External ID: SNCA-p-activity-luciferase

Protocol: PROTOCOL TABLE
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION

1; Cells; 4 uL; Dispense 1500 HEK-293-SNCA-luc cells/well into Greiner 1536-well white / solid bottom tissue culture treated plate. The plate was covered with metal lids with gas-exchange holes.
2; Incubate; 24 hours; Incubate at 37C, 5% CO2, 95% RH.
3; Compounds; 23 nL; Compounds and controls were transferred via a Kalypsys Pin Tool (Wako USA) equipped with a 1536-slotted pin array. The plate was covered with metal lids with gas-exchange holes.
4; Incubate; 24 hours; Incubate at 37C, 5% CO2, 95% RH.
5; Dispense; 1 uL; Dispense Gly-Phe-7-amino-4-trifluoromethylcoumarin (GF-AFC, prepared at 125 uM in PBS) was added. The plate was covered with metal lids with gas-exchange holes.
6; Incubate; 30 min; Incubate at 37C, 5% CO2.
7; Detector; Fluorescence; Measure fluorescence with ViewLux microplate reader (PerkinElmer) equipped with 405/10 excitation and 540/25 emission filters.
8; Dispense; 3 uL; Dispense ONE-Glo (PerkinElmer) lucifase detection reagent was added to each well. Plates were covered with metal lids with gas-exchange holes.
9; Incubate; 15 min; Incubate at room temperature.
10; Detector; Luminescence; Measure luminescence with ViewLux microplate reader (PerkinElmer) equipped with clear filters.

NOTES (numbers refer to sequence above)
1; HEK-293-SNCA-luc were cultured and suspended in phenol-red free DMEM (4.5 g/L glucose, 25 mM HEPES, cat #21063 (Thermo)).
3; Compounds were added to the assay plate in an 11-point intra plate dose response, 1:3 titration in DMSO with a final concentration range of xxx - yyy uM. Vehicle-only plates, with DMSO being pin-transferred to every well, were inserted at the beginning of screening runs to confirm expected assay performance. Activity was normalized to wells containing medium only (-100% activity, full inhibition) and SNCA-luc cells treated with DMSO vehicle control (0% activity), contained on the same plate as test samples.
10; Signals were analyzed, and dose-response curves were fit using the Hill equation. Compounds in curve classes -1.1, -1.2, -2.1, -2.2 in the SNCA-luc assay were considered active. Compounds were eliminated from further consideration if also active (curve class -1.1, -1.2, -1.3, -1.4, -2.1, -2.2, -2.3, -2.4) in the GF-AFC cytotoxicity assay.

Comment: Disclaimer:
Although all reasonable efforts have been made to ensure the accuracy and reliability of the data, caution should be exercised when interpreting the results as artifacts are possible from nonspecific effects such as assay signal interference. The curve fitting and activity calls presented here are based on the NCATS analysis methods.

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. For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, with a ratio activity curve class of 4, 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. For a ratio activity curve class = -1.1, score = 80+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 && abs(ratio.max_response) > 6*10, score = 60+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -2.1 || ( ratio.curve_class==-2.2 && abs(ratio.max_response) > 6*10), score = 40+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 || ratio.curve_class == -2.2, score = 20+abs((log_ac50+4.5)*inf_activity/20). Inconclusive compounds, with a donor curve class other than 4, have PUBCHEM_ACTIVITY_SCORE of 10. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39.

External ID: CHRM1_AG_FLUO8_1536_1X%ACT PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that act as agonists of the human M1 muscarinic receptor (CHRM1; M1). In this assay, CHO-K1 cells stably expressing human M1 are loaded, intracellularly with the calcium indicator dye, Fluo-8, followed by treatment with agonist control or test compounds. As designed, compounds that act as CHRM1 agonists will increase intracellular calcium mobilization, resulting in increased relative fluorescence of the indicator dye and well fluorescence. Compounds are tested in singlicate at a final nominal concentration of 3 uM.

Protocol Summary:

The CHO-hM1 cell line was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of Ham's F-12 Nutrient Media (F-12) supplemented with 10% v/v heat-inactivated qualified fetal bovine serum, 20 mM HEPES, 50 ug/mL Geneticin, and 1X antibiotic mix (penicillin and streptomycin).

The day before the assay 3000 cells in 3 uL of growth media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 C, 5% CO2, and 95 % RH for 17-24 hours. Next, 2 uL of the fluorogenic Fluo-8 intracellular calcium indicator mixture (prepared according to the manufacturer's protocol) was added to each well. Plates were then incubated for 1 hour at 37 C, 5% CO2, and 95 % RH, followed by 30 minute incubation at room temperature. Then, 15 nL of test compound in DMSO were dispensed to appropriate wells. The assay was started by performing a basal read of plate fluorescence (470-495 nm excitation and 515-575 nm emission) for 5 seconds on the FLIPR Tetra (Molecular Devices). Then a real time fluorescence measurement was immediately performed for the remaining 140 seconds of the assay. A ratio for each well was calculated to normalize assay data, according to the following mathematical expression:

Ratio = I_Max / I_Min

Where:

I_Max represents the maximum measured fluorescence emission intensity over the 140 second read.
I_Min represents the minimum (basal) measured fluorescence emission intensity before compound was added.

