External ID: PDBbind-Kd for protein-ligand complexes

Protocol:

Comment:

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: TRND-SARS-cytotox-48hr-p1-npc

Protocol: PROTOCOL TABLE
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION.
1; Dispense; 2 uL cells; white, 1536-well Greiner assay plate.
2; Incubate; 16 hr; 37 C, 5% CO2
3; Dispense; 23 nL; compounds in DMSO
4; Incubate; 1 hr; 37 C, 5% CO2
5; Dispense; 2 uL; media
6; Incubate; 48 hr; 37C, 5% CO2
7; Centrifuge; 10 sec; Blue Washer gentle centrifugation
8; Dispense; 4 uL; ATPLite reagent
9; Incubate; 5 min; room temperature
10; Read; Luminescence; ViewLux plate reader

NOTES (numbers refer to Sequence numbers above)
1. Seed 1000 Vero E6 cells (ATCC #CRL-1586) in 2 uL/well media (EMEM 10% FetalClone II, 1x Pen/Strep) in white 1536-well assay plates (Greiner #782073).
2. Incubate at 37 C with 5% CO2 overnight (~16 h).
3. Dispense 23 nL/well compounds in DMSO via pin transfer.
4. Incubate for 1 hr at 37C 5% CO2.
5. Dispense 2 uL/well of media (EMEM 10% FetalClone II, 1x Pen/Strep).
6. Incubate at for 48 hr at 37C 5% CO2.
7. Remove supernatant with gentle centrifugation using a Blue Washer (BlueCat Bio).
8. Dispense 4 uL/well of ATPLite 1step luminescence assay reagent (PerkinElmer #6016739).
9.Incubate for 5 min at room temperature.
10. Read luminescence signal (Viewlux plate reader, PerkinElmer). Data was normalized with wells containing cells as 100%, and wells containing media as 0%.

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 apparent 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: GPx1-biochemical-p4-p7

Protocol: PROTOCOL TABLE
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION.
1; Reagent 1; 3 uL; GPx1 enzyme or no enzyme control in 1536-well, black, solid Greiner assay plate.
2; Compound; 25 nL; Kalypsis pin tool (Wako USA) equipped with a 1536-well pin head.
3; Incubation; 30 min; room temperature.
4; Reagent 2; 1 uL; 10 nM GPX1, 1 mM GSH, 0.5 mM CHP, 0.5 mM NADPH, 100 nM GR, 0.01% BSA substrate
5; Detection; Fluorescence; ViewLux microplate reader, excitation 340 nm and emission 450 nm.
6; Incubation; 15 min; room temperature.
7; Detection; Fluorescence; ViewLux microplate reader, excitation 340 nm and emission 450 nm.

NOTES (numbers refer to Sequence numbers above)
1. 3 uL of 16.66 nM GPX1 and 0.016% BSA in assay buffer (50 mM Tris-HCl, 2 mM EDTA (pH 7.5), 150 mM NaCl) was added to columns 3-48 of 1536-well assay plates using a BioRAPTR Flying Reagent Dispenser (Let's Go Robotics (LGR), Carlsbad, CA). A no-enzyme control (0.01% BSA in TES assay buffer) was added to columns 1-2.
2. 25 nL of test compounds and DMSO controls were added to each well with a pin tool (Kalypsys).
3. The assay plates were incubated for 30 minutes at room temperature.
4. 1 uL of master mix (500 nM glutathione reductase (GR), 5 mM reduced glutathione (GSH), 2.5 mM NADPH in assay buffer) was added. 1 uL of cumene hydroperoxide (CHP) ([2.5 mM] in 50% EtOH) was then added within 5 minutes to initiate the reaction. Final concentrations were: 10 nM GPX1, 1 mM GSH, 0.5 mM CHP, 0.5 mM NADPH, 100 nM GR, 0.01% BSA.
5. Initial fluorescence readout (t=0) at 340/450 nm was measured using a ViewLux multimodal detector (PerkinElmer, Waltham, MA).
7. Endpoint fluorescence readout (t=15) at 340/450 nm was measured 15 min after room temperature incubation.

