External ID: DSHEA-v1-HepG2-viability-CTG-ATP

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; Cells; 5 uL; HepG2 cells, white solid-bottom tissue treated Greiner plate.
2; Incubation; 4 hrs; 37C, 95% humidity, 5% CO2.
3; Compounds; 23 nL; Kalypsis pin tool (Wako USA) equipped with a 1536-well pin head to transfer to the assay plates.
4; Incubation; 24 hrs; 37C, 95% humidity, 5% CO2.
5; Reagent; 1 uL; GF-AFC substrate.
6; Incubation; 30 min; 37C, 95% humidity, 5% CO2.
7; Read 1; Fluorescence; EnVision plate reader excitation 380 nm, emission 510 nm.
8; Reagent; 3 uL; CellTiter-Glo (Promega).
9; Centrifuge; 1000 RPM; 15 seconds.
10; Incubation; 30 min; room temperature.
11; Read 2; Luminescence; ViewLux plate reader.

NOTES (numbers refer to Sequence numbers above)
1. 5 uL of HepG2 cells at 2 x 105 cells/mL (1,000 cells/well) were dispensed into a 1536-well white solid-bottom tissue treated plate (Greiner Bio-One) using a Multidrop Combi dispenser (Thermo Scientific). This assay use both GF-AFC substrate and Cell-Titer Glo (CTG), measuring cellular protease activity and ATP content, respectively.
2. Plates were incubated at 37C, 95% humidity, and 5% CO2 for ~4 hours to allow for cell attachment.
3. Compounds were transferred (16 nL) via pintool (Wako Automation), for a final concentration range for most substances of 15.6 nM - 31.3 uM.
4. Plates were incubated for ~24 hours.
5. 1 uL addition of GF-AFC substrate (final concentration 25 uM; MP Biomedicals).
6. Plates were incubated at 37C, 95% humidity, and 5% CO2 for 30 minutes.
7. After incubation, plates were read for fluorescence intensity (Ex/Em = 380 nm/510 nm) on an EnVision detector (PerkinElmer, Shelton, Connecticut).
8. Once the detection for fluorescence was completed, plates were dispensed with 3 uL of CellTiter-Glo (Promega, Madison, WI).
9. Plates were centrifuged for 15 seconds at 1,000 RPM's.
10. Plates were incubated for 30 minutes at room temperature.
11. Plates were then read for luminescence intensity on a ViewLux detector.

REFERENCES:
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.

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: CHEMBL5225614

Protocol: N/A

Comment: Journal: ACS Med Chem Lett
Year: 2023
Volume: 14
Issue: 1.0
First Page: 59
Last Page: 65
DOI: 10.1021/acsmedchemlett.2c00425

Target ChEMBL ID: CHEMBL391
ChEMBL Target Name: Vero
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: 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-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: 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: HMS1482

Protocol: Prior to screening, cells were passaged in high glucose DMEM supplemented with 10% FBS, 1% P/S, 1 mM pyruvate, and HEPES. Media was changed every 1-2 days to prevent starvation and maintain cellular health.

On the day of screening, 384-well plates (Corning 3765) were filled with 30 uL of control cybrid cell suspension (33.3 cells/ul). Media for resuspension was DMEM supplemented with 10% FBS, 1% P/S, 1 mM pyruvate, and HEPES. Positive control columns were 23 and 24 for most plate setups. Initially, tunicamycin (15 nM) and galactose media (10 mM + 1 mM glucosamine) were used in columns 23 and 24, respectively. Over the course of the screen, galactose was substituted with Vidofludimus (11 uM). Next, 33 nL of each compound were pin-transferred to each plate in duplicate. Final assay well volume was 30 uL. Plates were stacked 2 high, covered with lids, and incubated at 37 degrees C for 2 days.

After 2 days of incubation, Hoescht (10 ul/well) was added to assay plates and fluorescence was read using the Acumen laser scanning cytometer. After, the bottom of plates were sealed with two layers of Brightmax Sealing Films. Then Promega NanoGlo Live cell reagent (20 ul/well) was added to each well and luminescence was read on a PerkinElmer EnVision plate reader after 5 minute of shaking.

