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: 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: s-my-keats_OPM1-m4-1

Protocol: SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE and DESCRIPTION.
1; Cells; 5 uL; white, solid bottom 1536-well Greiner assay plate.
2; Compounds; 23 nL; Kalypsis pin tool (Wako USA) equipped with a 1536-well pin head to transfer to the assay plates.
3; Incubation; 48 hours; 37C incubation.
4; Reagent;5uL; CellTiter Glo (Promega).
5; Detector; 425V; Luminescence; Viewlux PerkinElmer.

NOTES (numbers refer to Sequence numbers above)
1. The cell-based screening methods employed in this study were like those previously published [1]. Briefly, the corresponding myeloma cell lines were screened in 1,536-well plates with 500 cells, 5-uL per well for inhibition of cell viability (assessed by measuring ATP levels with CellTiterGlo).
2. NCATS MIPE 4.0 library of approved and investigational drugs were added using the Kalypsis pin tool. Wells treated with DMSO only for 100% viability and media only for 0% viability were used as controls.
3. The assay plates were covered and incubated for 48 hours in a 37C, 5% CO2 incubator with controlled humidity.
4. After 48 hours, CellTilter-GloTM luminescent substrate mix (Promega) reagent was added to the assay plate and plates we incubated for 5 min at room temperature.
5. Signal was measured as median relative luminescence units on a ViewLux (PerkinElmer) multiplate reader.

Reference:
[1] Heske CM, Davis MI, Baumgart JT, Wilson K, Gormally MV, Chen L, Zhang X, Ceribelli M, Duveau DY, Guha R, Ferrer M, Arnaldez FI, Ji J, Tran HL, Zhang Y, Mendoza A, Helman LJ, Thomas CJ. Matrix Screen Identifies Synergistic Combination of PARP Inhibitors and Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibitors in Ewing Sarcoma. Clin Cancer Res. 2017 Dec 1;23(23):7301-7311. doi: 10.1158/1078-0432.CCR-17-1121. Epub 2017 Sep 12. PMID: 28899971; PMCID: PMC6636827.

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 cytotoxic compounds 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: s-my-OC1MY5-m4-1

Protocol: SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE and DESCRIPTION.
1; Cells; 5 uL; white, solid bottom 1536-well Greiner assay plate.
2; Compounds; 23 nL; Kalypsis pin tool (Wako USA) equipped with a 1536-well pin head to transfer to the assay plates.
3; Incubation; 48 hours; 37C incubation.
4; Reagent;5uL; CellTiter Glo (Promega).
5; Detector; 425V; Luminescence; Viewlux PerkinElmer.

NOTES (numbers refer to Sequence numbers above)
1. The cell-based screening methods employed in this study were like those previously published [1]. Briefly, the corresponding myeloma cell lines were screened in 1,536-well plates with 500 cells, 5-uL per well for inhibition of cell viability (assessed by measuring ATP levels with CellTiterGlo).
2. NCATS MIPE 4.0 library of approved and investigational drugs were added using the Kalypsis pin tool. Wells treated with DMSO only for 100% viability and media only for 0% viability were used as controls.
3. The assay plates were covered and incubated for 48 hours in a 37C, 5% CO2 incubator with controlled humidity.
4. After 48 hours, CellTilter-GloTM luminescent substrate mix (Promega) reagent was added to the assay plate and plates we incubated for 5 min at room temperature.
5. Signal was measured as median relative luminescence units on a ViewLux (PerkinElmer) multiplate reader.

Reference:
[1] Heske CM, Davis MI, Baumgart JT, Wilson K, Gormally MV, Chen L, Zhang X, Ceribelli M, Duveau DY, Guha R, Ferrer M, Arnaldez FI, Ji J, Tran HL, Zhang Y, Mendoza A, Helman LJ, Thomas CJ. Matrix Screen Identifies Synergistic Combination of PARP Inhibitors and Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibitors in Ewing Sarcoma. Clin Cancer Res. 2017 Dec 1;23(23):7301-7311. doi: 10.1158/1078-0432.CCR-17-1121. Epub 2017 Sep 12. PMID: 28899971; PMCID: PMC6636827.

