External ID: SNCA-p-activity-luciferase

Protocol: PROTOCOL TABLE
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION

1; Cells; 4 uL; Dispense 1500 HEK-293-SNCA-luc cells/well into Greiner 1536-well white / solid bottom tissue culture treated plate. The plate was covered with metal lids with gas-exchange holes.
2; Incubate; 24 hours; Incubate at 37C, 5% CO2, 95% RH.
3; Compounds; 23 nL; Compounds and controls were transferred via a Kalypsys Pin Tool (Wako USA) equipped with a 1536-slotted pin array. The plate was covered with metal lids with gas-exchange holes.
4; Incubate; 24 hours; Incubate at 37C, 5% CO2, 95% RH.
5; Dispense; 1 uL; Dispense Gly-Phe-7-amino-4-trifluoromethylcoumarin (GF-AFC, prepared at 125 uM in PBS) was added. The plate was covered with metal lids with gas-exchange holes.
6; Incubate; 30 min; Incubate at 37C, 5% CO2.
7; Detector; Fluorescence; Measure fluorescence with ViewLux microplate reader (PerkinElmer) equipped with 405/10 excitation and 540/25 emission filters.
8; Dispense; 3 uL; Dispense ONE-Glo (PerkinElmer) lucifase detection reagent was added to each well. Plates were covered with metal lids with gas-exchange holes.
9; Incubate; 15 min; Incubate at room temperature.
10; Detector; Luminescence; Measure luminescence with ViewLux microplate reader (PerkinElmer) equipped with clear filters.

NOTES (numbers refer to sequence above)
1; HEK-293-SNCA-luc were cultured and suspended in phenol-red free DMEM (4.5 g/L glucose, 25 mM HEPES, cat #21063 (Thermo)).
3; Compounds were added to the assay plate in an 11-point intra plate dose response, 1:3 titration in DMSO with a final concentration range of xxx - yyy uM. Vehicle-only plates, with DMSO being pin-transferred to every well, were inserted at the beginning of screening runs to confirm expected assay performance. Activity was normalized to wells containing medium only (-100% activity, full inhibition) and SNCA-luc cells treated with DMSO vehicle control (0% activity), contained on the same plate as test samples.
10; Signals were analyzed, and dose-response curves were fit using the Hill equation. Compounds in curve classes -1.1, -1.2, -2.1, -2.2 in the SNCA-luc assay were considered active. Compounds were eliminated from further consideration if also active (curve class -1.1, -1.2, -1.3, -1.4, -2.1, -2.2, -2.3, -2.4) in the GF-AFC cytotoxicity assay.

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

Compound Ranking:
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, with a ratio activity curve class of 4, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. For a ratio activity curve class = -1.1, score = 80+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 && abs(ratio.max_response) > 6*10, score = 60+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -2.1 || ( ratio.curve_class==-2.2 && abs(ratio.max_response) > 6*10), score = 40+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 || ratio.curve_class == -2.2, score = 20+abs((log_ac50+4.5)*inf_activity/20). Inconclusive compounds, with a donor curve class other than 4, have PUBCHEM_ACTIVITY_SCORE of 10. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39.

External ID: CYP273

Protocol: Tox21 Assay Protocol Summary:

Two ul of enzyme-substrate mix was dispensed into medium binding white/solid 1536-well plates (Greiner Bio-One North America Inc., Monroe, NC) using a BioRaptr Flying Reagent Dispenser (FRD, Beckman Coulter, Brea, CA). Compounds dissolved in DMSO and positive control (furafyllline) were transferred to the assay plates at 23 nl using a Pintool station (Wako, San Diego, CA). The assay plates were incubated at room temperature for 10 min. Then 2 ul of NADPH regeneration solution was added to each well of the assay plates using an FRD and incubated at room temperature for 1 h. The reaction was stopped by adding 4 ul of detection reagent using an FRD and after 20 min incubation at room temperature the luminescence signal was measured using a ViewLux plate reader (Perkin Elmer, Shelton, CT). Data were expressed as relative luminescence units.

Comment: Disclaimer:

Although all reasonable efforts have been made to ensure the accuracy and reliability of the data, caution should be exercised when interpreting the results as artifacts are possible from nonspecific effects such as assay signal interference. The curve fitting and activity calls presented here are based on the NCATS analysis methods. Alternative analysis methods and interpretations of the data are available at EPA (http://actor.epa.gov) and NTP (http://tools.niehs.nih.gov/cebs3/ui/).

