External ID: SERCaMPGLuc-p1-antagonist

Protocol: PROTOCOL TABLES
SEQUENCE No. (1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE and DESCRIPTION.
1; Reagent; 5 uL; 1000 SH-SY5Y cells per wells
2; Time; 5 hour; 37C, 5% CO2
3; Compound; 23 nL; Control inhibitor / compound library
4; Time; 16 hour; 37C, 5% CO2
5; Reagent; 100 nM; Thapsigargin
6; Time; 4 hour; 37C, 5% CO2
7; Reagent; 1 uL; 0.5x coelenterazine
8; Detection; Luminescence; ViewLux imaging system

NOTES (numbers refer to sequence above)
1; SH-SY5Y human neuroblastoma cells stably expressing GLuc-SERCaMP (SH-SY5Y-GLuc-ASARTDL) cells were seeded in 1,536 well white tissue culture treated plates (Corning, Cat# 7464) in DMEM-high glucose-sodium pyruvate (ThermoFisher Scientific, Cat #10569) supplemented with 10% bovine growth serum (Hyclone), 10 U/ml penicillin (Gibco), 10 ug/ml streptomycin (Gibco), and 20 mM HEPES.
2; Assay plates were incubated for 5 hour at 37C in a humidified incubator containing 5% CO2.
3; qHTS libraries (23 nl, final concentrations of 1.53 uM, 7.67 uM, 38.3 uM) or controls (neutral control: DMSO, positive control: dantrolene) were added using a Kalypsis pin-tool Robotic System equipped with 1536 pinheads.
4; Cells were then incubated for 16 hours at 37C, 5% CO2.
5; Thapsigargin was added at 100 nM to deplete ER calcium stores.
6; Cells were incubated for 4 hour (37oC, 5% CO2)
7; Gaussia luciferase in the medium was measured by adding 1 ul of 0.5x coelenterazine (final concentration 0.07x) prepared in Gaussia Luciferase Glow Assay Buffer (Pierce), without addition of the Cell Lysis Buffer Reagent.
8; Luminescence was measured using a ViewLux high-432 throughput CCD imaging system (Perkin Elmer) equipped with clear filters. Compounds exhibiting inhibitory activity (defined as curve class -1.1, -1.2, -1.3, -1.4, -2.1, -2.2, -2.3, -2.4, -3) were identified by normalizing plate-wise to corresponding intra-plate controls (neutral control = Tg only; positive control (100% inhibition) = DMSO vehicle) with percent activity derived using in-house software (https://tripod.nih.gov/curvefit). The same controls were also used for the calculation of the Z' factor, a measure of assay quality control, as previously described (Zhang et al., 1999). For the initial validation of activity in the SERCaMP assays, hits from the primary screen were assayed again at 11-concentrations (1.3 nM - 76.6 uM). SH-SY5Y-GLuc-SERCaMP cells were assayed for ER Ca2+ depletion as outlined above.

Reference:
1. Zhang, J.H., Chung, T.D., and Oldenburg, K.R. (1999). A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen 4, 67-73.

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: 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: huntington-HTTAS8-p1-FF-overN

Protocol: PROTOCOL TABLES
SEQUENCE No. (1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE and DESCRIPTION.
1; Reagent; 4 uL; 2,500 Hek293 luciferase reporter cells per wells
2; Time; 24 hour; 37C, 5% CO2
3; Compound; 23 nL; Control inhibitor / compound library
4; Time; 24 hour; 37C, 5% CO2
5; Reagent; 2.5 uL; Firefly Luc reagent; Dual-Glo Luciferase Reporter Assay System (Promega)
6; Time; 10 min; room temperature incubation
7; Detection; Luminescence; FLuc

NOTES (numbers refer to sequence above)
1; High-throughput screening, measuring luminescence from a Hek293 luciferase reporter, was performed in 1,536 well white-walled tissue-culture treated plates. 2,500 cells were plated in 4 muL of Opti-MEM per well using a Multidrop Combi Reagent Dispenser (ThermoFisher).
2; Assay plates were cultured for 24 hours.
3; Control inhibitor (PTC124; final concentration of 50 uM) and compound library (at final concentrations of 460 nM - 57.5 uM) were added to the assay plate using the Kalypsis Pintool Robotic System equipped with 1536 pinheads.
4; Cells were then incubated for 24 hours at 37 degrees C prior to luminescence measurements.
5; In all cases, luminescence was measured using the Dual-Glo Luciferase Reporter Assay System (Promega) following the manufacturer's instructions.
6; Room temperature incubation.
7; Luminescence signal was obtained using the PerkinElmer ViewLux plate reader. Results were normalized to vehicle control (0% activity) and zero RLU (-100% activity).

