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: HAPV101

Protocol: NCGC Assay Protocol Summary:

The CellTiter-Glo# Luminescent Cell Viability Assay (Promega) is a homogeneous method to determine the number of viable cells in culture based on quantitation of the ATP present, which signals the presence of metabolically active cells. The homogeneous assay procedure involves adding a single reagent directly to cells cultured in serum-supplemented medium. Addition and mixing of the reagent results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present.

- Chemicals were dissolved with dimethyl sulfoxide (DMSO) into 12 different concentrations ranging from 0.26 nM to 46.0 uM and were aliquoted to 1536-well plate format via pin tool (Kalypsys, San Diego, CA). Negative control was established using vehicle, while positive control was established using staurosporine.
- Cells were grown to a concentration of at least 106 cells/ml, volume of at least 100 ml, and viability of 85% before treatment. For treatment, cells were filtered, diluted with fresh media to 3-4E5cells/ml, and plated to a tissue-culture treated 1,536 well white/solid bottom assay plates (Grenier Bio-One North Americal, NC) at 2000 cells/5uL/well using a flying reagent dispenser (Aurora discovery, Carlsbad, CA).
- ViewLux plate reader (PerkinElmer, Shelton, CT) was used to detect luminescent intensity in each well 40 hours post chemical treatment.

Comment: This is a profiling assay where compounds were screened against a panel of 81 immortalized human lymphoblast cell lines. Ranking of compounds within each cell line is described below.

Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description".
2. For all inactive 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: HAPC101

Protocol: NCGC Assay Protocol Summary:

The Caspase-Glo 3/7 Assay (Promega) provides a homogeneous luminescent assay that measures caspase-3/7 activities. The assay provides a proluminescent caspase-3/7 DEVD-aminoluciferin substrate and a proprietary thermostable luciferase in a reagent optimized for caspase-3/7 activity, luciferase activity and cell lysis. Adding the single Caspase-Glo 3/7 Reagent in an "add-mix-measure" format results in cell lysis, followed by caspase cleavage of the substrate. This liberates free aminoluciferin, which is consumed by the luciferase, generating a "glow-type" luminescent signal. The signal is proportional to caspase-3/7 activity. The stabilized luciferase and proprietary buffer system improve assay performance across a wide range of assay conditions, and the assay is less likely to be affected by compound interference unlike fluorescent- or colorimetric-based assays. The Caspase-Glo 3/7 Assay is designed for use with multiwell plate formats using either purified enzyme or cells in culture.
- Chemicals were dissolved with dimethyl sulfoxide (DMSO) into 12 different concentrations ranging from 0.26 nM to 46.0 uM and were aliquoted to 1536-well plate format via pin tool (Kalypsys, San Diego, CA). Negative control was established using vehicle, while positive control was established using staurosporine.
- Cells were grown to a concentration of at least 106 cells/ml, volume of at least 100 ml, and viability of 85% before treatment. For treatment, cells were filtered, diluted with fresh media to 3-4*105cells/ml, and plated to a tissue-culture treated 1,536 well white/solid bottom assay plates (Grenier Bio-One North Americal, NC) at 2000 cells/5uL/well using a flying reagent dispenser (Aurora discovery, Carlsbad, CA).
- ViewLux plate reader (PerkinElmer, Shelton, CT) was used to detect luminescent intensity in each well 40 hours post chemical treatment.

Comment: This is a profiling assay where compounds were screened against a panel of 81 immortalized human lymphoblast cell lines. Ranking of compounds within each cell line is described below.