Compound Ranking:

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

External ID: CHEMBL3761240

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: 2016
Volume: 24
Issue: 5
First Page: 1115
Last Page: 1120
DOI: 10.1016/j.bmc.2016.01.037

External ID: CHEMBL3760464

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: 2016
Volume: 24
Issue: 5
First Page: 1115
Last Page: 1120
DOI: 10.1016/j.bmc.2016.01.037

External ID: CHEMBL3761242

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: 2016
Volume: 24
Issue: 5
First Page: 1115
Last Page: 1120
DOI: 10.1016/j.bmc.2016.01.037

Target ChEMBL ID: CHEMBL261
ChEMBL Target Name: Carbonic anhydrase I
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL3761241

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: 2016
Volume: 24
Issue: 5
First Page: 1115
Last Page: 1120
DOI: 10.1016/j.bmc.2016.01.037

Target ChEMBL ID: CHEMBL205
ChEMBL Target Name: Carbonic anhydrase II
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL3270415

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: 2014
Volume: 22
Issue: 11
First Page: 2939
Last Page: 2946
DOI: 10.1016/j.bmc.2014.04.006

Target ChEMBL ID: CHEMBL261
ChEMBL Target Name: Carbonic anhydrase I
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL3270416

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: 2014
Volume: 22
Issue: 11
First Page: 2939
Last Page: 2946
DOI: 10.1016/j.bmc.2014.04.006

Target ChEMBL ID: CHEMBL205
ChEMBL Target Name: Carbonic anhydrase II
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL3270419

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: 2014
Volume: 22
Issue: 11
First Page: 2939
Last Page: 2946
DOI: 10.1016/j.bmc.2014.04.006

External ID: CHEMBL3270418

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: 2014
Volume: 22
Issue: 11
First Page: 2939
Last Page: 2946
DOI: 10.1016/j.bmc.2014.04.006

External ID: CHEMBL3270420

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: 2014
Volume: 22
Issue: 11
First Page: 2939
Last Page: 2946
DOI: 10.1016/j.bmc.2014.04.006

External ID: CHEMBL3407118

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: 8
First Page: 1728
Last Page: 1734
DOI: 10.1016/j.bmc.2015.02.045

External ID: CHEMBL3407119

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: 8
First Page: 1728
Last Page: 1734
DOI: 10.1016/j.bmc.2015.02.045

External ID: CHEMBL3407116

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: 8
First Page: 1728
Last Page: 1734
DOI: 10.1016/j.bmc.2015.02.045

Target ChEMBL ID: CHEMBL261
ChEMBL Target Name: Carbonic anhydrase I
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL3407117

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: 8
First Page: 1728
Last Page: 1734
DOI: 10.1016/j.bmc.2015.02.045

Target ChEMBL ID: CHEMBL205
ChEMBL Target Name: Carbonic anhydrase II
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL3407120

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: 8
First Page: 1728
Last Page: 1734
DOI: 10.1016/j.bmc.2015.02.045

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: CHEMBL5144455

Protocol: N/A

Comment: Journal: Eur J Med Chem
Year: 2022
Volume: 227
First Page: 113956
Last Page: 113956
DOI: 10.1016/j.ejmech.2021.113956

External ID: CHEMBL5144456

Protocol: N/A

Comment: Journal: Eur J Med Chem
Year: 2022
Volume: 227
First Page: 113956
Last Page: 113956
DOI: 10.1016/j.ejmech.2021.113956

External ID: CHEMBL5144457

Protocol: N/A

Comment: Journal: Eur J Med Chem
Year: 2022
Volume: 227
First Page: 113956
Last Page: 113956
DOI: 10.1016/j.ejmech.2021.113956

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: 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: CHEMBL3789760

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. Lett.
Year: 2016
Volume: 26
Issue: 8
First Page: 1941
Last Page: 1946
DOI: 10.1016/j.bmcl.2016.03.014

External ID: CHEMBL1063127

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: 2009
Volume: 17
Issue: 13
First Page: 4503
Last Page: 4509
DOI: 10.1016/j.bmc.2009.05.002

Target ChEMBL ID: CHEMBL205
ChEMBL Target Name: Carbonic anhydrase II
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: H - Homologous protein target assigned
Confidence: Homologous single protein target assigned

External ID: CHEMBL1063128

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: 2009
Volume: 17
Issue: 13
First Page: 4503
Last Page: 4509
DOI: 10.1016/j.bmc.2009.05.002

Target ChEMBL ID: CHEMBL261
ChEMBL Target Name: Carbonic anhydrase I
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL1063125

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: 2009
Volume: 17
Issue: 13
First Page: 4503
Last Page: 4509
DOI: 10.1016/j.bmc.2009.05.002

Target ChEMBL ID: CHEMBL5337
ChEMBL Target Name: Carbonic anhydrase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: H - Homologous protein target assigned
Confidence: Homologous single protein target assigned

External ID: CHEMBL1063126

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: 2009
Volume: 17
Issue: 13
First Page: 4503
Last Page: 4509
DOI: 10.1016/j.bmc.2009.05.002

External ID: BindingDB_3445_1

Protocol: N/A

Comment: Compounds with any of Ki, IC50, Kd, or EC50 activity value <= 10uM are labeled as "Active".
If multiple measurements are available for a given compound, it is labeled as "Active" if any of the measurements meet the criterion. Activity values are checked in the order of Ki, IC50, Kd, and EC50. The first entry that meets the above activity threshold is used to determine "Standard Type", "Standard Relation", and "PubChem Standard Value". Otherwise, the first non-empty entry will be used to set those values.

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.