External ID: adst_MBL_Abs_LOPAC_o1

Protocol: PROTOCOL TABLE (as described by Inglese et al., 2007)
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., enzyme, Incubation, Reagent, etc.); VALUE; DESCRIPTION
1; NDM-1; 4 uL of 3 nM enzyme in 50 mM HEPES (pH 7.0), 1 uM Zn2SO4, 0.01% Tween20; Use BioRaptr to dispense into Aurora black, clear bottom low base 1536-well plates
2; Compounds; 23 nL; Use Pin Tool (Wako) to transfer DMSO control and library compounds in DMSO
3; Incubate; >30 min
4; Reagent; 2 uL of 50 uM nitrocefin
5; Incubate; 15 min
6; Detector; Absorbance at 485 nm (bandwidth = 5 nm, 25 flashes/well) on Tecan Infinity

NOTES (numbers refer to sequence above)
1; Column 1 of each plate received assay buffer without enzyme and columns 2-48 received enzyme for a final assay concentration of 2 nM.
2; Control plate contained DMSO in columns 1, 2, 3, and 4.
3; Compound interaction with enzyme.
4; Nitrocefin substrate was prepared in the same assay buffer as step 1 for a final assay concentration of 16.7 uM and added to all wells across the assay plates.
5; Substrate interaction with enzyme, plateau phase of kinetics.
6; Default reader settings were applied for absorbance read at 485 nm, bandwidth = 5 nm, 25 flashes/well.

References:
Inglese J, Shamu CE, Guy RK. 2007. Reporting data from high-throughput screening of small-molecule libraries. Nat Chem Biol. doi:10.1038/nchembio0807-438

Yasgar A, Shinn P, Jadhav A, Auld D, Michael S, Zheng W, Austin CP, Inglese J, Simeonov A. 2008. Compound Management for Quantitative High-Throughput Screening. J Lab Autom 13:79 - 89. doi:10.1016/j.jala.2007.12.004

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: SERCaMPGLuc-p1-antagonist

Protocol: PROTOCOL TABLES
SEQUENCE No. (1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE and DESCRIPTION.
1; Reagent; 5 uL; 1000 SH-SY5Y cells per wells
2; Time; 5 hour; 37C, 5% CO2
3; Compound; 23 nL; Control inhibitor / compound library
4; Time; 16 hour; 37C, 5% CO2
5; Reagent; 100 nM; Thapsigargin
6; Time; 4 hour; 37C, 5% CO2
7; Reagent; 1 uL; 0.5x coelenterazine
8; Detection; Luminescence; ViewLux imaging system

NOTES (numbers refer to sequence above)
1; SH-SY5Y human neuroblastoma cells stably expressing GLuc-SERCaMP (SH-SY5Y-GLuc-ASARTDL) cells were seeded in 1,536 well white tissue culture treated plates (Corning, Cat# 7464) in DMEM-high glucose-sodium pyruvate (ThermoFisher Scientific, Cat #10569) supplemented with 10% bovine growth serum (Hyclone), 10 U/ml penicillin (Gibco), 10 ug/ml streptomycin (Gibco), and 20 mM HEPES.
2; Assay plates were incubated for 5 hour at 37C in a humidified incubator containing 5% CO2.
3; qHTS libraries (23 nl, final concentrations of 1.53 uM, 7.67 uM, 38.3 uM) or controls (neutral control: DMSO, positive control: dantrolene) were added using a Kalypsis pin-tool Robotic System equipped with 1536 pinheads.
4; Cells were then incubated for 16 hours at 37C, 5% CO2.
5; Thapsigargin was added at 100 nM to deplete ER calcium stores.
6; Cells were incubated for 4 hour (37oC, 5% CO2)
7; Gaussia luciferase in the medium was measured by adding 1 ul of 0.5x coelenterazine (final concentration 0.07x) prepared in Gaussia Luciferase Glow Assay Buffer (Pierce), without addition of the Cell Lysis Buffer Reagent.
8; Luminescence was measured using a ViewLux high-432 throughput CCD imaging system (Perkin Elmer) equipped with clear filters. Compounds exhibiting inhibitory activity (defined as curve class -1.1, -1.2, -1.3, -1.4, -2.1, -2.2, -2.3, -2.4, -3) were identified by normalizing plate-wise to corresponding intra-plate controls (neutral control = Tg only; positive control (100% inhibition) = DMSO vehicle) with percent activity derived using in-house software (https://tripod.nih.gov/curvefit). The same controls were also used for the calculation of the Z' factor, a measure of assay quality control, as previously described (Zhang et al., 1999). For the initial validation of activity in the SERCaMP assays, hits from the primary screen were assayed again at 11-concentrations (1.3 nM - 76.6 uM). SH-SY5Y-GLuc-SERCaMP cells were assayed for ER Ca2+ depletion as outlined above.

