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: TGF788

Protocol: Tox21 Assay Protocol Summary:

SBE-bla HEK 293T cells were dispensed at 4,000/well in 6 uL of assay medium into black wall/clear-bottom 1536-well plates using a Multidrop Combi (ThermoFisher Scientific, Waltham, MA) dispenser. After the assay plates were incubated at 37 C and 5% CO2 for an overnight, 23 nL of compounds dissolved in DMSO, positive controls or DMSO only was transferred to the assay plate by a Pintool station (Kalypsys, San Diego, CA). The assay plates were incubated at 37 C and 5% CO2 for 5 hr. One uL of LiveBLAzer B/G FRET substrate (Life Technologies) was added using a Flying Reagent Dispenser (FRD, Aurora Discovery, San Diego, CA) and the plates were incubated at room temperature for 2 hr. Fluorescence intensity (405 nm excitation, 460 nm and 530 nm emissions) was measured using an Envision plate reader (PerkinElmer, Shelton, CT). For cytotoxicity read, 4 uL of CellTiter-Glo reagent (Promega Corporation, Madison, WI) was added to each well of the assay plates using FRD. After 30min incubation at room temperature, the luminescence intensity was measured using ViewLux plate reader (PerkinElmer).

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: TGF602

Protocol: Tox21 Assay Protocol Summary:

SBE-bla HEK 293T cells were dispensed at 4,000/well in 5 uL of assay medium into black wall/clear-bottom 1536-well plates using a Multidrop Combi (ThermoFisher Scientific, Waltham, MA) dispenser. After the assay plates were incubated at 37 C and 5% CO2 for an overnight, 23 nL of compounds dissolved in DMSO, positive controls or DMSO only was transferred to the assay plate by a Pintool station (Kalypsys, San Diego, CA). Compound transfer was followed by the addition of 1 uL of 0.25 ng/mL TGF-beta1 or assay buffer using two separate tips of Flying Reagent Dispenser (FRD, Aurora Discovery, San Diego, CA). The assay plates were incubated at 37 C and 5% CO2 for 5 hr. One uL of LiveBLAzer B/G FRET substrate (Life Technologies) was added using a Flying Reagent Dispenser (FRD, Aurora Discovery, San Diego, CA) and the plates were incubated at room temperature for 2 hr. Fluorescence intensity (405 nm excitation, 460 nm and 530 nm emissions) was measured using an Envision plate reader (PerkinElmer, Shelton, CT). For cytotoxicity read, 4 uL of CellTiter-Glo reagent (Promega Corporation, Madison, WI) was added to each well of the assay plates using FRD. After 30 min incubation at room temperature, the luminescence intensity was measured using ViewLux plate reader (PerkinElmer).

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: P53600

Protocol: Tox21 Assay Protocol Summary:

The p53RE-bla cells were dispensed at 4,000 cells/5 ul/well in 1536-well black wall/clear bottom plates using a Multidrop dispenser. After the assay plates were incubated at a 37 C/5% CO2 incubator for 5 hours, 23 nL of compounds dissolved in DMSO, positive controls or DMSO only was transferred to the assay plate by a pin tool. The plates were incubated at 37 C for 16 hours. After 1 uL of LiveBLAzerTM B/G FRET substrate was added using a Flying Reagent Dispenser, the plates were incubated at room temperature for 2 hours, and fluorescence intensity was measured by an Envision plate reader. For cell viability readout that measures cytotoxicity, 4 ul/well of CellTiter-Glo reagent was added into the assay plates using a Flying Reagent Dispenser. After 30 min incubation at room temperature, the luminescence intensity in the plates was measured using a ViewLux plate reader.