The percent activation was calculated from the median ratio as follows:

%_Activation = ( ( Ratio_Test_Compound - Median_Ratio_Low_Control ) / ( Median_Ratio_High_Control - Median_Ratio_Low_Control ) ) * 100

Where:

Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing DMSO.
High_Control is defined as wells containing Acetylcholine (EC100) and DMSO.

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated for each assay plate: (1) the average percent activation of test compound wells and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for the entire run, i.e. any compound that exhibited greater % activation than the entire screen's cutoff parameter was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary activation. Negative % activation values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-1, and for inactive compounds 1-0.

List of Reagents:

Cell line: Chinese Hamster Ovary (CHO) cells containing hM1 receptor; (Conn Lab)
Calcium sensitive dye: Fluo-8 No Wash Calcium Assay Kit; (AAT Bioquest, part 36316)
Growth media: Ham's F-12; 10% FBS, 20mM HEPES, 50 ug/mL G418
Assay media: Ham's F-12, 10% FBS, 20 mM HEPES
Assay plates: Aurora black/clear 1536well FLIPR plate; (Aurora, part 00019326)
Probenecid: 250 mM (pH 8.0); (Sigma P8761)
Agonist: Acetylcholine (50 mM stock in water); Sigma A9187

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: CYP273

Protocol: Tox21 Assay Protocol Summary:

Two ul of enzyme-substrate mix was dispensed into medium binding white/solid 1536-well plates (Greiner Bio-One North America Inc., Monroe, NC) using a BioRaptr Flying Reagent Dispenser (FRD, Beckman Coulter, Brea, CA). Compounds dissolved in DMSO and positive control (furafyllline) were transferred to the assay plates at 23 nl using a Pintool station (Wako, San Diego, CA). The assay plates were incubated at room temperature for 10 min. Then 2 ul of NADPH regeneration solution was added to each well of the assay plates using an FRD and incubated at room temperature for 1 h. The reaction was stopped by adding 4 ul of detection reagent using an FRD and after 20 min incubation at room temperature the luminescence signal was measured using a ViewLux plate reader (Perkin Elmer, Shelton, CT). Data were expressed as relative luminescence units.

Comment: Disclaimer:

Although all reasonable efforts have been made to ensure the accuracy and reliability of the data, caution should be exercised when interpreting the results as artifacts are possible from nonspecific effects such as assay signal interference. The curve fitting and activity calls presented here are based on the NCATS analysis methods. Alternative analysis methods and interpretations of the data are available at EPA (http://actor.epa.gov) and NTP (http://tools.niehs.nih.gov/cebs3/ui/).

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: CHEMBL875369

Protocol: N/A

Comment: Journal: J. Med. Chem.
Year: 1982
Volume: 25
Issue: 3
First Page: 315
Last Page: 320
DOI: 10.1021/jm00345a016

External ID: CHEMBL3381331

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2014
Volume: 22
Issue: 21
First Page: 6250
Last Page: 6255
DOI: 10.1016/j.bmc.2014.08.020

Target ChEMBL ID: CHEMBL612848
ChEMBL Target Name: Leishmania infantum
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL2091726

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: Bioorg. Med. Chem. Lett.
Year: 2012
Volume: 22
Issue: 10
First Page: 3535
Last Page: 3539
DOI: 10.1016/j.bmcl.2012.03.055

Target ChEMBL ID: CHEMBL5160
ChEMBL Target Name: Transient receptor potential cation channel subfamily A member 1
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: CHEMBL3381330

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2014
Volume: 22
Issue: 21
First Page: 6250
Last Page: 6255
DOI: 10.1016/j.bmc.2014.08.020

Target ChEMBL ID: CHEMBL612848
ChEMBL Target Name: Leishmania infantum
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL2091727

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem. Lett.
Year: 2012
Volume: 22
Issue: 10
First Page: 3535
Last Page: 3539
DOI: 10.1016/j.bmcl.2012.03.055

Target ChEMBL ID: CHEMBL5011
ChEMBL Target Name: Transient receptor potential cation channel subfamily M member 8
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: CHEMBL2091724

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem. Lett.
Year: 2012
Volume: 22
Issue: 10
First Page: 3535
Last Page: 3539
DOI: 10.1016/j.bmcl.2012.03.055

Target ChEMBL ID: CHEMBL5160
ChEMBL Target Name: Transient receptor potential cation channel subfamily A member 1
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: CHRM1_PAM_FLUO8_1536_1X%ACT PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that act as positive allosteric modulators (PAMs) and increase activity of the human M1 muscarinic receptor (CHRM1; M1) in cells pre-treated with a known agonist. In this assay, CHO-K1 cells stably expressing human M1 are loaded with the Fluo-8 calcium indicator dye, followed by addition of test compounds and subsequent treatment with the activator acetylcholine at a concentration that results in 20% activation (EC20). As designed, compounds that act as CHRM1 PAMs will increase calcium mobilization, resulting in increased intracellular calcium and relative fluorescence of the indicator dye beyond that of the EC20 of acetylcholine. Compounds are tested in singlicate at a final nominal concentration of 3 micromolar.