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 activators are ranked higher than compounds that showed apparent inhibition.
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: ZIK159

Protocol: Assay Protocol Summary:

The medium for SNB-19 cells is composed of RPMI 1640 (ATCC, Cat.# 30-2001), 10% fetal bovine serum (FBS) (GE healthcare Life Sciences, Cat.# SH30071.03), and 1% Pen/Strep (Gibco, Cat.# 15140-122). A Caspase-Glo 3/7 assay kit (catalog number G8092; Promega, Madison, WI) was used to detect caspase-3 activity induced by Zika virus infection in human cells. The reagent mixture was reconstituted as described in the protocol from the manufacturer. Polystyrene tissue culture treated and PDL coated white plates were obtained from Greiner Bio-One (Monroe, NC). Cells were seeded in 384- or 1536-well assay plates and cultured at 37 C with 5% CO2 for 16 to 20 hours. The typical cell seeding density in the 1536-well plate assay is 250 cells/well in 3ul medium for SNB-19 cells in tissue culture treated plates. Compounds were added to cells and incubated for one hour before addition of ZIKV solution to cells (2 FFU/cell). After incubation at 37 C with 5% CO2 for 6 hours, the reagent mixture of Caspase-Glo 3/7 assay kit was added to each well, followed by incubation at room temperature for 30 minutes. The luminescence intensity of the assay plates was measured using a ViewLux plate reader (PerkinElmer). Data were normalized by using the cell-containing wells without ZIKV as a negative control (0% induction of caspase 3/7 activity) and wells containing ZIKV infected cells (Caspase-3 activity induced) as a positive control (100% induction of caspase 3 activity). The percentage inhibitions of the increased Caspase-3 activity by small molecule inhibitors were then calculated.

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: HMS1315

Protocol: Cybrid cells were washed with PBS, trypsinized, spun down at 300g for 5 minutes, and resuspended in 10 mLs of PBS. Cells were counted, and the appropriate amount of cells were added to a vial containing DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose, DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 0.3% DMSO, DMEM No Glucose + 2% FBS + 1% P/S + 10mM Galactose + 1uM I-BET GSK 525762A, or DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 1.25mM Glucose. Cells were then seeded in a Corning 3570 384-well plate (40 ul/well,1500 cells/well). Two replicates were prepared as described at the same time per library plate.

100 nl of compound was added to each well via pin transfer immediately after seeding. After the addition of compound, cells were incubated for 72 hours at 37 degrees C with 5% CO2.

Following the 72 hour incubation period, the 384 well plates were centrifuged for 1 minute at 1000 rpm. The media was aspirated using an aspiration wand and fresh media DMEM only, was added to the cells (40uL/ well) using a well-mate. Fifteen-microliters of Cell Titer Glow substrate was then added to each well using the well-mate. The plates were gently vortexed for 10 seconds and then centrifuged for 1 minute at 1000 rpm. The plates were then immediately read on the Envision instrument and the luminescence was detected for each individual well.

Positive control (strong): Cells seeded in DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 1.25mM Glucose
Positive control (weak): Cells seeded in DMEM No Glucose + 2% FBS + 1% P/S + 10mM Galactose + 1uM I-BET GSK 525762A
Negative control: Cells seeded in DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 0.3% DMSO

Comment: Data analysis method and criteria for scoring active compounds:

Z-scores were calculated for both replicates separately using the plate average and standard deviation of experimental well luminescence (Z = (x - mu)/sigma). Compounds were considered active if both replicate Z-scores >= 1.8. Activity scores were determined by scaling replicate average Z-scores from 0 (activity score = 0) to 4 (activity score = 100), with activity score > 45 being considered active. Z-scores < 0 were set to activity score = 0; Z-scores > 4 were set to activity score = 100 (100% activity).