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

Normalized luminescence values (luminescence / cell number) for each replicate were calculated (based on average of 2 luminescence reads per replicate). Z-scores were calculated using the plate average and standard deviation of experimental well normalized values. Each plate replicate was individually analyzed to measure reproducibility across plates. Z-scores were then averaged across replicates and a Z-score threshold of 1.96 was selected for positive hits, indicating increased mitochondrial supercomplex formation. Wells were excluded from further consideration based on low cell number (1 or both replicates with number of objects count < 1500). Activity scores were determined by scaling Z-scores from 0 (activity score = 0) to 4 (activity score = 100), with activity score > 50 being considered active. Z-scores < 0 were set to activity score = 0; Z-scores > 4 were set to activity score = 100 (100% activity).

External ID: 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: DSHEA-v1-PXR-antagonist-CTF

Protocol: For PXR luciferase reporter gene assays, we used hPXR-LucHepG2cells provided by Dr. Taosheng Chen (Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital). Cells were cultured in EMEM supplemented with 10% fetal bovine serum, 100U/mL penicillin and 100ug/mL streptomycin, and 500ug/mL of geneticin.

PROTOCOL TABLE (as described by Inglese J, Shamu CE and Guy RK. 2007) [1]
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION.
1; Cells; 5 uL; hPXR-LucHepG2; white solid-bottom Greiner plate.
2; Incubation; 5h; 37C, 95% humidity, 5% CO2
3; Compounds and control; 23 nL; Kalypsis pintool (Wako USA).
4; Reagent; 1uL; rifampicin (RIF) in the antagonist mode.
5; Incubation; 24 hr; 37C, 5% CO2.
6; Reagent; 1 uL; CellTiter-Fluor detection reagent.
7; Incubation; 1 hr; 37C, 5% CO2.
8; Read; Fluorescence; ViewLux plate reader.
9; Reagent; 4 uL; ONE-Glo Luciferase detection reagent.
10; Incubation; 30 min; room temperature.
11; Read; Luminescence; ViewLux plate reader.

NOTES (numbers refer to Sequence numbers above)
1. Briefly, 5uL of hPXR-LucHepG2 cells in phenol red-free DMEM containing 5% charcoal-stripped FBS, 1 mM sodium pyruvate (Invitrogen), 2 mM L-glutamine (Invitrogen), and 100 U/mL penicillin and 100ug/mL streptomycin were dispensed using a BioRAPTR FRD at 4 x 105cells/mL (2000cells/well) in tissue culture-treated 1536-well white assay plates (GreinerBio-One).
2. Plates were incubated for 5h at 37C, 95% humidity, and 5% CO2 to allow for cell attachment.
3. Compounds (final concentration for most substances ranged from 15.6 nM to 45.9uM), positive SPA70 (final concentration range of 2.8 nM to 92uM for the antagonist mode), and cytotoxicity control tetraoctyl ammonium bromide (final concentration of 92uM) were transferred (23 nL) via pintool (Wako Automation).
4-5. After compound transfer, plates were dispensed with 1uL of RIF (final concentration of 2uM) in the antagonist mode, using a BioRAPTR FRD and incubated for 24 h at 37C and 5% CO2.
6-8. Following incubation, 1uL of CellTiter-Fluor (Promega, Madison, Wisconsin) was added using a BioRAPTR FRD, after which all plates were incubated (37C/ 5% CO2) for1 h and then measured for fluorescence intensity (Ex/Em= 405/540 nm) on a ViewLux plate reader to determine cell viability.
9-11. Directly after this fluorescence reading, 4uL of ONE-Glo Luciferasere agent (Promega) was added to each well using the BioRAPTR FRD, followed by a 30 min incubation (RT) and read for luminescence intensity on a ViewLux detector.

REFERENCES:
[1] 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.
[2] Lin W, Wu J, Dong H, Bouck D, Zeng FY, Chen T. Cyclin-dependent kinase 2 negatively regulates human pregnane X receptor-mediated CYP3A4 gene expression in HepG2 liver carcinoma cells. J Biol Chem. 2008 Nov 7;283(45):30650-7. doi: 10.1074/jbc.M806132200. Epub 2008 Sep 9. PMID: 18784074; PMCID: PMC2662154.