Protocol Summary:

The CHO-hM1 cell line was routinely cultured in T-175 sq cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media consisted of Ham's F-12 Nutrient Media (F-12) supplemented with 10% v/v heat-inactivated qualified fetal bovine serum, 20 mM HEPES, 50 micrograms/mL Geneticin, and 1X antibiotic mix (penicillin and streptomycin).

The day before the assay 3000 cells in 3 microliters of growth media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 degrees C, 5% CO2, and 95 % RH for 17-24 hours. Next, 2 microliters of the fluorogenic Fluo-8 intracellular calcium indicator mixture (prepared according to the manufacturer's protocol) was added to each well. Plates were then incubated for 1 hour at 37 degrees C, 5% CO2, and 95 % RH, followed by 30 minute incubation at room temperature. Then, 15 nL of test compound in DMSO were transferred to appropriate wells. The assay was started by performing a basal read of plate fluorescence (470-495 nm excitation and 515-575 nm emission) for 5 seconds on the FLIPR Tetra (Molecular Devices) prior to all wells being treated with an EC20 concentration of acetylcholine. Then a real time fluorescence measurement was immediately performed for the remaining 140 seconds of the assay. A ratio for each well was calculated to normalize assay data, according to the following mathematical expression:

Ratio = I_Max / I_Min

Where:

I_Max represents the maximum measured fluorescence emission intensity over the 140 second read and;
I_Min represents the minimum (basal) measured fluorescence emission intensity before compound was added.

The percent activation was calculated from the median ratio as follows:

% Activation = ( ( Ratio_Test_Compound - Median_Ratio_Low_Control ) / ( Median_Ratio_High_Control - Median_Ratio_Low_Control ) ) * 100

Where:

Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing Acetylcholine at EC20 and DMSO.
High_Control is defined as wells containing Acetylcholine (EC100) and DMSO.

A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated for each assay plate: (1) the average percent activation of test compound wells and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter on an individual plate basis, i.e. any compound that exhibited greater % activation than the plate based cutoff parameter was declared active.

PubChem Activity Outcome and Score:

The inactive compounds of this assay have an activity score range of 0 to 78 and the active compounds have an activity score range of 50 to 100.

The reported PubChem Activity Score has been normalized to 100% observed primary activation. Negative % activation values are reported as activity score zero.

List of Reagents:

Cell line: Chinese Hamster Ovary (CHO) cells containing hM1 receptor; (Conn Lab)
Calcium sensitive dye: Fluo-8 No Wash Calcium Assay Kit; (AAT Bioquest, part 36316)
Growth media: Ham's F-12; 10% FBS, 20mM HEPES, 50?g/mL G418
Assay media: Ham's F-12, 10% FBS, 20 mM HEPES
Assay plates: Aurora black/clear 1536well FLIPR plate; (Aurora, part 00019326)
Probenecid: 250mM (pH 8.0); (Sigma P8761)
Potentiator: Acetylcholine (50mM stock in water); Sigma A9187

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: CHEMBL3381332

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2014
Volume: 22
Issue: 21
First Page: 6250
Last Page: 6255
DOI: 10.1016/j.bmc.2014.08.020

Target ChEMBL ID: CHEMBL612557
ChEMBL Target Name: RAW264.7
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL2091725

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: Bioorg. Med. Chem. Lett.
Year: 2012
Volume: 22
Issue: 10
First Page: 3535
Last Page: 3539
DOI: 10.1016/j.bmcl.2012.03.055

Target ChEMBL ID: CHEMBL5160
ChEMBL Target Name: Transient receptor potential cation channel subfamily A member 1
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: CHEMBL2091728

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: Bioorg. Med. Chem. Lett.
Year: 2012
Volume: 22
Issue: 10
First Page: 3535
Last Page: 3539
DOI: 10.1016/j.bmcl.2012.03.055

Target ChEMBL ID: CHEMBL5011
ChEMBL Target Name: Transient receptor potential cation channel subfamily M member 8
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: DSHEA-v1-PXR-antagonist-CTF

Protocol: For PXR luciferase reporter gene assays, we used hPXR-LucHepG2cells provided by Dr. Taosheng Chen (Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital). Cells were cultured in EMEM supplemented with 10% fetal bovine serum, 100U/mL penicillin and 100ug/mL streptomycin, and 500ug/mL of geneticin.

PROTOCOL TABLE (as described by Inglese J, Shamu CE and Guy RK. 2007) [1]
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION.
1; Cells; 5 uL; hPXR-LucHepG2; white solid-bottom Greiner plate.
2; Incubation; 5h; 37C, 95% humidity, 5% CO2
3; Compounds and control; 23 nL; Kalypsis pintool (Wako USA).
4; Reagent; 1uL; rifampicin (RIF) in the antagonist mode.
5; Incubation; 24 hr; 37C, 5% CO2.
6; Reagent; 1 uL; CellTiter-Fluor detection reagent.
7; Incubation; 1 hr; 37C, 5% CO2.
8; Read; Fluorescence; ViewLux plate reader.
9; Reagent; 4 uL; ONE-Glo Luciferase detection reagent.
10; Incubation; 30 min; room temperature.
11; Read; Luminescence; ViewLux plate reader.