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 cytotoxic compounds 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: s-my-KMS_34-m4-1

Protocol: SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE and DESCRIPTION.
1; Cells; 5 uL; white, solid bottom 1536-well Greiner assay plate.
2; Compounds; 23 nL; Kalypsis pin tool (Wako USA) equipped with a 1536-well pin head to transfer to the assay plates.
3; Incubation; 48 hours; 37C incubation.
4; Reagent;5uL; CellTiter Glo (Promega).
5; Detector; 425V; Luminescence; Viewlux PerkinElmer.

NOTES (numbers refer to Sequence numbers above)
1. The cell-based screening methods employed in this study were like those previously published [1]. Briefly, the corresponding myeloma cell lines were screened in 1,536-well plates with 500 cells, 5-uL per well for inhibition of cell viability (assessed by measuring ATP levels with CellTiterGlo).
2. NCATS MIPE 4.0 library of approved and investigational drugs were added using the Kalypsis pin tool. Wells treated with DMSO only for 100% viability and media only for 0% viability were used as controls.
3. The assay plates were covered and incubated for 48 hours in a 37C, 5% CO2 incubator with controlled humidity.
4. After 48 hours, CellTilter-GloTM luminescent substrate mix (Promega) reagent was added to the assay plate and plates we incubated for 5 min at room temperature.
5. Signal was measured as median relative luminescence units on a ViewLux (PerkinElmer) multiplate reader.

Reference:
[1] Heske CM, Davis MI, Baumgart JT, Wilson K, Gormally MV, Chen L, Zhang X, Ceribelli M, Duveau DY, Guha R, Ferrer M, Arnaldez FI, Ji J, Tran HL, Zhang Y, Mendoza A, Helman LJ, Thomas CJ. Matrix Screen Identifies Synergistic Combination of PARP Inhibitors and Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibitors in Ewing Sarcoma. Clin Cancer Res. 2017 Dec 1;23(23):7301-7311. doi: 10.1158/1078-0432.CCR-17-1121. Epub 2017 Sep 12. PMID: 28899971; PMCID: PMC6636827.

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 cytotoxic compounds 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: s-my-keats_L363-m4-1

Protocol: SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE and DESCRIPTION.
1; Cells; 5 uL; white, solid bottom 1536-well Greiner assay plate.
2; Compounds; 23 nL; Kalypsis pin tool (Wako USA) equipped with a 1536-well pin head to transfer to the assay plates.
3; Incubation; 48 hours; 37C incubation.
4; Reagent;5uL; CellTiter Glo (Promega).
5; Detector; 425V; Luminescence; Viewlux PerkinElmer.

NOTES (numbers refer to Sequence numbers above)
1. The cell-based screening methods employed in this study were like those previously published [1]. Briefly, the corresponding myeloma cell lines were screened in 1,536-well plates with 500 cells, 5-uL per well for inhibition of cell viability (assessed by measuring ATP levels with CellTiterGlo).
2. NCATS MIPE 4.0 library of approved and investigational drugs were added using the Kalypsis pin tool. Wells treated with DMSO only for 100% viability and media only for 0% viability were used as controls.
3. The assay plates were covered and incubated for 48 hours in a 37C, 5% CO2 incubator with controlled humidity.
4. After 48 hours, CellTilter-GloTM luminescent substrate mix (Promega) reagent was added to the assay plate and plates we incubated for 5 min at room temperature.
5. Signal was measured as median relative luminescence units on a ViewLux (PerkinElmer) multiplate reader.

Reference:
[1] Heske CM, Davis MI, Baumgart JT, Wilson K, Gormally MV, Chen L, Zhang X, Ceribelli M, Duveau DY, Guha R, Ferrer M, Arnaldez FI, Ji J, Tran HL, Zhang Y, Mendoza A, Helman LJ, Thomas CJ. Matrix Screen Identifies Synergistic Combination of PARP Inhibitors and Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibitors in Ewing Sarcoma. Clin Cancer Res. 2017 Dec 1;23(23):7301-7311. doi: 10.1158/1078-0432.CCR-17-1121. Epub 2017 Sep 12. PMID: 28899971; PMCID: PMC6636827.