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 antagonists 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: DSHEA-v1-PXR-antagonist-ONEGlo

Protocol: For PXR luciferase reporter gene assays, we used hPXR-LucHepG2cells provided by Dr. Taosheng Chen (Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital). Cells were cultured in EMEM supplemented with 10% fetal bovine serum, 100U/mL penicillin and 100ug/mL streptomycin, and 500ug/mL of geneticin.

PROTOCOL TABLE (as described by Inglese J, Shamu CE and Guy RK. 2007) [1]
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION.
1; Cells; 5 uL; hPXR-LucHepG2; white solid-bottom Greiner plate.
2; Incubation; 5h; 37C, 95% humidity, 5% CO2
3; Compounds and control; 23 nL; Kalypsis pintool (Wako USA).
4; Reagent; 1uL; rifampicin (RIF) in the antagonist mode.
5; Incubation; 24 hr; 37C, 5% CO2.
6; Reagent; 1 uL; CellTiter-Fluor detection reagent.
7; Incubation; 1 hr; 37C, 5% CO2.
8; Read; Fluorescence; ViewLux plate reader.
9; Reagent; 4 uL; ONE-Glo Luciferase detection reagent.
10; Incubation; 30 min; room temperature.
11; Read; Luminescence; ViewLux plate reader.

NOTES (numbers refer to Sequence numbers above)
1. Briefly, 5uL of hPXR-LucHepG2 cells in phenol red-free DMEM containing 5% charcoal-stripped FBS, 1 mM sodium pyruvate (Invitrogen), 2 mM L-glutamine (Invitrogen), and 100 U/mL penicillin and 100ug/mL streptomycin were dispensed using a BioRAPTR FRD at 4 x 105cells/mL (2000cells/well) in tissue culture-treated 1536-well white assay plates (GreinerBio-One).
2. Plates were incubated for 5h at 37C, 95% humidity, and 5% CO2 to allow for cell attachment.
3. Compounds (final concentration for most substances ranged from 15.6 nM to 45.9uM), positive SPA70 (final concentration range of 2.8 nM to 92uM for the antagonist mode), and cytotoxicity control tetraoctyl ammonium bromide (final concentration of 92uM) were transferred (23 nL) via pintool (Wako Automation).
4-5. After compound transfer, plates were dispensed with 1uL of RIF (final concentration of 2uM) in the antagonist mode, using a BioRAPTR FRD and incubated for 24 h at 37C and 5% CO2.
6-8. Following incubation, 1uL of CellTiter-Fluor (Promega, Madison, Wisconsin) was added using a BioRAPTR FRD, after which all plates were incubated (37C/ 5% CO2) for1 h and then measured for fluorescence intensity (Ex/Em= 405/540 nm) on a ViewLux plate reader to determine cell viability.
9-11. Directly after this fluorescence reading, 4uL of ONE-Glo Luciferasere agent (Promega) was added to each well using the BioRAPTR FRD, followed by a 30 min incubation (RT) and read for luminescence intensity on a ViewLux detector.

REFERENCES:
[1] 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.
[2] Lin W, Wu J, Dong H, Bouck D, Zeng FY, Chen T. Cyclin-dependent kinase 2 negatively regulates human pregnane X receptor-mediated CYP3A4 gene expression in HepG2 liver carcinoma cells. J Biol Chem. 2008 Nov 7;283(45):30650-7. doi: 10.1074/jbc.M806132200. Epub 2008 Sep 9. PMID: 18784074; PMCID: PMC2662154.

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 antagonists 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: HMS1262

Protocol: NEC is stored at -80 degrees at a concentration of 15mg/ml in single use aliquots.

On the day of the screen, 20ul of purified NEC is aliquoted using a Multidrop Combi reagent dispenser into 384 well plates (Corning 3824). 100nl of compound dissolved in DMSO was transferred to each well of the assay plated via pin transfer. The plates (NEC + compound) are incubated at room temperature for 3 hours. Acceptor and donor reagents (CisBio 620/665 pair) are combined then added to each well at 5 microL volumes at a concentration of 8 nM and 80nM respectively. The plates are spun at 1k rpm for 1 min and incubated overnight at 4 degrees, then for one hour the subsequent day at room temperature.

Flourescent measurements are read on the Envision 1 plate reader at ICCB-L. The raw data consists of two fluorescence readings - at 665 nm and 620 nm for the acceptor and donor respectively.