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: TRND-MERS-PP-p1-npc

Protocol: PROTOCOL TABLE
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION.
1; Dispense; 2 uL cells; white, 1536-well Greiner assay plate.
2; Incubate; 16 hr; 37C, 5% CO2
3; Dispense; 23 nL; compounds in DMSO
4; Incubate; 1 hr; 37C, 5% CO2
5; Dispense; 2 uL; SARS-S MLVpp
6; Centrifuge; 45 min; 1500 rpm, room temperature
7; Incubate; 48 hr; 37C, 5% CO2
8; Centrifuge; 10 sec; Blue Washer gentle centrifugation
9; Dispense; 4 uL; Bright-Glo Luciferase reagent
10; Incubate; 5 min; room temperature
11; Read; Luminescence; ViewLux plate reader

NOTES (numbers refer to Sequence numbers above)
1. Seed 2000 Huh7 cells (JCRB cell bank # JCRB0403) in 2 microL/well media (DMEM 10% FetalClone II, 1x Pen/Strep) in white 1536-well assay plates (Greiner #782073).
2. Incubate at 37 degrees C with 5% CO2 overnight (~16 h).
3. Dispense 23 nL/well compounds in DMSO via pin transfer.
4. Incubate for 1 h at 37C 5% CO2.
5. Dispense 2 microL/well of MERS-S pseudotyped particles (MERS-S MLVpp).
6. Spin-inoculate by centrifugation at 1500 rpm (453 xg) for 45 min at room temperature.
7. Incubate at for 48 h at 37C 5% CO2.
8. Remove supernatant with gentle centrifugation using a Blue Washer (BlueCat Bio).
9. Dispense 4 microL/well of Bright-Glo Luciferase detection reagent (Cat#E2620, Promega).
10. Incubate for 5 min at room temperature.
11. Read luminescence signal (Viewlux plate reader, PerkinElmer). Data was normalized with wells containing MERS-S MLVpp as 100%, and wells containing control MLVpp (no fusion protein) as 0%.

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: TRND-SARS-CoV-2-cytotox-48hr

Protocol: PROTOCOL TABLE
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE and DESCRIPTION.
1. Cells. Seed 1500 HEK293-ACE2 cells (Expi293F with stable expression of human ACE2) in 2 uL/well media (DMEM, 10% FBS, 1x L-glutamine, 1x Pen/Strep, 1 ug/ml puromycin) in white 1536-well assay plates (Greiner #782073).
2. Incubation. Incubate at 37 C with 5% CO2 overnight (~16 h).
3. Compounds. Dispense 23 nL/well compounds in DMSO via pin transfer.
4. Incubation. Incubate for 1 h at 37C 5% CO2.
5. Reagent. Dispense 2 uL/well of media (DMEM, 10% FBS, 1x L-glutamine, 1x Pen/Strep, 1 ug/ml puromycin).
6. Incubation. Incubate at for 48h at 37C 5% CO2
7. Reagent. Dispense 4 uL/well of ATPLite 1step luminescence assay reagent (PerkinElmer #6016739).
8. Incubation. Incubate for 15 min at room temperature.
9. Detection. Read luminescence signal (Viewlux plate reader, PerkinElmer). Data was normalized with wells containing cells as 100%, and wells without cells (media only control) as 0%.

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: TRND-MERS-cytotox-48hr-p1-npc

Protocol: PROTOCOL TABLE
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION.
1; Dispense; 2 uL cells; white, 1536-well Greiner assay plate.
2; Incubate; 16 hr; 37C, 5% CO2
3; Dispense; 23 nL; compounds in DMSO
4; Incubate; 1 hr; 37C, 5% CO2
5; Dispense; 2 uL; media
6; Incubate; 48 hr; 37C, 5% CO2
7; Centrifuge; 10 sec; Blue Washer gentle centrifugation
8; Dispense; 4 uL; ATPLite reagent
9; Incubate; 5 min; room temperature
10; Read; Luminescence; ViewLux plate reader

NOTES (numbers refer to Sequence numbers above)
1. Seed 2000 Huh7 cells (JCRB cell bank # JCRB0403) in 2 uL/well media (DMEM 10% FetalClone II, 1x Pen/Strep) in white 1536-well assay plates (Greiner #782073).
2. Incubate at 37C with 5% CO2 overnight (~16 h).
3. Dispense 23 nL/well compounds in DMSO via pin transfer.
4. Incubate for 1 hr at 37C 5% CO2.
5. Dispense 2 uL/well of media (DMEM 10% FetalClone II, 1x Pen/Strep).
6. Incubate at for 48 hr at 37C 5% CO2.
7. Remove supernatant with gentle centrifugation using a Blue Washer (BlueCat Bio).
8. Dispense 4 uL/well of ATPLite 1step luminescence assay reagent (PerkinElmer #6016739).
9. Incubate for 5 min at room temperature.
10. Read luminescence signal (Viewlux plate reader, PerkinElmer). Data was normalized with wells containing cells as 100%, and wells containing media as 0%.