NOTES (numbers refer to Sequence numbers above)
1. Briefly, 5uL of hPXR-LucHepG2 cells in phenol red-free DMEM containing 5% charcoal-stripped FBS, 1 mM sodium pyruvate (Invitrogen), 2 mM L-glutamine (Invitrogen), and 100 U/mL penicillin and 100ug/mL streptomycin were dispensed using a BioRAPTR FRD at 4 x 105cells/mL (2000cells/well) in tissue culture-treated 1536-well white assay plates (GreinerBio-One).
2. Plates were incubated for 5h at 37C, 95% humidity, and 5% CO2 to allow for cell attachment.
3. Compounds (final concentration for most substances ranged from 15.6 nM to 45.9uM), positive SPA70 (final concentration range of 2.8 nM to 92uM for the antagonist mode), and cytotoxicity control tetraoctyl ammonium bromide (final concentration of 92uM) were transferred (23 nL) via pintool (Wako Automation).
4-5. After compound transfer, plates were dispensed with 1uL of RIF (final concentration of 2uM) in the antagonist mode, using a BioRAPTR FRD and incubated for 24 h at 37C and 5% CO2.
6-8. Following incubation, 1uL of CellTiter-Fluor (Promega, Madison, Wisconsin) was added using a BioRAPTR FRD, after which all plates were incubated (37C/ 5% CO2) for1 h and then measured for fluorescence intensity (Ex/Em= 405/540 nm) on a ViewLux plate reader to determine cell viability.
9-11. Directly after this fluorescence reading, 4uL of ONE-Glo Luciferasere agent (Promega) was added to each well using the BioRAPTR FRD, followed by a 30 min incubation (RT) and read for luminescence intensity on a ViewLux detector.

REFERENCES:
[1] Inglese J, Shamu CE and Guy RK, Reporting data from high throughput screening of small molecule libraries, Nature Chemical Biology, 2007, 3(8): 438-441. doi.org/10.1038/nchembio0807-438.
[2] Lin W, Wu J, Dong H, Bouck D, Zeng FY, Chen T. Cyclin-dependent kinase 2 negatively regulates human pregnane X receptor-mediated CYP3A4 gene expression in HepG2 liver carcinoma cells. J Biol Chem. 2008 Nov 7;283(45):30650-7. doi: 10.1074/jbc.M806132200. Epub 2008 Sep 9. PMID: 18784074; PMCID: PMC2662154.

Comment: Disclaimer:
Although all reasonable efforts have been made to ensure the accuracy and reliability of the data, caution should be exercised when interpreting the results as artifacts are possible from nonspecific effects such as assay signal interference. The curve fitting and activity calls presented here are based on the NCATS analysis methods.

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. For this assay, apparent antagonists are ranked higher than compounds that showed no activity.
2. For all inactive compounds, with a ratio activity curve class of 4, 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. For a ratio activity curve class = -1.1, score = 80+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 && abs(ratio.max_response) > 6*10, score = 60+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -2.1 || ( ratio.curve_class==-2.2 && abs(ratio.max_response) > 6*10), score = 40+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 || ratio.curve_class == -2.2, score = 20+abs((log_ac50+4.5)*inf_activity/20). Inconclusive compounds, with a donor curve class other than 4, have PUBCHEM_ACTIVITY_SCORE of 10. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39.

External ID: DSHEA-v1-PXR-antagonist-ONEGlo

Protocol: For PXR luciferase reporter gene assays, we used hPXR-LucHepG2cells provided by Dr. Taosheng Chen (Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital). Cells were cultured in EMEM supplemented with 10% fetal bovine serum, 100U/mL penicillin and 100ug/mL streptomycin, and 500ug/mL of geneticin.

PROTOCOL TABLE (as described by Inglese J, Shamu CE and Guy RK. 2007) [1]
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION.
1; Cells; 5 uL; hPXR-LucHepG2; white solid-bottom Greiner plate.
2; Incubation; 5h; 37C, 95% humidity, 5% CO2
3; Compounds and control; 23 nL; Kalypsis pintool (Wako USA).
4; Reagent; 1uL; rifampicin (RIF) in the antagonist mode.
5; Incubation; 24 hr; 37C, 5% CO2.
6; Reagent; 1 uL; CellTiter-Fluor detection reagent.
7; Incubation; 1 hr; 37C, 5% CO2.
8; Read; Fluorescence; ViewLux plate reader.
9; Reagent; 4 uL; ONE-Glo Luciferase detection reagent.
10; Incubation; 30 min; room temperature.
11; Read; Luminescence; ViewLux plate reader.

NOTES (numbers refer to Sequence numbers above)
1. Briefly, 5uL of hPXR-LucHepG2 cells in phenol red-free DMEM containing 5% charcoal-stripped FBS, 1 mM sodium pyruvate (Invitrogen), 2 mM L-glutamine (Invitrogen), and 100 U/mL penicillin and 100ug/mL streptomycin were dispensed using a BioRAPTR FRD at 4 x 105cells/mL (2000cells/well) in tissue culture-treated 1536-well white assay plates (GreinerBio-One).
2. Plates were incubated for 5h at 37C, 95% humidity, and 5% CO2 to allow for cell attachment.
3. Compounds (final concentration for most substances ranged from 15.6 nM to 45.9uM), positive SPA70 (final concentration range of 2.8 nM to 92uM for the antagonist mode), and cytotoxicity control tetraoctyl ammonium bromide (final concentration of 92uM) were transferred (23 nL) via pintool (Wako Automation).
4-5. After compound transfer, plates were dispensed with 1uL of RIF (final concentration of 2uM) in the antagonist mode, using a BioRAPTR FRD and incubated for 24 h at 37C and 5% CO2.
6-8. Following incubation, 1uL of CellTiter-Fluor (Promega, Madison, Wisconsin) was added using a BioRAPTR FRD, after which all plates were incubated (37C/ 5% CO2) for1 h and then measured for fluorescence intensity (Ex/Em= 405/540 nm) on a ViewLux plate reader to determine cell viability.
9-11. Directly after this fluorescence reading, 4uL of ONE-Glo Luciferasere agent (Promega) was added to each well using the BioRAPTR FRD, followed by a 30 min incubation (RT) and read for luminescence intensity on a ViewLux detector.