Reference:
1. Zhang, J.H., Chung, T.D., and Oldenburg, K.R. (1999). A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen 4, 67-73.

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: EYA2477

Protocol: The Eya2 enzyme was kindly provided by the laboratory of Dr. Rui Zhao. The 3-O-methyl-fluorescein phosphate (OMFP) substrate and all buffer reagents were purchased from Sigma Aldrich.
qHTS Assay Setup:
1. Buffer- 50 mM HEPES pH 7.5, 50 mM NaCl, 5 uM MgCl2, 0.05% BSA, 1 mM DTT.
2. OMFP Stock- 10 mM solution prepared in 100% DMSO and stored in single use aliquots at -20C.

Assay Procedure-
1. Dispense 1.5 ul per well of Eya2 at 0.2 uM in black medium binding 1536 well Greiner plate
2. Transfer 23 nl of compound and control compound (EGTA) to plate
3. Incubate at ambient for 10 minutes
4. Dispense 1.5 ul per well of OMFP at 50 uM
5. Incubate at ambient for 10 minutes
6. Read on ViewLux using Excitation = 485 nm, Emission = 525 nm

Comment: Compound Ranking:

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

External ID: 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: UBCH001

Protocol: NCGC Assay Protocol Summary:

The assay buffer, prepared fresh at the day of the assay, contains 20mM Tris-HCl pH 8.0, 2mM CaCl2, 2mM beta-Mercaptoethanol, 0.05% CHAPS. 3ul of 20nM (10nM final) USP2 core, which is stored on ice, is dispensed into a medium-binding black solid Kalypsys 1,536 well plate using the Kalypsys dispenser. The assay plate is then pinned with 23nL compound with the Kalypsys pintool in columns 5-48. The controls are pinned as follows: column 1 two-fold dilutions of 2M NEM in duplicate; column 2 2M NEM (final concentration 38.2mM); column 3&4 DMSO. The assay plate is incubated at RT for 30min. Subsequently, 1.5uL of 100nM (25nM final) Ub-CHOP2 and 1.5uL of 100nM (25nM final) Reporter Substrate (both stored on ice and protected from light) were dispensed into the plate using the Kalypsys dispenser. Plates were read after 60min RT incubation on a ViewLux (Perkin Elmer) plate reader using the following wavelengths Ex 485nm /Em 531nm.

Data were normalized to the to AC100 inhibition (NEM). Concentration-response curves were fitted to the normalized data and the concentration-response curves were then classified based on curve quality (r2), response magnitude and degree of measured activity. The time zero reading was used to flag fluorescence artifacts.

Keywords: ubiquitination, deubiquitination, proteases, profluorescent, MLSMR, MLPCN, NIH Roadmap, qHTS, NCGC

Comment: Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description".
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 20 and 39. Fluoresent compounds have PUBCHEM_ACTIVITY_SCORE 10. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

External ID: ZIK159

Protocol: Assay Protocol Summary:

