same assay plate using a FRD. After 30 min incubation at room temperature, the luminescence intensity in the plates was measured using a ViewLux (PerkinElmer) plate reader.

Comment: Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

External ID: RXRA10

Protocol: NCGC Assay Protocol Summary

Using a beta-lactamase reporter-gene under control of Retinoid X receptor--alpha (RXR-alpha) response element, a cell-based assay [GeneBLAzer (RXR-alpha -UAS-bla assay developed by Invitrogen] was used to measure RXR-alpha ligands signaling. GeneBLAzer RXR-alpha -UAS-bla HEK293T cells were used with assay medium containing 2% charcoal-stripped FBS. The assay was performed in clear bottom black Greiner 1536-well plates. 9-cis-retinoic acid was used in the screen as the positive control. Library compounds were measured for their ability to stimulate the reporter gene activity. Compounds were screened in a titration series in 1536-well format. The fluorescence intensity (405 nm excitation, 460/530 nm emission) was measured using an EnVision plate reader. Data was normalized relative to 9-cis-retinoic acid control (500 nM, 100%), and DMSO only wells (basal, 0%). Concentration-response titration points for each compound were fitted to the Hill equation yielding concentrations of half-maximal stimulation (EC50) and maximal response (efficacy) values.

qHTS protocol for RXR-alpha beta-lactamase assay in agonist mode

[Step] [Parameter] [Value] [Description]
1. Reagent; 6 uL; 2500 cells/well
2. Incubation; 37 C for 4 hrs
3. Compounds; 23 nL; 0.59 nM to 92 uM
4. Controls (9-cis-retinoic acid); 23 nL,
- One column with dose response, start 38 uM, 1:3 dilutions in duplicates
- Final concentrations of 500 nM and 50 nM in two columns
5. Incubation; 37 C for 18 hrs
6. Detection reagent; 1 uL of CCF4-dye
7. Incubation; RT for 2 hrs
8. Detection; Fluorescence; Envision plate reader

Comment: Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, active compounds are expected to have the activator phenotype and apparent activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

External ID: TRBA10

Protocol: NCGC Assay Protocol Summary

Using a beta-lactamase reporter-gene under control of the TR response element, a cell-based assay [GeneBLAzer TR-UAS-bla-GripTite assay developed by Invitrogen] was used to measure TR-beta signaling in agonist mode. TR-UAS-bla-GripTite HEK 293 cells were used with assay medium containing 2% charcoal/dextran FBS, 0.1 mM NEAA and 1 mM sodium pyruvate. The assay was performed in clear bottom black Greiner 1536-well plates. T3 was used as a positive control in the screen. Library compounds were measured for their ability to stimulate the reporter gene activity. Compounds were screened in a titration series in 1536-well format. The fluorescence intensity (405 nm excitation, 460/530 nm emission) was measured using an EnVision plate reader. Data was normalized relative to T3 control (10 nM, 100%), and DMSO only wells (basal, 0%). Concentration-response titration points for each compound were fitted to the Hill equation yielding concentrations of half-maximal stimulation (EC50) and maximal response (efficacy) values.

qHTS protocol for TR-beta beta-lactamase assay in agonist mode

[Step] [Parameter] [Value] [Description]
1. Reagent; 6 uL; 2500 cells/well
2. Incubation; 37 C for 4 hrs
3. Compounds; 23 nL; 0.59 nM to 92 uM
4. Controls (T3); 23 nL;
- Dose response; start 3.8 uM, 1:3 dilutions, duplicates in first column
- Two columns of each 10 and 0.4 nM (finals) concentrations.
5. Incubation; 37 C for 18 hrs
6. Detection reagent; 1 uL of CCF4-dye
7. Incubation; RT for 2 hrs
8. Detection; Fluorescence; Envision plate reader

Comment: Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, active compounds are expected to have the activator phenotype and apparent activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