REFERENCES:
[1] Inglese J, Shamu CE and Guy RK, Reporting data from high throughput screening of small molecule libraries, Nature Chemical Biology, 2007, 3(8): 438-441. doi.org/10.1038/nchembio0807-438.
[2] Lin W, Wu J, Dong H, Bouck D, Zeng FY, Chen T. Cyclin-dependent kinase 2 negatively regulates human pregnane X receptor-mediated CYP3A4 gene expression in HepG2 liver carcinoma cells. J Biol Chem. 2008 Nov 7;283(45):30650-7. doi: 10.1074/jbc.M806132200. Epub 2008 Sep 9. PMID: 18784074; PMCID: PMC2662154.

Comment: Disclaimer:
Although all reasonable efforts have been made to ensure the accuracy and reliability of the data, caution should be exercised when interpreting the results as artifacts are possible from nonspecific effects such as assay signal interference. The curve fitting and activity calls presented here are based on the NCATS analysis methods.

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. For this assay, apparent antagonists are ranked higher than compounds that showed no activity.
2. For all inactive compounds, with a ratio activity curve class of 4, 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. For a ratio activity curve class = -1.1, score = 80+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 && abs(ratio.max_response) > 6*10, score = 60+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -2.1 || ( ratio.curve_class==-2.2 && abs(ratio.max_response) > 6*10), score = 40+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 || ratio.curve_class == -2.2, score = 20+abs((log_ac50+4.5)*inf_activity/20). Inconclusive compounds, with a donor curve class other than 4, have PUBCHEM_ACTIVITY_SCORE of 10. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39.

External ID: DSHEA-v1-PXR-agonist-ONEGlo

Protocol: For PXR luciferase reporter gene assays, we used hPXR-LucHepG2cells provided by Dr. Taosheng Chen (Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital). Cells were cultured in EMEM supplemented with 10% fetal bovine serum, 100U/mL penicillin and 100ug/mL streptomycin, and 500ug/mL of geneticin.

PROTOCOL TABLE (as described by Inglese J, Shamu CE and Guy RK. 2007) [1]
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION.
1; Cells; 5 uL; hPXR-LucHepG2; white solid-bottom Greiner plate.
2; Incubation; 5h; 37C, 95% humidity, 5% CO2
3; Compounds and control; 23 nL; Kalypsis pintool (Wako USA).
4; Reagent; 1uL; rifampicin (RIF) in the antagonist mode.
5; Incubation; 24 hr; 37C, 5% CO2.
6; Reagent; 1 uL; CellTiter-Fluor detection reagent.
7; Incubation; 1 hr; 37C, 5% CO2.
8; Read; Fluorescence; ViewLux plate reader.
9; Reagent; 4 uL; ONE-Glo Luciferase detection reagent.
10; Incubation; 30 min; room temperature.
11; Read; Luminescence; ViewLux plate reader.

NOTES (numbers refer to Sequence numbers above)
1. Briefly, 5uL of hPXR-LucHepG2 cells in phenol red-free DMEM containing 5% charcoal-stripped FBS, 1 mM sodium pyruvate (Invitrogen), 2 mM L-glutamine (Invitrogen), and 100 U/mL penicillin and 100ug/mL streptomycin were dispensed using a BioRAPTR FRD at 4 x 105cells/mL (2000cells/well) in tissue culture-treated 1536-well white assay plates (GreinerBio-One).
2. Plates were incubated for 5h at 37C, 95% humidity, and 5% CO2 to allow for cell attachment.
3. Compounds (final concentration for most substances ranged from 15.6 nM to 45.9uM), positive control rifampicin (RIF; final concentration range of 2.8 nM to 92uM for the agonist mode), and cytotoxicity control tetraoctyl ammonium bromide (final concentration of 92uM) were transferred (23 nL) via pintool (Wako Automation).
4-5. After compound transfer, plates were dispensed with 1uL of the media in the agonist mode using a BioRAPTR FRD and incubated for 24 h at 37C and 5% CO2.
6-8. Following incubation, 1uL of CellTiter-Fluor (Promega, Madison, Wisconsin) was added using a BioRAPTR FRD, after which all plates were incubated (37C/ 5% CO2) for1 h and then measured for fluorescence intensity (Ex/Em= 405/540 nm) on a ViewLux plate reader to determine cell viability.
9-11. Directly after this fluorescence reading, 4uL of ONE-Glo Luciferasere agent (Promega) was added to each well using the BioRAPTR FRD, followed by a 30 min incubation (RT) and read for luminescence intensity on a ViewLux detector.