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, active compounds are expected to have the activator phenotype and apparent activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

External ID: 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: HMS1625

Protocol: The fix-reporter plasmid was conjugated into this strain which the stably integrates into the chromosome using the mini-Tn7 system (PMID: 15908923). This plasmid consists of the fixK promoter, which is a target of the fixLJ pathway driving expression of GFP.
The day before screen an overnight culture of B. multivorans strain VC7102, with fix reporter, is set up in tryptic soy broth (TSB) at 37 degrees C with shaking. The day of the screen the overnight culture is diluted 1:100 in fresh tryptic soy broth.
The day of the screen, 30 microL of tryptic soy broth (TSB) is pipetted into columns 1-22 of a 384-well plate (Corning 3764) using the Combi multidrop dispenser. To column 24, 30 microL of TSB containing 78 microM of Benserazide TSB is added for a positive control. To column 23, 30 microL of TSB containing with DMSO (0.078% v/v in TSB) is added for a negative control. 300 nL of each compound are pin-transferred to each plate. For every compound plate 2 replicate assay plates are set up. 30 microL of the 1:100 diluted-B. multivorans strain VC7102 is added to each well using the Combi. Initial OD600 and GFP are measured using the PerkinElmer EnVision. Plates are stacked 5 high, covered with lids and incubated at 37 degrees C overnight (~18 h).
The following day, assay plates are read using a PerkinElmer EnVision (600 nm filter and GFP).
Positive control: Benserazide 39 microM in column 24 (No positive control was used for plates 2089 & 2090; Plates 2091-2093: columns 1 & 24)
Negative control: DMSO 300 nL/well in column 23 (For plates 2089 & 2090 negative controls were located in columns 2 & 24; plates 2091-2093: columns 2 & 23)

Comment: Analysis method:
For each compound well, the initial GFP value was subtracted from overnight GFP value to calculate the deltaGFP. The mean and standard deviation of the negative control wells deltaGFP (column 23) on both replicate plates was calculated. Wells with replicate average deltaGFP at least 3 standard deviations above the plate negative control deltaGFP average were scored as potential positives.

To determine activity scores, Z-scores were calculated for each replicate separately based on the replicate negative control deltaGFP plate average and standard deviation. The replicate average deltaGFP Z-scores were used for activity scores, with activity score of 100 set to Z-score = 4 and activity score = 0 (no activity) set to Z-score = 0. Z-scores > 4 were considered activity score = 100, and Z-scores < 0 were considered activity score = 0.

External ID: CHEMBL1015624

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Journal: J. Nat. Prod.
Year: 2002
Volume: 65
Issue: 4
First Page: 509
Last Page: 512
DOI: 10.1021/np010445g

Target ChEMBL ID: CHEMBL368
ChEMBL Target Name: Trypanosoma cruzi
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: HERG01

Protocol: NCGC Assay Protocol Summary:

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

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

qHTS protocol for hERG-U2OS cellular assay

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

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

Comment: Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

External ID: CHEMBL3631891

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Journal: Bioorg. Med. Chem.
Year: 2015
Volume: 23
Issue: 23
First Page: 7478
Last Page: 7486
DOI: 10.1016/j.bmc.2015.10.048

Target ChEMBL ID: CHEMBL368
ChEMBL Target Name: Trypanosoma cruzi
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL5130483

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Journal: J Nat Prod
Year: 2022
Volume: 85
Issue: 9.0
First Page: 2184
Last Page: 2191
DOI: 10.1021/acs.jnatprod.2c00478

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

External ID: CHEMBL5130484

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Journal: J Nat Prod
Year: 2022
Volume: 85
Issue: 9.0
First Page: 2184
Last Page: 2191
DOI: 10.1021/acs.jnatprod.2c00478

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

External ID: CHEMBL3631893

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2015
Volume: 23
Issue: 23
First Page: 7478
Last Page: 7486
DOI: 10.1016/j.bmc.2015.10.048

External ID: CHEMBL3631892

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Journal: Bioorg. Med. Chem.
Year: 2015
Volume: 23
Issue: 23
First Page: 7478
Last Page: 7486
DOI: 10.1016/j.bmc.2015.10.048

Target ChEMBL ID: CHEMBL614123
ChEMBL Target Name: J774
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: CHEMBL5303715