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 cytotoxic compounds 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: TargetID_659_CEMA

Protocol: Black, standard capacity streptavidin-coated 384-well plates (Pierce 15407) were first washed with 50 L of phosphate buffer (100 mM, pH 7.0; PB7) three times using a Biotek 405 ELX plate washer. Subsequently, 5 L of biotinylated pre-miRNA substrate (500 nM final) was dispensed into the plate using a Multidrop Combi Reagent Dispenser (Thermo Scientific). Plates were then centrifuged for 1 min at 1,000 RPM (223 g), sealed with plate tape, and incubated overnight at 4 C. The following morning, plates were washed three times with 50 L of PB7, followed by the addition of 5 L of Dicer digest buffer (20 mM Tris, 12 mM NaCl, 2.5 mM MgCl2, 1 mM fresh DTT, and 4.5% DMSO) and centrifugation. Compounds (50 nL of 5 mM DMSO stock, 25 M final) were then added into the sample wells using a Sciclone (Caliper) liquid handler with V&P pintool; the same volume of DMSO was added to the control wells. The plates were incubated at 25 C for 15 min before addition of 5 L of digest buffer containing 217 g/nL Dicer (108 g/mL Dicer, 5% glycerol and 0.01% Triton X-100 final, excess with respect to pre-miRNA). For the positive control wells, digest buffer without Dicer was added. The plates were centrifuged again and resealed before being placed in a 37 C incubator for 5 h. After Dicer cleavage, plates were washed three times with 50 L of PB7. mTet-HRP in PB7 (10 L, 750 nM final) was then dispensed into each well. The plates were subsequently centrifuged, sealed, and incubated at 25 C for 2 h. Plates were then washed three times with 50 L of wash buffer (2 mM imidazole, 260 mM NaCl, 0.5 mM EDTA, 0.1% Tween-20, pH 7.0), followed by washing three additional times with 50 L of PB7. Finally, SuperSignal West Pico (25 L; Pierce) was added, the plates were incubated at 25 C for 5 min, and chemiluminescence signal was detected using a PHERAstar plate reader using LUM plus module (BMG Labtech).

Comment: The activity outcome is based on a Z-score (number of standard deviations from the negative control mean) of 3 or higher on at least 50% times that the sample was screened.

For instance, if the sample was screened in n=4 runs, it would be considered active only if it had a Z-score of 3 or above in at least 2 runs.

External ID: s-my-keats_OCIMY7-m4-1

Protocol: SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE and DESCRIPTION.
1; Cells; 5 uL; white, solid bottom 1536-well Greiner assay plate.
2; Compounds; 23 nL; Kalypsis pin tool (Wako USA) equipped with a 1536-well pin head to transfer to the assay plates.
3; Incubation; 48 hours; 37C incubation.
4; Reagent;5uL; CellTiter Glo (Promega).
5; Detector; 425V; Luminescence; Viewlux PerkinElmer.

NOTES (numbers refer to Sequence numbers above)
1. The cell-based screening methods employed in this study were like those previously published [1]. Briefly, the corresponding myeloma cell lines were screened in 1,536-well plates with 500 cells, 5-uL per well for inhibition of cell viability (assessed by measuring ATP levels with CellTiterGlo).
2. NCATS MIPE 4.0 library of approved and investigational drugs were added using the Kalypsis pin tool. Wells treated with DMSO only for 100% viability and media only for 0% viability were used as controls.
3. The assay plates were covered and incubated for 48 hours in a 37C, 5% CO2 incubator with controlled humidity.
4. After 48 hours, CellTilter-GloTM luminescent substrate mix (Promega) reagent was added to the assay plate and plates we incubated for 5 min at room temperature.
5. Signal was measured as median relative luminescence units on a ViewLux (PerkinElmer) multiplate reader.

Reference:
[1] Heske CM, Davis MI, Baumgart JT, Wilson K, Gormally MV, Chen L, Zhang X, Ceribelli M, Duveau DY, Guha R, Ferrer M, Arnaldez FI, Ji J, Tran HL, Zhang Y, Mendoza A, Helman LJ, Thomas CJ. Matrix Screen Identifies Synergistic Combination of PARP Inhibitors and Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibitors in Ewing Sarcoma. Clin Cancer Res. 2017 Dec 1;23(23):7301-7311. doi: 10.1158/1078-0432.CCR-17-1121. Epub 2017 Sep 12. PMID: 28899971; PMCID: PMC6636827.