REFERENCES:
[1] Inglese J, Shamu CE and Guy RK, Reporting data from high throughput screening of small molecule libraries, Nature Chemical Biology, 2007, 3(8): 438-441. doi.org/10.1038/nchembio0807-438.
[2] Lin W, Wu J, Dong H, Bouck D, Zeng FY, Chen T. Cyclin-dependent kinase 2 negatively regulates human pregnane X receptor-mediated CYP3A4 gene expression in HepG2 liver carcinoma cells. J Biol Chem. 2008 Nov 7;283(45):30650-7. doi: 10.1074/jbc.M806132200. Epub 2008 Sep 9. PMID: 18784074; PMCID: PMC2662154.

Comment: Disclaimer:
Although all reasonable efforts have been made to ensure the accuracy and reliability of the data, caution should be exercised when interpreting the results as artifacts are possible from nonspecific effects such as assay signal interference. The curve fitting and activity calls presented here are based on the NCATS analysis methods.

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. For this assay, apparent agonists are ranked higher than compounds that showed apparent antagonists.
2. For all inactive compounds, with a ratio activity curve class of 4, 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. For a ratio activity curve class = 1.1, score = 80+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == 1.2 && abs(ratio.max_response) > 6*10, score = 60+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == 2.1 || ( ratio.curve_class==-2.2 && abs(ratio.max_response) > 6*10), score = 40+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == 1.2 || ratio.curve_class == 2.2, score = 20+abs((log_ac50+4.5)*inf_activity/20). Inconclusive compounds, with a donor curve class other than 4, have PUBCHEM_ACTIVITY_SCORE of 10. Inconclusive compounds also have PUBCHEM_ACTIVITY_SCORE between 1 and 39.

External ID: DSHEA-v1-PXR-agonist-CTF

Protocol: For PXR luciferase reporter gene assays, we used hPXR-LucHepG2cells provided by Dr. Taosheng Chen (Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital). Cells were cultured in EMEM supplemented with 10% fetal bovine serum, 100U/mL penicillin and 100ug/mL streptomycin, and 500ug/mL of geneticin.

PROTOCOL TABLE (as described by Inglese J, Shamu CE and Guy RK. 2007) [1]
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION.
1; Cells; 5 uL; hPXR-LucHepG2; white solid-bottom Greiner plate.
2; Incubation; 5h; 37C, 95% humidity, 5% CO2
3; Compounds and control; 23 nL; Kalypsis pintool (Wako USA).
4; Reagent; 1uL; rifampicin (RIF) in the antagonist mode.
5; Incubation; 24 hr; 37C, 5% CO2.
6; Reagent; 1 uL; CellTiter-Fluor detection reagent.
7; Incubation; 1 hr; 37C, 5% CO2.
8; Read; Fluorescence; ViewLux plate reader.
9; Reagent; 4 uL; ONE-Glo Luciferase detection reagent.
10; Incubation; 30 min; room temperature.
11; Read; Luminescence; ViewLux plate reader.

NOTES (numbers refer to Sequence numbers above)
1. Briefly, 5uL of hPXR-LucHepG2 cells in phenol red-free DMEM containing 5% charcoal-stripped FBS, 1 mM sodium pyruvate (Invitrogen), 2 mM L-glutamine (Invitrogen), and 100 U/mL penicillin and 100ug/mL streptomycin were dispensed using a BioRAPTR FRD at 4 x 105cells/mL (2000cells/well) in tissue culture-treated 1536-well white assay plates (GreinerBio-One).
2. Plates were incubated for 5h at 37C, 95% humidity, and 5% CO2 to allow for cell attachment.
3. Compounds (final concentration for most substances ranged from 15.6 nM to 45.9muM), positive control rifampicin (RIF; final concentration range of 2.8 nM to 92muM for the agonist mode), and cytotoxicity control tetraoctyl ammonium bromide (final concentration of 92muM) were transferred (23 nL) via pintool (Wako Automation).
4-5. After compound transfer, plates were dispensed with 1muL of the media in the agonist mode using a BioRAPTR FRD and incubated for approximately 24 h at 37 degrees C and 5% CO2.
6-8. Following incubation, 1uL of CellTiter-Fluor (Promega, Madison, Wisconsin) was added using a BioRAPTR FRD, after which all plates were incubated (37C/ 5% CO2) for 1 h and then measured for fluorescence intensity (Ex/Em= 405/540 nm) on a ViewLux plate reader to determine cell viability.
9-11. Directly after this fluorescence reading, 4uL of ONE-Glo Luciferasere agent (Promega) was added to each well using the BioRAPTR FRD, followed by a 30 min incubation (RT) and read for luminescence intensity on a ViewLux detector.

REFERENCES:
[1] Inglese J, Shamu CE and Guy RK, Reporting data from high throughput screening of small molecule libraries, Nature Chemical Biology, 2007, 3(8): 438-441. doi.org/10.1038/nchembio0807-438.
[2] Lin W, Wu J, Dong H, Bouck D, Zeng FY, Chen T. Cyclin-dependent kinase 2 negatively regulates human pregnane X receptor-mediated CYP3A4 gene expression in HepG2 liver carcinoma cells. J Biol Chem. 2008 Nov 7;283(45):30650-7. doi: 10.1074/jbc.M806132200. Epub 2008 Sep 9. PMID: 18784074; PMCID: PMC2662154.