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Target ChEMBL ID: CHEMBL395
ChEMBL Target Name: HepG2
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: EU-OPENSCREEN

External ID: CHEMBL1282981

Protocol: N/A

Comment: Journal: Antimicrob. Agents Chemother.
Year: 2008
Volume: 52
Issue: 10
First Page: 3604
Last Page: 3611
DOI: 10.1128/aac.00661-08

Target ChEMBL ID: CHEMBL3879801
ChEMBL Target Name: NON-PROTEIN TARGET
ChEMBL Target Type: NON-MOLECULAR - Target has not been defined at a molecular level, only the non-molecular entity which is affected (e.g., organism, cell line etc)
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: APP-Toga-CHIKV-nsp2-p

Protocol: PROTOCOL TABLE (as described by Inglese J, Shamu CE and Guy RK. 2007)
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION.
1; Control / Compound; 20 nL; Echo 655 acoustic dispenser, Greiner 1536-well solid bottom black plate.
2; Enzyme; 4 uL; BioRAPTR FRD liquid dispenser (Beckman Coulter).
3; Incubation; 15 min; room temperature.
4; Reagent; 4 uL; 2.5 uM Peptide 2 substrate.
5; Incubation; 1 hr; room temperature.
6; Detection; Fluorescence; WiewLux microplate reader (PerkinElmer), 525 nm excitation, 598/25 nm emission.

NOTES (numbers refer to sequence numbers above).
1. Briefly, 20 nL DMSO, positive control ZnAc (20nM final concentration), and test compounds were transferred into a 1,536-well solid bottom black plate (789176-F, Greiner One) via Echo 655 acoustic dispenser (Beckman Coulter). For primary screens, compounds were tested at 7 concentrations, 1:3 dilution points ranging from 25 uM to 34 nM. Follow-up confirmatory screens were carried out at 11 concentrations, 1:3 dilution points from 25 uM to 0.42 nM.
2. Four uL nsP2pro enzyme mix (150 nM final concentration) in 10 mM Tris-HCl pH 8.0 with 0.01% Tween 20 assay buffer was dispensed into the plate using a BioRAPTR FRD liquid dispenser (Beckman Coulter).
3. The plate was incubated at room temperature (protected from light) for 15 min
4. Four microliter of peptide 2 substrate (2.5 uM final concentration) in assay buffer was added to the plate.
5. After 1 hour, plates were immediately read on a ViewLux high-throughput CCD imager (Exposure = 10 sec, Gain = High, Speed = Slow, Binning = 2X). The above assay was also incorporated in the NCATS HTS facility41, which allowed for robotic liquid and compound dispensing, microplate handling, and fluorescence reading..

REFERENCE:
Inglese J, Shamu CE and Guy RK, Reporting data from high throughput screening of small molecule libraries, Nature Chemical Biology, 2007, 3(8): 438-441. doi.org/10.1038/nchembio0807-438.

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 [1].

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.

Reference:
1. Inglese J, Auld DS, Jadhav A, et al. Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries. Proc Natl Acad Sci U S A. 2006;103(31):11473-11478.

External ID: Ebola screen2_EMI141217_green

Protocol: This 1536-well plate assay was adapted from the original 6-wells assay with a modification that eliminated cell wash steps. HeLa cells were plated at 750 cells/well in 3 microl of assay medium (DMEM + 10% FBS) in 1536-well assay plates and incubated for 16 hours at 37 degrees C and 5% CO2. Compounds in the 1536-well drug source plates were added to the 1536-well assay plates in a volume of 23 nl/well via a NX-TR pintool station (WAKO Scientific Solutions, San Diego, CA). Following 1-hour incubation at 37 degrees C with 5% CO2, 1 microl/well of VLP solution was added to the assay plates using a BioRapTR FRD dispenser (the VLP solution was diluted in Opti-MEM to a final concentration of 1:16). Plates were then spinoculated by centrifugation at 1500 rpm at 4 degrees C for 45 minutes followed by incubation at 37 degrees C with 5% CO2 for 4.5 hours. The CCF2-AM beta-lactamase substrate was prepared at 6x concentration following the manufacturer's instruction which was added to assay plates at 1 microl/well. Following a 2-hour incubation at room temperature, dual fluorescence intensities (Ex1 = 405+/-20, Em1 = 460+/-20, and Ex2 = 405+/-20, Em2 = 530+/-20 nm) were measured in an EnVision plate reader (PerkinElmer, Boston, MA). The ratio of fluorescence intensities (Em1/Em2) was calculated to represent the beta-lactamase activity that is proportional to the amount of VLP entry into the host cells.