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 (https://www.epa.gov/comptox-tools/comptox-chemicals-dashboard) and NTP (https://cebs.niehs.nih.gov/cebs/).
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: P53MS958

Protocol: Tox21 Assay Protocol Summary:

The p53RE-bla cells were dispensed at 4,000cells/3ul/well in 1536-well black-clear bottom plates. After the assay plates were incubated at 37 C, 5% CO2 incubator for 5 hours, 23 nL of compounds dissolved in DMSO, positive controls or DMSO only was transferred to the assay plate. Following compound addition, 3 uL of rat liver microsomes (Molecular Toxicology, Boone, NC) at final concentration of 0.5 mg/mL and 1uL of B-Nicotinamide adenine dinucleotide 2'-phosphate (NADPH) at final concentration of 0.5 mg/mL were transferred to the assay plate. The plates were incubated at 37 C for 16 hours. 1 uL of LiveBLAzer detection mixture was added to each well and the plates were incubated at room temperature in the dark for 2 h. Fluorescence intensity at 460 and 530 nm emission and 405 nm excitation were measured by an Envision plate reader. For cell viability readout that measures cytotoxicity, 4 ul/well of CellTiter-Glo reagent was added into the assay plates using a Flying Reagent Dispenser. After 30 min incubation at room temperature, the luminescence intensity in the plates was measured using a ViewLux plate reader.

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 (https://www.epa.gov/comptox-tools/comptox-chemicals-dashboard) and NTP (https://cebs.niehs.nih.gov/cebs/).

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: P53MS482

Protocol: Please refer to other AIDs 1963578, 1963580, 720687, 720685, 720678 and 720681 for detailed assay protocols.

Comment: This summary is written for the purposes of summarizing the compound activities from the project combining the results from both the p53 with rat microsomes agonist mode assay (AID 1963578), cell viability counter screen (AID 1963580), and auto fluorescence counter screens (AID 720687, 720685, 720678, and 720681). For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Potency and efficacy were used for determining relative score. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 5 and 30 determined by phenotype.

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 (https://www.epa.gov/comptox-tools/comptox-chemicals-dashboard) and NTP (https://cebs.niehs.nih.gov/cebs/).

External ID: P53344

Protocol: Tox21 Assay Protocol Summary:

The p53RE-bla cells were dispensed at 4,000 cells/5 ul/well in 1536-well black wall/clear bottom plates using a Multidrop dispenser. After the assay plates were incubated at a 37 C/5% CO2 incubator for 5 hours, 23 nL of compounds dissolved in DMSO, positive controls or DMSO only was transferred to the assay plate by a pin tool. The plates were incubated at 37 C for 16 hours. After 1 uL of LiveBLAzerTM B/G FRET substrate was added using a Flying Reagent Dispenser, the plates were incubated at room temperature for 2 hours, and fluorescence intensity was measured by an Envision plate reader.

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 (https://www.epa.gov/comptox-tools/comptox-chemicals-dashboard) and NTP (https://cebs.niehs.nih.gov/cebs/).

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 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: P53MS233

Protocol: Tox21 Assay Protocol Summary:

The p53RE-bla cells were dispensed at 4,000cells/3ul/well in 1536-well black-clear bottom plates. After the assay plates were incubated at 37 C, 5% CO2 incubator for 5 hours, 23 nL of compounds dissolved in DMSO, positive controls or DMSO only was transferred to the assay plate. Following compound addition, 3 uL of human liver microsomes (XenoTech, Kansas City, KS) at final concentration of 0.5 mg/mL and 1uL of Beta-Nicotinamide adenine dinucleotide 2-phosphate (NADPH) at final concentration of 0.5 mg/mL were transferred to the assay plate. The plates were incubated at 37 C for 16 hours. 1 uL of LiveBLAzer detection mixture was added to each well and the plates were incubated at room temperature in the dark for 2 h. Fluorescence intensity at 460 and 530 nm emission and 405 nm excitation were measured by an Envision plate reader.

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 (https://www.epa.gov/comptox-tools/comptox-chemicals-dashboard) and NTP (https://cebs.niehs.nih.gov/cebs/).

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 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.