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 apparent 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: TRND-SARS-CoV-2-PP

Protocol: PROTOCOL TABLE (format 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 and DESCRIPTION.

1. Cells. Seed 1500 HEK293-ACE2 cells (Expi293F with stable expression of human ACE2) in 2 uL/well media (DMEM, 10% FBS, 1x L-glutamine, 1x Pen/Strep, 1 ug/ml puromycin) in white 1536-well assay plates (Greiner #782073).
2. Incubation. Incubate at 37C with 5% CO2 overnight (~16 h).
3. Compounds. Dispense 23 nL/well compounds in DMSO via pin transfer.
4. Incubation. Incubate for 1 hr at 37C 5% CO2.
5. Reagent. Dispense 2 uL/well of SARS-CoV-2-S pseudotyped particles. [a] PPs are produced with murine leukemia virus pseudotyping. [b] SARS-CoV-2-S is Wuhan-Hu-1 sequence (BEI #NR-52420) with C-terminal 19 amino acid truncation.
6. Centrifuge. Spin-inoculate by centrifugation at 1500 rpm (453 xg) for 45 min at room temperature.
7. Incubation. Incubate at for 48 hr at 37C 5% CO2
8. Centrifuge. Remove supernatant with gentle centrifugation using a Blue Washer (BlueCat Bio).
9. Reagent. Dispense 4 uL/well of Bright-Glo Luciferase detection reagent (Promega #E2620).
10. Incubation. Incubate for 5 min at room temperature.
11. Detection. Read luminescence signal (Viewlux plate reader, PerkinElmer). Data was normalized with wells containing SARS-CoV-2-S PP as 100%, and wells containing bald PP (no fusion protein) as 0%.

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: TRND-SARS-PP-p1-npc

Protocol: Protocol:
PROTOCOL TABLE
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION.
1; Dispense; 2 uL cells; white, 1536-well Greiner assay plate.
2; Incubate; 16 hr; 37 C, 5% CO2
3; Dispense; 23 nL; compounds in DMSO
4; Incubate; 1 hr; 37 C, 5% CO2
5; Dispense; 2 uL; SARS-S MLVpp
6; Centrifuge; 45 min; 1500 rpm, room temperature
7; Incubate; 48 hr; 37C, 5% CO2
8; Centrifuge; 10 sec; Blue Washer gentle centrifugation
9; Dispense; 4 uL; Bright-Glo Luciferase reagent
10; Incubate; 5 min; room temperature
11; Read; Luminescence; ViewLux plate reader

NOTES (numbers refer to Sequence numbers above)
1. Seed 2000 Vero E6 cells (ATCC #CRL-1586) in 2 uL/well media (EMEM 10% FetalClone II, 1x Pen/Strep) in white 1536-well assay plates (Greiner #782073).
2. Incubate at 37 C with 5% CO2 overnight (~16 h).
3. Dispense 23 nL/well compounds in DMSO via pin transfer.
4. Incubate for 1 hr at 37C 5% CO2.
5. Dispense 2 uL/well of SARS-S pseudotyped particles (SARS-S MLVpp).
6. Spin-inoculate by centrifugation at 1500 rpm (453 xg) for 45 min at room temperature.
7. Incubate at for 48 hr at 37C 5% CO2.
8. Remove supernatant with gentle centrifugation using a Blue Washer (BlueCat Bio).
9. Dispense 4 uL/well of Bright-Glo Luciferase detection reagent (Promega #E2620).
10. Incubate for 5 min at room temperature.
11. Read luminescence signal (Viewlux plate reader, PerkinElmer). Data was normalized with wells containing SARS-S MLVpp as 100%, and wells containing control MLVpp (no fusion protein) as 0%.

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.