The medium for SNB-19 cells is composed of RPMI 1640 (ATCC, Cat.# 30-2001), 10% fetal bovine serum (FBS) (GE healthcare Life Sciences, Cat.# SH30071.03), and 1% Pen/Strep (Gibco, Cat.# 15140-122). A Caspase-Glo 3/7 assay kit (catalog number G8092; Promega, Madison, WI) was used to detect caspase-3 activity induced by Zika virus infection in human cells. The reagent mixture was reconstituted as described in the protocol from the manufacturer. Polystyrene tissue culture treated and PDL coated white plates were obtained from Greiner Bio-One (Monroe, NC). Cells were seeded in 384- or 1536-well assay plates and cultured at 37 C with 5% CO2 for 16 to 20 hours. The typical cell seeding density in the 1536-well plate assay is 250 cells/well in 3ul medium for SNB-19 cells in tissue culture treated plates. Compounds were added to cells and incubated for one hour before addition of ZIKV solution to cells (2 FFU/cell). After incubation at 37 C with 5% CO2 for 6 hours, the reagent mixture of Caspase-Glo 3/7 assay kit was added to each well, followed by incubation at room temperature for 30 minutes. The luminescence intensity of the assay plates was measured using a ViewLux plate reader (PerkinElmer). Data were normalized by using the cell-containing wells without ZIKV as a negative control (0% induction of caspase 3/7 activity) and wells containing ZIKV infected cells (Caspase-3 activity induced) as a positive control (100% induction of caspase 3 activity). The percentage inhibitions of the increased Caspase-3 activity by small molecule inhibitors were then calculated.

Comment: Compound Ranking:

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

External ID: HMS1315

Protocol: Cybrid cells were washed with PBS, trypsinized, spun down at 300g for 5 minutes, and resuspended in 10 mLs of PBS. Cells were counted, and the appropriate amount of cells were added to a vial containing DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose, DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 0.3% DMSO, DMEM No Glucose + 2% FBS + 1% P/S + 10mM Galactose + 1uM I-BET GSK 525762A, or DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 1.25mM Glucose. Cells were then seeded in a Corning 3570 384-well plate (40 ul/well,1500 cells/well). Two replicates were prepared as described at the same time per library plate.

100 nl of compound was added to each well via pin transfer immediately after seeding. After the addition of compound, cells were incubated for 72 hours at 37 degrees C with 5% CO2.

Following the 72 hour incubation period, the 384 well plates were centrifuged for 1 minute at 1000 rpm. The media was aspirated using an aspiration wand and fresh media DMEM only, was added to the cells (40uL/ well) using a well-mate. Fifteen-microliters of Cell Titer Glow substrate was then added to each well using the well-mate. The plates were gently vortexed for 10 seconds and then centrifuged for 1 minute at 1000 rpm. The plates were then immediately read on the Envision instrument and the luminescence was detected for each individual well.

Positive control (strong): Cells seeded in DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 1.25mM Glucose
Positive control (weak): Cells seeded in DMEM No Glucose + 2% FBS + 1% P/S + 10mM Galactose + 1uM I-BET GSK 525762A
Negative control: Cells seeded in DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 0.3% DMSO

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

Z-scores were calculated for both replicates separately using the plate average and standard deviation of experimental well luminescence (Z = (x - mu)/sigma). Compounds were considered active if both replicate Z-scores >= 1.8. Activity scores were determined by scaling replicate average Z-scores from 0 (activity score = 0) to 4 (activity score = 100), with activity score > 45 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: ZIK097

Protocol: Assay Protocol Summary:

The medium for hNPCs consists of DMEM/F12, N2 supplement (ThermoFisher, Cat.# 17502048), NEAA (ThermoFisher, Cat. # 11140050), 2 ug/ml heparin, 2 uM cyclopamine, and B27 (ThermoFisher, Cat. # 17504044). A Caspase-Glo 3/7 assay kit (catalog number G8092; Promega, Madison, WI) was used to detect caspase-3 activity induced by Zika virus infection in human cells. The reagent mixture was reconstituted as described in the protocol from the manufacturer. Polystyrene tissue culture treated and PDL coated white plates were obtained from Greiner Bio-One (Monroe, NC). Cells were seeded in 384- or 1536-well assay plates and cultured at 37 C with 5% CO2 for 16 to 20 hours. The typical cell seeding density in the 1536-well plate assay is 350 cells/well in 3 ul medium for hNPCs in tissue culture treated plates. Compounds were added to cells and incubated for one hour before addition of ZIKV solution to cells (2 FFU/cell). After incubation at 37 C with 5% CO2 for 6 hours, the reagent mixture of Caspase-Glo 3/7 assay kit was added to each well, followed by incubation at room temperature for 30 minutes. The luminescence intensity of the assay plates was measured using a ViewLux plate reader (PerkinElmer). Data were normalized by using the cell-containing wells without ZIKV as a negative control (0% induction of caspase 3/7 activity) and wells containing ZIKV infected cells (Caspase-3 activity induced) as a positive control (100% induction of caspase 3 activity). The percentage inhibitions of the increased Caspase-3 activity by small molecule inhibitors were then calculated.