Comment: Data analysis:
The raw data consists of two fluorescence readings - at 665 and 620 nm for the acceptor and donor respectively. The data is processed as a ratio of the emission from the acceptor over the donor (homogeneous time resolved fluorescence ratio). Normalized percent inhibition (NPI) for all experimental wells is calculated based on plate averages for negative and positive control HTRF ratio. Positives are scored as any ratio with a 50% or greater inhibition as compared with the positive control (i.e. NEC + Untagged UL50). To be considered a hit, both replicates need to score as positive. Activity scores are derived from NPI, with 100 = 100% inhibition (> 100% set to 100) and 0 = no inhibition (< 0% set to 0). Note that some compounds with NPI <50% (activity scores < 50) are classified as potential hits based on additional criteria (typically by selecting wells with low ratios compared to other experimental wells on the plate).

External ID: DSHEA-v1-PXR-agonist-ONEGlo

Protocol: For PXR luciferase reporter gene assays, we used hPXR-LucHepG2cells provided by Dr. Taosheng Chen (Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital). Cells were cultured in EMEM supplemented with 10% fetal bovine serum, 100U/mL penicillin and 100ug/mL streptomycin, and 500ug/mL of geneticin.

PROTOCOL TABLE (as described by Inglese J, Shamu CE and Guy RK. 2007) [1]
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION.
1; Cells; 5 uL; hPXR-LucHepG2; white solid-bottom Greiner plate.
2; Incubation; 5h; 37C, 95% humidity, 5% CO2
3; Compounds and control; 23 nL; Kalypsis pintool (Wako USA).
4; Reagent; 1uL; rifampicin (RIF) in the antagonist mode.
5; Incubation; 24 hr; 37C, 5% CO2.
6; Reagent; 1 uL; CellTiter-Fluor detection reagent.
7; Incubation; 1 hr; 37C, 5% CO2.
8; Read; Fluorescence; ViewLux plate reader.
9; Reagent; 4 uL; ONE-Glo Luciferase detection reagent.
10; Incubation; 30 min; room temperature.
11; Read; Luminescence; ViewLux plate reader.

NOTES (numbers refer to Sequence numbers above)
1. Briefly, 5uL of hPXR-LucHepG2 cells in phenol red-free DMEM containing 5% charcoal-stripped FBS, 1 mM sodium pyruvate (Invitrogen), 2 mM L-glutamine (Invitrogen), and 100 U/mL penicillin and 100ug/mL streptomycin were dispensed using a BioRAPTR FRD at 4 x 105cells/mL (2000cells/well) in tissue culture-treated 1536-well white assay plates (GreinerBio-One).
2. Plates were incubated for 5h at 37C, 95% humidity, and 5% CO2 to allow for cell attachment.
3. Compounds (final concentration for most substances ranged from 15.6 nM to 45.9uM), positive control rifampicin (RIF; final concentration range of 2.8 nM to 92uM for the agonist mode), and cytotoxicity control tetraoctyl ammonium bromide (final concentration of 92uM) were transferred (23 nL) via pintool (Wako Automation).
4-5. After compound transfer, plates were dispensed with 1uL of the media in the agonist mode using a BioRAPTR FRD and incubated for 24 h at 37C and 5% CO2.
6-8. Following incubation, 1uL of CellTiter-Fluor (Promega, Madison, Wisconsin) was added using a BioRAPTR FRD, after which all plates were incubated (37C/ 5% CO2) for1 h and then measured for fluorescence intensity (Ex/Em= 405/540 nm) on a ViewLux plate reader to determine cell viability.
9-11. Directly after this fluorescence reading, 4uL of ONE-Glo Luciferasere agent (Promega) was added to each well using the BioRAPTR FRD, followed by a 30 min incubation (RT) and read for luminescence intensity on a ViewLux detector.

REFERENCES:
[1] 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.
[2] Lin W, Wu J, Dong H, Bouck D, Zeng FY, Chen T. Cyclin-dependent kinase 2 negatively regulates human pregnane X receptor-mediated CYP3A4 gene expression in HepG2 liver carcinoma cells. J Biol Chem. 2008 Nov 7;283(45):30650-7. doi: 10.1074/jbc.M806132200. Epub 2008 Sep 9. PMID: 18784074; PMCID: PMC2662154.

