External ID: OPRM1-OPRD1_AG_LUMI_1536_1X%ACT PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that activate heterodimer formation between the mu (OPRM1) and delta (OPRD1) opioid receptors, resulting in membrane recruitment of beta-arrestin. The assay monitors GPCR-beta-arrestin proximity using low affinity fragment complementation of beta-galactosidase (beta-gal). This assay employs U2OS cells which express OPRD1, OPRM1 fused to a beta-gal peptide fragment (enzyme donor), and beta-arrestin fused to the complementary beta-gal fragment (enzyme acceptor). Cells are incubated with test compound, followed by measurement of well luminescence. As designed, compounds that induce formation of OPRD1 homodimers or OPRM1-OPRD1 (Mu-Delta) heterodimers will cause beta-arrestin recruitment, resulting in reconstitution of the beta-gal holoenzyme. The reconstituted holoenzyme can then catalyze the hydrolysis of a substrate which yields a chemiluminescent signal, resulting in increased well luminescence. Deltorphin B will be used as the high (100% RLU) control for agonists, and wells containing cells treated with DMSO will be used as the low (0% RLU) control. Compounds were tested in singlicate at a final nominal concentration of 9.3 uM.

Protocol Summary:

The U2OS-OPRM1-OPRD1 (Mu-Delta) cell line was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of a 1:1 mixture of Ham's F-12 Nutrient Media (F-12) and Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% v/v heat-inactivated certified fetal bovine serum, 25 mM HEPES, 250 ug/mL Geneticin, 250 ug/mL Hygromycin B, 0.25 ug/mL Puromycin and 1X antibiotic mix (penicillin, streptomycin, and neomycin).

The day before the assay 1000 cells in 3 uL of cell plating media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 C, 5% CO2, and 95 % RH for 23 hours. Next, 28 nL of test compound in DMSO, Deltorphin B (0.9 uM final concentration) in DMSO, or DMSO alone were dispensed to the appropriate wells. The plates were then incubated for 3 hours at 37 C, 5% CO2, and 95 % RH. The assay was started by adding 2 uL of PathHuntertrade mark reagent (prepared according to the manufacturer's protocol); followed by 1 hour incubation at room temperature. Then, Well Luminescence was read on the ViewLux plate reader

The percent activation for each compound was calculated as follows:

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

Where:

High_Control is defined as wells containing cells, Deltorphin B and DMSO.
Test_Compound is defined as wells containing cells, test compounds and DMSO.
Low_Control is defined as wells containing cells and DMSO.

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

PubChem Activity Outcome and Score:

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 100-2, and for inactive compounds 2-0.

List of Reagents:

PathHuntertrade mark B-arrestin recruitment assay, containing the U2OS OPRM1 OPRD1 Beta-arrestin cell line; PathHunter Detection Kit (DiscoveRx, part, 93-0558C3)
Ham's F-12 media (Invitrogen, part 11765-054)
DMEM media (Invitrogen, part 11995-073)
Detachin (Genlantis, part T100100)
Heat Inactivated Fetal Bovine Serum (Invitrogen, part 10082-147)
Puromycin (Invitrogen, part A11138-02)
Hygromycin B (Invitrogen, part 10687-010)
Geneticin (Invitrogen, part 10131-027)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
T-175 tissue culture flasks (Nunc, part 159910)
Agonist: Deltorphin B (Anaspec, part 62683)
1536-well plates (Greiner, part 789173)

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: 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: 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: 7124-01_Inhibitor_SinglePoint_HTS_Activity

Protocol:
Protocol:
1) Plate 10 uL BL2-NFkB-luc cells at 12.5K cells/well, using a Multidrop Combi reagent dispenser (1,250 cells/uL).
2) Add 25 nL compound or positive control, by pin transfer; IKK inhibitor VII will be used at 50 uM.
3) Incubate at 37 masculineC for 1 hour (allowing compounds to enter cells for action).
4) Add 5 uL 192 ng/mL tCD40L per well for a final concentration of 64 ng/ml using a Thermo Multidrop Combi.
5) Incubate at 37 masculineC for 4 hrs, then leave plate at RT for 30 minutes.
6) Add 15 uL SteadyGlo luciferase substrate (Promega) per well using a Thermo Multidrop Combi.
7) Incubate at RT for 5min, then read Luminescence using LJL Analyst HT plate reader.

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

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
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.10.0.2) 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 at -50.

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 55.

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 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

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: 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: CHEMBL955283

Protocol: N/A

Comment: Journal: J Nat Prod
Year: 1982
Volume: 45
Issue: 4
First Page: 418
Last Page: 426
DOI: 10.1021/np50022a010

Target ChEMBL ID: CHEMBL398
ChEMBL Target Name: KB
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: 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.

External ID: s-my-KMS-28PE-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.