Comment: Compound Ranking:

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

External ID: CGM data for Cell Systems paper Dec 2015

Protocol: Yeast strains and Media:
All S. cerevisiae deletion strains were obtained from the Euroscarf deletion collection. The wild type parental strain for this collection is BY 4741 MATa his31 leu20 met150 ura30. An isogenic pdr1pdr3 strain (MT2481) was created using PDR1::nat and PDR::URA3 deletion cassettes. All strains were grown and screened in synthetic complete (SC) medium with 2% glucose.

Handling:
Strains were seeded at 50,000 cells per well in a volume of 100 L in 96 well plates followed by addition of 2 L of 1 mM compound stock for final concentration of 20 M. Screens were conducted in technical duplicate using a Biomek FX liquid handling workstation with an integrated stacker carousel. DMSO solvent only controls and 10 uM cycloheximide positive controls were seeded in columns 1 and 12 of each assay plate. Plates were incubated at 30 C without shaking for approximately 18 h or until culture saturation was achieved for the solvent controls. Cultures were resuspended by shaking on the robotic platform prior to reading OD600 values on either Tecan M1000 or Tecan Sunrise plate readers.

Analysis procedure:
All data was subjected to the following workflow:
1. Consistent spatial effects on growth across plates were corrected by Lowess regression using an empirically estimated sliding window of 1/3 and normalized based on plate median.
2. If more than 30% of compounds were active within a plate, data was normalized to DMSO controls.
3. Median normalization was applied to all plates and experiments.
4. Z-factors for growth inhibition were calculated using the median and the interquartile range (IQR) by fitting a normal distribution with N(1,IQR) to the experimental data. In addition, we calculated the Z-factor, percent inhibition and normalized OD values for manual validation.
5. Data points with high variation between replicates (> 3 MAD) were removed as inconsistent outliers.

BioAssay submission file header:

Pubchem_ext_datasource_regid: compound identifier used in on chemgrid.org/cgm
Orf yeast gene deletion (open reading frame)
Sym# yeast gene deletion gene symbol
Raw OD read 1 first replicate
Raw OD read 2 second replicate
Normalized OD average normalized read based on workflow described above
Z_score Z-Score calculated based on kernel density distribution from normalized average reads per screen
P_value p-value calculated based on kernel density distribution calculated from average reads per screen
Non replicate test for non-replicates between first and second replicate
Pubchem_activity_outcome pubchem activity outcome
Cryptagen if compound identifier fulfilled cryptagen rule of activity in at least 4 and not more than 2/3rds of screens per library
Bioactivity sensitive if compound decreases fitness or resistant if compound increases fitness compared to negative control

Comment:

External ID: asa1-v2

Protocol: Four microliter (~8,000 cells/well) of SV40-transformed skin fibroblasts from an MLD patient (ASA-I179S) were seeded into a 1,536 solid white plate. Cells were left to adhere for 6 hrs, followed by addition of 23 nL of compounds. Cells were incubated with compounds for 48h at 37 degree C and 5% CO2. Four microliter of 1 mM pNCS substrate/lysis buffer (0.5mM pNCS final concentration) was added and incubated for 14h at room temperature. To stop the reaction, 5 uL of 5 N of NaOH (1N NaOH final concentration) was then added and fluorescence was measured using the ViewLux plate reader (Ex=525 nm; Em=598 nm).

Comment: Compound Ranking:

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

External ID: GPR119_PHUNTER_AG_LUMI_1536_3X%ACT CSRUN1

Protocol: Assay Overview:
A counterscreen assay, employing GPR119 cells, was also implemented by Scripps to identify compounds that non-specifically affect the PathHunter detection method. The purpose of this assay is to counterscreen and determine the selective activity of compounds identified as active in a set of previous experiments entitled, "Cell-based high throughput primary assay to identify activators of GPR151". In this assay, stably transfected cells expressing GPR119 and the B-arrestin pathhunter reporter are incubated with test compounds. As designed, test compounds that act as activators of GPR119 activity will increase luminescence signal. Compounds are tested in triplicate at a nominal test concentration of 11.2 micromolar.