Comment: The activity score was determined using both AC50, Efficacy and curve class. For a given compound j:
Activity is defined as the ability to inhibit viral entry.

If curve class = 4, activity score = 0 and outcome is set to 1. Compounds of this class have no significant activity points.
If efficacy is negative, activity score = 0 and outcome is set to 1. Compounds with negative efficacies, promote viral entry.

For all other curve classes:

Activity score(j) = [100*((max(Ac50)-Ac50(j))/(max(Ac50)-min(Ac50))) + 100*(1-(max(efficacy)-efficacy(j))/(max(efficacy)-min(efficacy))) ]/2

Compounds with Activity scores > 80% are deemed active, and the activity outcome is set to 2.

Refer to the following recent publication for more detail regarding active compounds:

Identification of 53 compounds that block Ebola virus-like particle entry via a repurposing screen of approved drugs

Jennifer Kouznetsova, Wei Sun, Carles Marti nez-Romero, Gregory Tawa, Paul Shinn, Catherine Z Chen,
Aaron Schimmer, Philip Sanderson, John C McKew, Wei Zheng and Adolfo Garci a-Sastre

Emerging Microbes and Infections (2014) 3, e84; doi:10.1038/emi.2014.88

External ID: Ebola screen2_EMI141217_ratio

Protocol: This 1536-well plate assay was adapted from the original 6-wells assay with a modification that eliminated cell wash steps. HeLa cells were plated at 750 cells/well in 3 microl of assay medium (DMEM + 10% FBS) in 1536-well assay plates and incubated for 16 hours at 37 degrees C and 5% CO2. Compounds in the 1536-well drug source plates were added to the 1536-well assay plates in a volume of 23 nl/well via a NX-TR pintool station (WAKO Scientific Solutions, San Diego, CA). Following 1-hour incubation at 37 degrees C with 5% CO2, 1 microl/well of VLP solution was added to the assay plates using a BioRapTR FRD dispenser (the VLP solution was diluted in Opti-MEM to a final concentration of 1:16). Plates were then spinoculated by centrifugation at 1500 rpm at 4 degrees C for 45 minutes followed by incubation at 37 degrees C with 5% CO2 for 4.5 hours. The CCF2-AM beta-lactamase substrate was prepared at 6x concentration following the manufacturer's instruction which was added to assay plates at 1 microl/well. Following a 2-hour incubation at room temperature, dual fluorescence intensities (Ex1 = 405+/-20, Em1 = 460+/-20, and Ex2 = 405+/-20, Em2 = 530+/-20 nm) were measured in an EnVision plate reader (PerkinElmer, Boston, MA). The ratio of fluorescence intensities (Em1/Em2) was calculated to represent the beta-lactamase activity that is proportional to the amount of VLP entry into the host cells.

Comment: The activity score was determined using both AC50, Efficacy and curve class. For a given compound j:
Activity is defined as the ability to inhibit viral entry.

If curve class = 4, activity score = 0 and outcome is set to 1. Compounds of this class have no significant activity points.
If efficacy is negative, activity score = 0 and outcome is set to 1. Compounds with negative efficacies, promote viral entry.