External ID: ERN101

Protocol: NCGC Assay Protocol Summary

Using a beta-lactamase reporter-gene under control of the ER response element, a cell-based assay [ER-alpha-UAS-bla GripTiteTM cell-Based Assay from Invitrogen] was used to measure ER-alpha signaling pathway in antagonist mode. ER-alpha-UAS-bla-GripTite HEK 293 cells were used with assay medium containing 2% charcoal/dextran treated FBS, 0.1 mM NEAA and 1 mM sodium pyruvate. Cells were cultured in this assay medium overnight in the flasks before the assay. The assay was performed in clear bottom black Greiner 1536-well plates. 17-beta-estradiol was used as a positive control in the screen. Library compounds were measured for their ability to inhibit the reporter gene activity. Compounds were screened in a titration series in 1536-well format. The fluorescence intensity (405 nm excitation, 460/530 nm emission) was measured using an EnVision plate reader. Data was normalized relative to 17-beta-estradiol control (0.5nM, 100%), and DMSO only wells (basal, 0%). Concentration-response titration points for each compound were fitted to the Hill equation yielding concentrations of half-maximal inhibition (IC50) and maximal response (efficacy) values.

qHTS protocol for ER-alpha beta-lactamase assay in antagonist mode

[Step] [Parameter] [Value] [Description]
1. Reagent; 5 uL; 5000 cells/well
2. Incubation; 37 C for 4 hrs
3. Compounds; 23 nL; 0.59 nM to 92 uM
4. Controls (4-hydroxytamoxifen); 23 nL
- Dose response, Start 38uM, 1:2 dilutions with duplicates
- Two columns with 38 and 19uM (final) concentration.
5. Agonist (Stimulator); 1uL; 17-beta-estradiol at 0.5nM (final) concentration
6. Incubation; 37 C for 18 hrs
7. Detection reagent; 1 uL of CCF4-dye
8. Incubation; RT for 2 hrs
9. Detection; Fluorescence; Envision plate reader or Acumen plate reader

Comment: Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, active compounds are expected to have the inhibitor phenotype and apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

External ID: ERA101

Protocol: NCGC Assay Protocol Summary

Using a beta-lactamase reporter-gene under control of the ER response element, a cell-based assay [ER-alpha-UAS-bla GripTiteTM cell-Based Assay from Invitrogen] was used to measure ER-alpha signaling pathway in agonist mode. ER-alpha-UAS-bla-GripTite HEK 293 cells were used with assay medium containing 2% charcoal/dextran treated FBS, 0.1 mM NEAA and 1 mM sodium pyruvate. Cells were cultured in this assay medium overnight in the flasks before the assay. The assay was performed in clear bottom black Greiner 1536-well plates. 17-beta-estradiol was used as a positive control in the screen. Library compounds were measured for their ability to either stimulate the reporter gene activity. Compounds were screened in a titration series in 1536-well format. The fluorescence intensity (405 nm excitation, 460/530 nm emission) was measured using an EnVision plate reader. Data was normalized relative to 17-beta-estradiol control (20 nM, 100%), and DMSO only wells (basal, 0%). Concentration-response titration points for each compound were fitted to the Hill equation yielding concentrations of half-maximal stimulation (EC50) and maximal response (efficacy) values.

qHTS protocol for ER-alpha beta-lactamase assay in agonist mode

[Step] [Parameter] [Value] [Description]
1. Reagent; 6 uL; 5000 cells/well
2. Incubation; 37 C for 4 hrs
3. Compounds; 23 nL; 0.59 nM to 92 uM
4. Controls (17-beta-estradiol); 23 nL
- Dose response, start 1uM, 1:3 dilutions with duplicates
- Two columns with 20 and 0.5 nM (final) concentration.
5. Incubation; 37 C for 18 hrs
6. Detection reagent; 1 uL of CCF4-dye
7. Incubation; RT for 2 hrs
8. Detection; Fluorescence; Envision plate reader or Acumen plate reader

Comment: Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, active compounds are expected to have the activator phenotype and apparent activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.