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 cytotoxic compounds 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: s-my-MM1R-m4-1

Protocol: SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE and DESCRIPTION.
1; Cells; 5 uL; white, solid bottom 1536-well Greiner assay plate.
2; Compounds; 23 nL; Kalypsis pin tool (Wako USA) equipped with a 1536-well pin head to transfer to the assay plates.
3; Incubation; 48 hours; 37C incubation.
4; Reagent;5uL; CellTiter Glo (Promega).
5; Detector; 425V; Luminescence; Viewlux PerkinElmer.

NOTES (numbers refer to Sequence numbers above)
1. The cell-based screening methods employed in this study were like those previously published [1]. Briefly, the corresponding myeloma cell lines were screened in 1,536-well plates with 500 cells, 5-uL per well for inhibition of cell viability (assessed by measuring ATP levels with CellTiterGlo).
2. NCATS MIPE 4.0 library of approved and investigational drugs were added using the Kalypsis pin tool. Wells treated with DMSO only for 100% viability and media only for 0% viability were used as controls.
3. The assay plates were covered and incubated for 48 hours in a 37C, 5% CO2 incubator with controlled humidity.
4. After 48 hours, CellTilter-GloTM luminescent substrate mix (Promega) reagent was added to the assay plate and plates we incubated for 5 min at room temperature.
5. Signal was measured as median relative luminescence units on a ViewLux (PerkinElmer) multiplate reader.

Reference:
[1] Heske CM, Davis MI, Baumgart JT, Wilson K, Gormally MV, Chen L, Zhang X, Ceribelli M, Duveau DY, Guha R, Ferrer M, Arnaldez FI, Ji J, Tran HL, Zhang Y, Mendoza A, Helman LJ, Thomas CJ. Matrix Screen Identifies Synergistic Combination of PARP Inhibitors and Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibitors in Ewing Sarcoma. Clin Cancer Res. 2017 Dec 1;23(23):7301-7311. doi: 10.1158/1078-0432.CCR-17-1121. Epub 2017 Sep 12. PMID: 28899971; PMCID: PMC6636827.

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 cytotoxic compounds 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: s-my-mm1s-m4-1

Protocol: SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE and DESCRIPTION.
1; Cells; 5 uL; white, solid bottom 1536-well Greiner assay plate.
2; Compounds; 23 nL; Kalypsis pin tool (Wako USA) equipped with a 1536-well pin head to transfer to the assay plates.
3; Incubation; 48 hours; 37C incubation.
4; Reagent;5uL; CellTiter Glo (Promega).
5; Detector; 425V; Luminescence; Viewlux PerkinElmer.

NOTES (numbers refer to Sequence numbers above)
1. The cell-based screening methods employed in this study were like those previously published [1]. Briefly, the corresponding myeloma cell lines were screened in 1,536-well plates with 500 cells, 5-uL per well for inhibition of cell viability (assessed by measuring ATP levels with CellTiterGlo).
2. NCATS MIPE 4.0 library of approved and investigational drugs were added using the Kalypsis pin tool. Wells treated with DMSO only for 100% viability and media only for 0% viability were used as controls.
3. The assay plates were covered and incubated for 48 hours in a 37C, 5% CO2 incubator with controlled humidity.
4. After 48 hours, CellTilter-GloTM luminescent substrate mix (Promega) reagent was added to the assay plate and plates we incubated for 5 min at room temperature.
5. Signal was measured as median relative luminescence units on a ViewLux (PerkinElmer) multiplate reader.

Reference:
[1] Heske CM, Davis MI, Baumgart JT, Wilson K, Gormally MV, Chen L, Zhang X, Ceribelli M, Duveau DY, Guha R, Ferrer M, Arnaldez FI, Ji J, Tran HL, Zhang Y, Mendoza A, Helman LJ, Thomas CJ. Matrix Screen Identifies Synergistic Combination of PARP Inhibitors and Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibitors in Ewing Sarcoma. Clin Cancer Res. 2017 Dec 1;23(23):7301-7311. doi: 10.1158/1078-0432.CCR-17-1121. Epub 2017 Sep 12. PMID: 28899971; PMCID: PMC6636827.

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 cytotoxic compounds 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.