For all other curve classes:

Activity score(j) = [100*((max(Ac50)-Ac50(j))/(max(Ac50)-min(Ac50))) + 100*(1-(max(efficacy)-efficacy(j))/(max(efficacy)-min(efficacy))) ]/2

Compounds with Activity scores > 80% are deemed active, and the activity outcome is set to 2.

Refer to the following recent publication for more detail regarding active compounds:

Identification of 53 compounds that block Ebola virus-like particle entry via a repurposing screen of approved drugs

Jennifer Kouznetsova, Wei Sun, Carles Marti nez-Romero, Gregory Tawa, Paul Shinn, Catherine Z Chen,
Aaron Schimmer, Philip Sanderson, John C McKew, Wei Zheng and Adolfo Garci a-Sastre

Emerging Microbes and Infections (2014) 3, e84; doi:10.1038/emi.2014.88

External ID: Ebola screen2_EMI141217_blue

Protocol: This 1536-well plate assay was adapted from the original 6-wells assay with a modification that eliminated cell wash steps. HeLa cells were plated at 750 cells/well in 3 microl of assay medium (DMEM + 10% FBS) in 1536-well assay plates and incubated for 16 hours at 37 degrees C and 5% CO2. Compounds in the 1536-well drug source plates were added to the 1536-well assay plates in a volume of 23 nl/well via a NX-TR pintool station (WAKO Scientific Solutions, San Diego, CA). Following 1-hour incubation at 37 degrees C with 5% CO2, 1 microl/well of VLP solution was added to the assay plates using a BioRapTR FRD dispenser (the VLP solution was diluted in Opti-MEM to a final concentration of 1:16). Plates were then spinoculated by centrifugation at 1500 rpm at 4 degrees C for 45 minutes followed by incubation at 37 degrees C with 5% CO2 for 4.5 hours. The CCF2-AM beta-lactamase substrate was prepared at 6x concentration following the manufacturer's instruction which was added to assay plates at 1 microl/well. Following a 2-hour incubation at room temperature, dual fluorescence intensities (Ex1 = 405+/-20, Em1 = 460+/-20, and Ex2 = 405+/-20, Em2 = 530+/-20 nm) were measured in an EnVision plate reader (PerkinElmer, Boston, MA). The ratio of fluorescence intensities (Em1/Em2) was calculated to represent the beta-lactamase activity that is proportional to the amount of VLP entry into the host cells.

Comment: The activity score was determined using both AC50, Efficacy and curve class. For a given compound j:
Activity is defined as the ability to inhibit viral entry.

If curve class = 4, activity score = 0 and outcome is set to 1. Compounds of this class have no significant activity points.
If efficacy is negative, activity score = 0 and outcome is set to 1. Compounds with negative efficacies, promote viral entry.

For all other curve classes:

Activity score(j) = [100*((max(Ac50)-Ac50(j))/(max(Ac50)-min(Ac50))) + 100*(1-(max(efficacy)-efficacy(j))/(max(efficacy)-min(efficacy))) ]/2

Compounds with Activity scores > 80% are deemed active, and the activity outcome is set to 2.

Refer to the following recent publication for more detail regarding active compounds:

Identification of 53 compounds that block Ebola virus-like particle entry via a repurposing screen of approved drugs

Jennifer Kouznetsova, Wei Sun, Carles Marti nez-Romero, Gregory Tawa, Paul Shinn, Catherine Z Chen,
Aaron Schimmer, Philip Sanderson, John C McKew, Wei Zheng and Adolfo Garci a-Sastre

Emerging Microbes and Infections (2014) 3, e84; doi:10.1038/emi.2014.88

External ID: CHEMBL1282988

Protocol: N/A

Comment: Journal: Antimicrob. Agents Chemother.
Year: 2008
Volume: 52
Issue: 10
First Page: 3604
Last Page: 3611
DOI: 10.1128/aac.00661-08