Protocol Summary:
Prior to the start of the assay, cells were resuspended in growth media at 2000cells/well in 1536 well plates. This was followed by an incubation of 18 hours at 37C+5%CO2. Then 1uL of Opti-Mem added to all wells except high controls to which 3X EA reagent was added to each of those wells (0.2X final in lysis buffer). Compounds were pinned at 11.20uM final concentration in 1.0% DMSO followed by a 90 minute incubation at 37C+5%CO2. At this stage 3uL of Promega Beta-Glo detection Buffer was added to all wells followed by a 1 hour incubation at room temperature. Finally the assay end point read was taken using the ViewLux imaging reader from PerkinElemer Life Sciences with a 5 second exposure. Raw assay data was imported into Scripps' corporate database and ascertained for Z' greater than 0.5 in order to be processed futher.

The percent activation for each compound was calculated as follows:

Percent Response of compound= 100 * ((Test Well-Median Data Wells)/(Median High Control # Median Data Wells))
Where:
Test_Well is defined as wells containing GPR151 cells in the presence of test compound
High_Control represents wells containing GPR151 cells stimulated with 0.2X EA reagent
Low_Control is defined as wells containing GPR151 cells and DMSO

PubChem Activity Outcome and Score:
Standard Cutoff
The average percent activation and standard deviation of each compound tested were calculated. Any compound that exhibited an average percent activity greater than the hit cutoff calculated for the counterscreen (29.09%) was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

List of Reagents:
GPR119 cells (supplied by Patricia McDonald)
Pen Strep (Invtirogen 15140)
FBS (Invtirogen 16140)
Hygromycin (Invtirogen 10687010)
Lysis buffer (CisBio 62CL2FDF)
EA Reagent (DiscoverX 30-411)
G418 (Gemini Bio 400-113)
Opti-MEM (Invtirogen 31985)
Trypsin (Invtirogen 15400054)
Beta Glo (Promega E4780)
1536-well plates (Greiner Bio-One, part 789173)

Comment: Due to the size of the Scripps Molecular Screening Center compound library, this assay have been run as multiple separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the Scripps Molecular Screening Center.

External ID: GPR151_PHUNTER_AG_LUMI_1536_3X%ACT CRUN1

Protocol: Assay Overview:
The purpose of this assay is to confirm activity of compounds identified as active in a set of previous experiments entitled, "Cell-based high throughput primary assay to identify activators of GPR151". In this assay, stably transfected cells expressing GPR151 and the B-arrestin pathhunter reporter are incubated with test compounds. As designed, test compounds that act as activators of GPR151 activity will increase luminescence signal. Compounds are tested in triplicate at a nominal test concentration of 11.2 micromolar.

Protocol Summary:
Prior to the start of the assay, cells were resuspended in growth media at 2000cells/well in 1536 well plates. This was followed by an incubation of 18 hours at 37C+5%CO2. Then 1uL of Opti-Mem added to all wells except high controls to which 3X EA reagent was added to each of those wells (0.2X final in lysis buffer). Compounds were pinned at 11.20uM final concentration in 1.0% DMSO followed by a 90 minute incubation at 37C+5%CO2. At this stage 3uL of Promega Beta-Glo detection Buffer was added to all wells followed by a 1 hour incubation at room temperature. Finally the assay end point read was taken using the ViewLux imaging reader from PerkinElemer Life Sciences with a 5 second exposure. Raw assay data was imported into Scripps' corporate database and ascertained for Z' greater than 0.5 in order to be processed futher.

The percent activation for each compound was calculated as follows:

Percent Response of compound= 100 * ((Test Well-Median Data Wells)/(Median High Control # Median Data Wells))
Where:
Test_Well is defined as wells containing GPR151 cells in the presence of test compound
High_Control represents wells containing GPR151 cells stimulated with 0.2X EA reagent
Low_Control is defined as wells containing GPR151 cells and DMSO
PubChem Activity Outcome and Score:

Standard Cutoff
The average percent activation and standard deviation of each compound tested were calculated. Any compound that exhibited an average percent activity greater than the hit cutoff calculated for the confirmation screen (4.91%) was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

List of Reagents:
GPR151 cells (supplied by Patricia McDonald)
Pen Strep (Invtirogen 15140)
FBS (Invtirogen 16140)
Hygromycin (Invtirogen 10687010)
Lysis buffer (CisBio 62CL2FDF)
EA Reagent (DiscoverX 30-411)
G418 (Gemini Bio 400-113)
Opti-MEM (Invtirogen 31985)
Trypsin (Invtirogen 15400054)
Beta Glo (Promega E4780)
1536-well plates (Greiner Bio-One, part 789173)

Comment: Due to the size of the Scripps Molecular Screening Center compound library, this assay have been run as multiple separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the Scripps Molecular Screening Center.