External ID: ZIK097

Protocol: Assay Protocol Summary:

The medium for hNPCs consists of DMEM/F12, N2 supplement (ThermoFisher, Cat.# 17502048), NEAA (ThermoFisher, Cat. # 11140050), 2 ug/ml heparin, 2 uM cyclopamine, and B27 (ThermoFisher, Cat. # 17504044). A Caspase-Glo 3/7 assay kit (catalog number G8092; Promega, Madison, WI) was used to detect caspase-3 activity induced by Zika virus infection in human cells. The reagent mixture was reconstituted as described in the protocol from the manufacturer. Polystyrene tissue culture treated and PDL coated white plates were obtained from Greiner Bio-One (Monroe, NC). Cells were seeded in 384- or 1536-well assay plates and cultured at 37 C with 5% CO2 for 16 to 20 hours. The typical cell seeding density in the 1536-well plate assay is 350 cells/well in 3 ul medium for hNPCs in tissue culture treated plates. Compounds were added to cells and incubated for one hour before addition of ZIKV solution to cells (2 FFU/cell). After incubation at 37 C with 5% CO2 for 6 hours, the reagent mixture of Caspase-Glo 3/7 assay kit was added to each well, followed by incubation at room temperature for 30 minutes. The luminescence intensity of the assay plates was measured using a ViewLux plate reader (PerkinElmer). Data were normalized by using the cell-containing wells without ZIKV as a negative control (0% induction of caspase 3/7 activity) and wells containing ZIKV infected cells (Caspase-3 activity induced) as a positive control (100% induction of caspase 3 activity). The percentage inhibitions of the increased Caspase-3 activity by small molecule inhibitors were then calculated.

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: HMS1149-MLP

Protocol: A 558 bp region upstream of hspX was PCR amplified and cloned upstream of the GFP-mut2 (Cormack et al., 1996). This construct was cloned into a modified version of the replicating plasmid pSE100 and transformed into CDC1551 to generate CDC1551(hspX'::GFP).

The M. tuberculosis CDC 1551 (hspX'::GFP) fluorescent biosensor was grown in pH 7.0 Middlebrook 7H9 medium to mid- to late-log phase. Black-walled, clear-bottom, tissue-culture treated 384-well microtiter plates (Corning #3712) were filled with 25 uL of buffered pH 7.0 7H9 medium using a Matrix Combi. 100 nL of compound was then transferred to each well via stainless steel pin array. Plates were manually sealed with DMSO-resistant aluminum seals and frozen at -80C until they were ready to be screened. The cultures were re-suspended in pH 7.0 7H9 and 25 uL was dispensed into each well of the 384-well assay plates already containing the perturbator utilizing a Cy-Bio Selma liquid handler robot to an OD595 of 0.05. The plates were then placed in a Ziploc bag with a moistened paper towel (to limit evaporation) and incubated for 6 days at 37C. Fluorescence and optical density (OD) readings were made on an EnSpire plate reader (Perkin Elmer, Inc.) at excitation and emission wavelengths of 488 and 509 nm as a top read, with the OD being taken at 595 nm as a bottom read.

The ~328,633 compound library was arrayed into black-walled, clear-bottom tissue-culture treated 384-well microtiter plates (Corning #3712) by pin transfer (100 nL), containing 25 uL of buffered pH 7.0 7H9 medium. In a previous screen utilizing the same compound library and screening method, four random compound plates were screened in duplicate to observe variation in hit robustness across plates and method. The result was that the duplicate plates did not vary in a statistically significant manner and due to the high volume of compounds to be screened (~328,633), replicates were not utilized.

Negative control: DMSO, 0.1% final concentration in buffered pH 7.0 7H9 in column 2 or column 23 of each plate

Positive control: 0.3 uM Rifampicin final concentration in buffered pH 7.0 7H9 in column 1 or column 24 of each plate

Comment: Analysis methods:
To calculate normalized fluorescence percent inhibition, plate average negative control fluorescence intensity (based on all plates within a screening run) was subtracted from well fluorescence intensity, divided by the difference between plate negative and positive control average fluorescence intensity (based on all plates within a screening run), and multiplied by 100. To calculate normalized growth inhibition, plate average negative control absorbance (based on all plates within a screening run) was subtracted from well absorbance, divided by the difference between plate negative and positive control average absorbance (based on all plates within a screening run), and multiplied by 100. Normalized fluorescence percent inhibition was divided by normalized growth inhibition to calculate a ratio. Wells were considered active if growth was not greatly decreased and showed either a 3-fold selectivity fluorescence to growth inhibition ratio (with 35-45% fluorescence inhibition) or a 1.5-fold selectivity fluorescence to growth inhibition ratio (with > 45% fluorescence inhibition). These compounds may be directly or indirectly inhibiting DosRS signaling. Wells were considered general inhibitors of M. tuberculosis growth if both fluorescence and growth inhibition > 50%. Activity scores were scaled from 100 to 0 and derived from normalized fluorescence percent inhibition, with 100 (or > 100) = 100% inhibition and 0 (or < 0) = no inhibition.

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.