Comment: Disclaimer:
Although all reasonable efforts have been made to ensure the accuracy and reliability of the data, caution should be exercised when interpreting the results as artifacts are possible from nonspecific effects such as assay signal interference. The curve fitting and activity calls presented here are based on the NCATS analysis methods.

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. For this assay, apparent agonists are ranked higher than compounds that showed apparent antagonist.
2. For all inactive compounds, with a ratio activity curve class of 4, 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. For a ratio activity curve class = 1.1, score = 80+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == 1.2 && abs(ratio.max_response) > 6*10, score = 60+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == 2.1 || ( ratio.curve_class==-2.2 && abs(ratio.max_response) > 6*10), score = 40+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == 1.2 || ratio.curve_class == 2.2, score = 20+abs((log_ac50+4.5)*inf_activity/20). Inconclusive compounds, with a donor curve class other than 4, have PUBCHEM_ACTIVITY_SCORE of 10. Inconclusive compounds also have PUBCHEM_ACTIVITY_SCORE between 1 and 39.

External ID: 2070-01_Inhibitor_SinglePoint_HTS_Activity

Protocol: Protocol:
ASSAY PROCEDURE FOR HEDGEHOG AUTOPROCESSING

Buffer A: 20 mM sodium phosphate, pH 7.5, 0.5 M NaCl

Buffer B: 20 mM sodium phosphate, pH 7.5, 0.5 M NaCl, 8 M urea
This is prepared by adding 48 g of urea (enzyme grade) to 50 ml of double-strength Buffer A and adjusting volume to 100 ml with H2O.


Buffer D: 20 mM sodium phosphate, pH 7.0, 0.5 M NaCl, and 0.5 M arginine
(2.0 M arginine, pH 7.0, is prepared by dissolving 211 g of L-arginine hydrochloride (obtained from Research Organics) in 300 ml of H2O, adjusting pH to 7.0 with NaOH, and adjusting volume to 500 ml)



REAGENTS:
Urea - American Bioanalytical AB02100
Arginine Hydrochloride - American Bioanalytical AB000164
TCEP [Tris(2-carboxyethyl)phosphine hydrochloride] - Sigma C4706
DM-MEA [2-(Dimethylamino)ethanethiol] - Aldrich D141003 (very hygroscopic)
Cholesterol - Sigma C8667
FLAsH-EDT2-Invitrogen T34561

CELL DISRUPTION:

Suspend the cells from 50 ml of E. coli [Rosetta 2 (DE3)/pET45Dmel710#2] in 3 ml of Buffer A +
1 mM PMSF and disrupt by passing twice through the small French pressure cell. Centrifuge in 12 ml Sorvall tubes at 12,000 rpm for 30 min. and collect the pellet (inclusion bodies). The pellet is washed twice by resuspending in 4 ml of Buffer A + 1 mM PMSF and centrifugation at 12,000 rpm for 20 min. The pellet is then then resuspended in 3 ml of Buffer B + 1 mM PMSF to extract the inclusion bodies and centrifuged at 12,000 rpm for 20 min. Save the supernatant and extract the pellet a second time with 3 ml of Buffer B + 1 mM PMSF and combine the supernatants. Centrifuge the combined supernatants in the Sorvall at 18,000 rpm for 60 min to remove insoluble material and yield the solubilized inclusion bodies (IB). Save inclusion bodies at 4 degrees C after diluting about 10% with Buffer A to avoid crystallization of urea.

Typically, the protein concentration of the inclusion bodies is 1 mg/ml. The material is stable on storage at 4 degrees C for at least a month and can be kept frozen at -80 degrees C in 1 ml portions indefinitely.


Dilution Buffer:
297 ml of Buffer D
3 ml of 0.1 M TCEP
300 ml Final

Substrate mix
6.5 ml of 26 mM cholesterol in 95% EtOH
2.0 ml of 1% Triton X-100
41.5 ml of H2O
50 ml + 200 ml Dilution Buffer (cholesterol = 680 microM) - Dilute immediately before use

Control mix
0.8 ml of 1% Triton X-100
19.2 ml of H2O
20 ml + 80 ml Dilution Buffer (No cholesterol)

IB mix
13.2 ml Hedgehog protein ( approximately 1.65 mg/ml) [12 ml form prep of 10.05.11, 2 ml from prep of 10.05.10]
198 ml Buffer D (
room temp)
2.2 ml of 0.1 M TCEP
2.2 ml of 0.1 M DM-MEA
2.2 ml of 0.17 mM FlAsH
2.2 ml of H2O
220 ml final




Incubate at 25*C for 2 h. Dialyze against 3 X 1 liter Buffer D + 1 mM TCEP (10 ml of 0.1 M TCEP per liter) for 6 h - 14h. Recommended dialysis tubing: SpectraPor MWCO 8,000, flat width = 12 mM (VWR 25223-650). Readjust volume to 220 ml after dialysis.The dialyzed Hh Protein Cocktail can be stored at 4 degrees C for at least 5 days with negligible loss of activity. It should be stable at room temperature for at least 12 h.