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 agonists are ranked higher than compounds that showed apparent antagonists.
2. For all inactive compounds, with a ratio activity curve class of 4, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. For a ratio activity curve class = 1.1, score = 80+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == 1.2 && abs(ratio.max_response) > 6*10, score = 60+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == 2.1 || ( ratio.curve_class==-2.2 && abs(ratio.max_response) > 6*10), score = 40+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == 1.2 || ratio.curve_class == 2.2, score = 20+abs((log_ac50+4.5)*inf_activity/20). Inconclusive compounds, with a donor curve class other than 4, have PUBCHEM_ACTIVITY_SCORE of 10. Inconclusive compounds also have PUBCHEM_ACTIVITY_SCORE between 1 and 39.

External ID: DSHEA-v1-PXR-agonist-CTF

Protocol: For PXR luciferase reporter gene assays, we used hPXR-LucHepG2cells provided by Dr. Taosheng Chen (Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital). Cells were cultured in EMEM supplemented with 10% fetal bovine serum, 100U/mL penicillin and 100ug/mL streptomycin, and 500ug/mL of geneticin.

PROTOCOL TABLE (as described by Inglese J, Shamu CE and Guy RK. 2007) [1]
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION.
1; Cells; 5 uL; hPXR-LucHepG2; white solid-bottom Greiner plate.
2; Incubation; 5h; 37C, 95% humidity, 5% CO2
3; Compounds and control; 23 nL; Kalypsis pintool (Wako USA).
4; Reagent; 1uL; rifampicin (RIF) in the antagonist mode.
5; Incubation; 24 hr; 37C, 5% CO2.
6; Reagent; 1 uL; CellTiter-Fluor detection reagent.
7; Incubation; 1 hr; 37C, 5% CO2.
8; Read; Fluorescence; ViewLux plate reader.
9; Reagent; 4 uL; ONE-Glo Luciferase detection reagent.
10; Incubation; 30 min; room temperature.
11; Read; Luminescence; ViewLux plate reader.

NOTES (numbers refer to Sequence numbers above)
1. Briefly, 5uL of hPXR-LucHepG2 cells in phenol red-free DMEM containing 5% charcoal-stripped FBS, 1 mM sodium pyruvate (Invitrogen), 2 mM L-glutamine (Invitrogen), and 100 U/mL penicillin and 100ug/mL streptomycin were dispensed using a BioRAPTR FRD at 4 x 105cells/mL (2000cells/well) in tissue culture-treated 1536-well white assay plates (GreinerBio-One).
2. Plates were incubated for 5h at 37C, 95% humidity, and 5% CO2 to allow for cell attachment.
3. Compounds (final concentration for most substances ranged from 15.6 nM to 45.9muM), positive control rifampicin (RIF; final concentration range of 2.8 nM to 92muM for the agonist mode), and cytotoxicity control tetraoctyl ammonium bromide (final concentration of 92muM) were transferred (23 nL) via pintool (Wako Automation).
4-5. After compound transfer, plates were dispensed with 1muL of the media in the agonist mode using a BioRAPTR FRD and incubated for approximately 24 h at 37 degrees C and 5% CO2.
6-8. Following incubation, 1uL of CellTiter-Fluor (Promega, Madison, Wisconsin) was added using a BioRAPTR FRD, after which all plates were incubated (37C/ 5% CO2) for 1 h and then measured for fluorescence intensity (Ex/Em= 405/540 nm) on a ViewLux plate reader to determine cell viability.
9-11. Directly after this fluorescence reading, 4uL of ONE-Glo Luciferasere agent (Promega) was added to each well using the BioRAPTR FRD, followed by a 30 min incubation (RT) and read for luminescence intensity on a ViewLux detector.

REFERENCES:
[1] 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.
[2] Lin W, Wu J, Dong H, Bouck D, Zeng FY, Chen T. Cyclin-dependent kinase 2 negatively regulates human pregnane X receptor-mediated CYP3A4 gene expression in HepG2 liver carcinoma cells. J Biol Chem. 2008 Nov 7;283(45):30650-7. doi: 10.1074/jbc.M806132200. Epub 2008 Sep 9. PMID: 18784074; PMCID: PMC2662154.