External ID: TargetID_659_CEMA

Protocol: Black, standard capacity streptavidin-coated 384-well plates (Pierce 15407) were first washed with 50 L of phosphate buffer (100 mM, pH 7.0; PB7) three times using a Biotek 405 ELX plate washer. Subsequently, 5 L of biotinylated pre-miRNA substrate (500 nM final) was dispensed into the plate using a Multidrop Combi Reagent Dispenser (Thermo Scientific). Plates were then centrifuged for 1 min at 1,000 RPM (223 g), sealed with plate tape, and incubated overnight at 4 C. The following morning, plates were washed three times with 50 L of PB7, followed by the addition of 5 L of Dicer digest buffer (20 mM Tris, 12 mM NaCl, 2.5 mM MgCl2, 1 mM fresh DTT, and 4.5% DMSO) and centrifugation. Compounds (50 nL of 5 mM DMSO stock, 25 M final) were then added into the sample wells using a Sciclone (Caliper) liquid handler with V&P pintool; the same volume of DMSO was added to the control wells. The plates were incubated at 25 C for 15 min before addition of 5 L of digest buffer containing 217 g/nL Dicer (108 g/mL Dicer, 5% glycerol and 0.01% Triton X-100 final, excess with respect to pre-miRNA). For the positive control wells, digest buffer without Dicer was added. The plates were centrifuged again and resealed before being placed in a 37 C incubator for 5 h. After Dicer cleavage, plates were washed three times with 50 L of PB7. mTet-HRP in PB7 (10 L, 750 nM final) was then dispensed into each well. The plates were subsequently centrifuged, sealed, and incubated at 25 C for 2 h. Plates were then washed three times with 50 L of wash buffer (2 mM imidazole, 260 mM NaCl, 0.5 mM EDTA, 0.1% Tween-20, pH 7.0), followed by washing three additional times with 50 L of PB7. Finally, SuperSignal West Pico (25 L; Pierce) was added, the plates were incubated at 25 C for 5 min, and chemiluminescence signal was detected using a PHERAstar plate reader using LUM plus module (BMG Labtech).

Comment: The activity outcome is based on a Z-score (number of standard deviations from the negative control mean) of 3 or higher on at least 50% times that the sample was screened.

For instance, if the sample was screened in n=4 runs, it would be considered active only if it had a Z-score of 3 or above in at least 2 runs.

External ID: cib1-v1-fp-lopac

Protocol: PROTOCOL TABLES
SEQUENCE No. (1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE and DESCRIPTION.
1; Reagent; 3 uL; Protein or buffer (4/3x)
2; Compound; 46 nL; Control inhibitor / compound library
3; Time; 15 min; Room temperature incubation
4; Reagent; 1 uL; Fluorescent labeled peptide (4x)
5; Time; 1000 rpm; Centrifuge
6; Time; 15 min; Room temperature incubation
7; Detection; Ex 480/ Em 540; ViewLux Fluorescence Read

NOTES (numbers refer to sequence above)
1; Protein Mixture: C1B1-GST (final concentrations of 1 uM). Buffer composition: 5 mM HEPES pH 7.4, 125 mM NaCl, 5 mM CaCl2, 0.01% Tween20.
2; Control Inhibitor: unlabeled peptide (final concentration range 17.4 nM to 572 uM). Compound Library final concentration range 18.3 nM to 114 uM.
3; Room temperature incubation.
4; Fluorescent Labeled Peptide: FITC-aIIb (final concentration of 100 nM). Sequence of alphaIIb peptide: Acetyl-LVLAMWKVGFFKRNRK-FITC (purity is 95.83%).
5; Centrifuge 1000 rpm (164 g) for 15 seconds.
6; Room temperature incubation.
7; ViewLux Fluorescent Polization Read: excitation = 480(20) / emission = 540(25) S and P; FITC Dichroic mirror.