Dispense 35 microl per well. Add IB mix first to entire plate. Wait 30 min before adding cholesterol, while control mix (35ul) is added to columns 23&24.

Then add substrate mix to columns 1-22, 40 min after adding protein. Fluorescence polarization was measured after 1 hour incubation at 25C using Tecan Safire 2 microplate reader (Tecan, Mannedorf, Switzerland) with excitation at 390 nm and emission at 550
nm.

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in increasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.

PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set to be three standard deviations above the mean neutral control value.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at -50.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

tSamples passing PAR_T only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

External ID: HERG01

Protocol: NCGC Assay Protocol Summary:

HERG assay (FluxORTM thallium flux assay) was initially developed by Invitrogen/Molecular Probes, and then miniaturized into 1536-well plate in a homogeneous format by NCGC. This assay measures the activity of potassium channel using thallium dye (FluxOR) flux as surrogate measurement for potassium into the cells with a FDSS-7000 kinetic plate reader (Hamamatsu Corp., Hamamatsu City, Japan). The hERG ion channel is transduced into mammalian cells (U2OS) using a baculovirus (Bacmam) construct harboring the hERG K+ ion channel. So far we screened LOPAC1280 library (Sigma), the NTP collection of 1408 compounds, and the NCGC Pharmaceutical Collection (NPC), in which many well defined HERG blockers are present. The rank order potencies of many of these compounds are similar to that of other HERG assays (membrane potential, Rb+ flux, patch clamp, etc). This quick and homogeneous assay is also found to be sensitive, specific, and robust.

Using the FluxORTM thallium flux assay, the activity of potassium channel using thallium dye (FluxOR) flux as surrogate measurement for potassium into the cells was measured in the U2OS cell line transduced with hERG K+ ion channel using a baculovirus (Bacmam) using Opti-MEM medium (Invitrogen) containing 2% fetal calf serum (FCS, HyCone) following loading buffer addition, compound treatment for around 10 minutes and finally adding stimulation buffer. The assay was performed in black clear Kalypsys 1536-well plates. In the screen, Astemizole was used as positive controls. Library compounds were measured for their ability to cause hERG channel blockage in the cell line, as reflected by a decrease in fluorescence intensity, in a concentration-dependent manner. Data were normalized to the controls for basal activity (DMSO only) and 100% inhibition (5uM Astemizole). AC50 values were determined from concentration-response data modeled with the standard Hill equation.

qHTS protocol for hERG-U2OS cellular assay

[Step] [Parameter] [Value] [Description]

1. Day 1: Replace medium in 70-80% confluent T225 flask with 2.5 mL of hERG-BacMam virus plus 12.5 mL of phosphate buffered saline (PBS) (corresponding roughly to a multiplicity of infection ratio of 100 virus particles/cell)
2. Incubation: 4 hrs @ room Temperature in Dark
3. Reagent; Remove virus; wash once with 25 ml DPBS
4. Reagent; 35 ml culture medium
5. Incubation; 37oC overnight
6. Day2: Reagent; 3 uL; 2000 U2OS cells/well
7. Time; 4 hr; 37oC incubation
8. Loading buffer; 1 uL; 0.7X;
9. Incubation: 1hr @ RT in Dark.
10. Compounds; 23 nL; 0.59 nM to 92 uM
11. Controls; 23 nL; Astemizole 1.4 nM to 92 uM
12. Time; 10min; 37oC incubation
13. Read fluorescence Intensity on FDSS for 10 Sec with 1 sec interval
14. Reagent; 1 uL; stimulation buffer
15. Read fluorescence Intensity on FDSS for 2 min with 1 sec interval
16. Detection; Fluorescence Intensity; FDSS

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: SMAD3201

Protocol: Suspensions of trypsinized HEPG2 CAGA-GFP cells were dispensed into white, tissue culture-treated, solid 1536-well plates at 5uL/well (1000 cells/well final concentration) in DMEM medium supplemented with 1% FBS. Plates were incubated at 37 degrees C for 2 hours, after which 23 nL of compounds or DMSO were delivered to each well using a pin tool. One uL of recombinant TGF-beta in DMEM (1% FBS) was then dispensed (500 pg/mL final concentration), and plates were incubated at 37 degrees C for 18 hours. Two uL of CellTiter Glo (Promega), a luminescence-based viability reagent, was dispensed, followed by a 10 minute room temperature incubation. The plates were then measured on a PerkinElmer ViewLux plate reader for luminescence (clear filter) using a 5 second exposure. The %Activity was determined from the corrected luminescence values. Wells containing media only (no cells) were used to normalize %Activity of identified toxic compounds; media-only wells corresponded to 100%Activity (complete cell-killing), while DMSO-dosed cell controls were used to normalize 0%Activity (no toxicity).

Concentration-response curves were fitted to the signals arising from the resulting luminescence. The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity, and compounds were subsequently categorized based on their curve class. Toxic compounds showed concentration-dependent decreases in luminescence, concordant with a decrease in intracellular ATP concentration (CellTiter Glo's marker of viability), and thus a decrease in the number of viable cells. Inactive (non-toxic) compounds showed no effect on luminescence signal. Active (toxic) compounds showed concentration dependent decrease in luminescence.

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".

2. For all inactive (non-toxic) compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active (toxic) compounds, a score range was given for each curve class type given above. Active (toxic) 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.