Target ChEMBL ID: CHEMBL3879801
ChEMBL Target Name: NON-PROTEIN TARGET
ChEMBL Target Type: NON-MOLECULAR - Target has not been defined at a molecular level, only the non-molecular entity which is affected (e.g., organism, cell line etc)
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: Ebola screen2_EM141217_green

Protocol: This 1536-well plate assay was adapted from the original 6-wells assay with a modification that eliminated cell wash steps. HeLa cells were plated at 750 cells/well in 3 microl of assay medium (DMEM + 10% FBS) in 1536-well assay plates and incubated for 16 hours at 37 degrees C and 5% CO2. Compounds in the 1536-well drug source plates were added to the 1536-well assay plates in a volume of 23 nl/well via a NX-TR pintool station (WAKO Scientific Solutions, San Diego, CA). Following 1-hour incubation at 37 degrees C with 5% CO2, 1 microl/well of VLP solution was added to the assay plates using a BioRapTR FRD dispenser (the VLP solution was diluted in Opti-MEM to a final concentration of 1:16). Plates were then spinoculated by centrifugation at 1500 rpm at 4 degrees C for 45 minutes followed by incubation at 37 degrees C with 5% CO2 for 4.5 hours. The CCF2-AM beta-lactamase substrate was prepared at 6x concentration following the manufacturer's instruction which was added to assay plates at 1 microl/well. Following a 2-hour incubation at room temperature, dual fluorescence intensities (Ex1 = 405+/-20, Em1 = 460+/-20, and Ex2 = 405+/-20, Em2 = 530+/-20 nm) were measured in an EnVision plate reader (PerkinElmer, Boston, MA). The ratio of fluorescence intensities (Em1/Em2) was calculated to represent the beta-lactamase activity that is proportional to the amount of VLP entry into the host cells.

Comment: The activity score was determined using both AC50, Efficacy and curve class. For a given compound j:
Activity is defined as the ability to inhibit viral entry.

If curve class = 4, activity score = 0 and outcome is set to 1. Compounds of this class have no significant activity points.
If efficacy is negative, activity score = 0 and outcome is set to 1. Compounds with negative efficacies, promote viral entry.

For all other curve classes:

Activity score(j) = [100*((max(Ac50)-Ac50(j))/(max(Ac50)-min(Ac50))) + 100*(1-(max(efficacy)-efficacy(j))/(max(efficacy)-min(efficacy))) ]/2

Compounds with Activity scores > 70% are deemed active, and the activity outcome is set to 2.

Note that this scoring scheme only applies to the ratio of blue/green fluorescence intensities because it is this ratio that represents the activity of Bla inside cells. Separate uploads of blue and green data exist in this assay group but information on activity should not be derived from these.

Refer to the following recent publication for more detail regarding active compounds:

Identification of 53 compounds that block Ebola virus-like particle entry via a repurposing screen of approved drugs

Jennifer Kouznetsova, Wei Sun, Carles Marti nez-Romero, Gregory Tawa, Paul Shinn, Catherine Z Chen,
Aaron Schimmer, Philip Sanderson, John C McKew, Wei Zheng and Adolfo Garci a-Sastre

Emerging Microbes and Infections (2014) 3, e84; doi:10.1038/emi.2014.88

External ID: 5mMGluc_INS1E_InsulinRelease_40mMGluc_SP

Protocol: N/A

Comment: N/A

External ID: ERR985

Protocol: Tox21 Assay Protocol Summary:

The ERR cells were dispensed at 2,000 cells/5 ul/well in 1536-well white plates using a Multidrop dispenser. After the assay plates were incubated at a 37 C/5% CO2 incubator for 6 hours, 23 nL of compounds dissolved in DMSO, positive and negative controls or DMSO only was transferred to the assay plate by a pin tool. The plates were incubated at 37 C for 18 hours. 4 ul/well of One-Glo reagent was added into the assay plates using a Flying Reagent Dispenser. After 30-minute incubation at room temperature, the luminescence intensity in the plates was measured using a ViewLux plate reader.

Comment: Disclaimer:

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