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-LACV-FDA-LOPAC

Protocol: LACV-induced cytopathicity assay

PROTOCOL TABLES
SEQUENCE No. (1,2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE and DESCRIPTION.
1. Reagent; 2 uL; Cell solution.
2. Time; 24 hr; 37C, 5% CO2.
3. Compounds; 23nL; Compound library
4. Reagent; 12 uL; LACV virus
5. Time; 6 days; 37C, 5% CO2
6. Reagent; 24 uL; ATPLite (PerkinElmer)
7. Time; 5 min; Room temperature
8. Detection; Luminescence; View

NOTES (numbers refer to sequence above)
1. Seed 6000 SH-SY5Y cells (ATCC CRL-2266) in 2 microL/well media (DMEM/F12 10% FBS, 1x L-Glutamine, 1x Pyruvate, 1x NEAA, 1x Pen/Strep) in 384-well assay plates (tissue culture treated, white, solid bottom).
2. Incubate at 37 degrees C with 5% CO2 for 24 hr.
3. Dispense 23 nL/well compounds in DMSO via pin transfer.
4. Dispense 12 microL/well of LACV (LACV/human/1978) in media at 0.01 MOI.
5. Incubate at for 6 days at 37C 5% CO2
6. Dispense 24 microL/well of ATPLite 1step Luminescence assay system (PerkinElmer).
7. Incubate for 5 min at room temperature.
8. Read luminescence signal (Viewlux plate reader, PerkinElmer). Data was normalized with DMSO control wells containing LACV as 0%, and DMSO control wells without virus as 100%.

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: TRXR101

Protocol: Assay protocol: 2 uL of reagents (buffer in column 4 as negative control and 90 nM rTrxR1 in columns 1-3 and 5-48) were dispensed into Greiner black solid-bottom 1,536-well assay plates, followed by 1 uL of NADPH (400 uM final concentration) to each well. The plates were centrifuged at 1000 rpm for 15 seconds and subsequently incubated for 5 min at room temperature (~22 deg C) to allow for rTrxR1 reduction. Compounds (23 nL) were then transferred via Kalypsys pin tool equipped with 1536-pin array (10 nL slotted pins, V&P Scientific, San Diego, CA). In addition, a duplicate 2-fold serial dilution of the control compounds auranofin, a known gold-based TrxR1 inhibitor, and juglone (5-hydroxy-1,4-naphthoquinone), a natural TrxR1 substrate, were pin-transferred to columns 2 and 3, respectively. After incubation for 15 min at room temperature (~22 deg C), 1 uL of selenite (400 uM final concentration) were dispensed to each well. The plate was immediately transferred to a ViewLux high-throughput CCD imager (PerkinElmer), wherein kinetic measurements of NADPH fluorescence (Ex 340 nm, Em 450 nm) were acquired (8 minute kinetic read, see Table 1). Read 1 was utilized to assess the capacity of a compound to serve as an rTrxR1 substrate, i.e. a decrease in NADPH fluorescence compared to the no-compound background is an indication of a substrate behavior for that particular compound. For inhibitory activity of a compound, delta values, computed as the difference in fluorescence intensity between the first and last reads of an 8-minute time kinetic window, were used. All reagents were diluted in an assay buffer consisting of 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, and 0.01% Tween-20.

Throughout the screen, reagent bottle and all liquid lines were made light-tight to minimize reagent degradation. All screening operations were performed on a fully integrated robotic system (Kalypsys, San Diego, CA) containing one RX-130 and two RX-90 anthropomorphic robotic arms (Staubli, Duncan, SC). Library plates were screened starting from the lowest and proceeding to the highest concentration, and a 'double-pinning' step of the highest concentration was required to access higher concentrations of compounds. Vehicle-only plates, with DMSO being pin-transferred to the columns 5-48, were inserted uniformly at the beginning and the end of each library in order to monitor and record any shifts in the background, which can be affected by reagent dispensers or loss in enzyme activity over time. Screening data were corrected, normalized, and concentration-effect relationships were derived by using publicly-available curve fitting algorithms developed in-house (http://ncgc.nih.gov/pub/openhts).

Comment: Compound Ranking:

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