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: CHEMBL1266185

Protocol: N/A

Comment: Journal: Nat Chem Biol
Year: 2007
Volume: 3
Issue: 5
First Page: 268
Last Page: 273
DOI: 10.1038/nchembio873

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: ERK5 transcriptional activity-HTS

Protocol: Stable cells plated on a 384-well plate (2500 cells/well) were treated with test compounds at the concentration of 5 muM for 18 hrs. The level of luciferase activity was assayed using a
Luciferase kit (Promega corporation, Madison, WI) and a series of positive and negative control compounds were used as references.

Comment:

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: 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: 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: CHEMBL1020493

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2008
Volume: 16
Issue: 24
First Page: 10205
Last Page: 10209
DOI: 10.1016/j.bmc.2008.10.058

Target ChEMBL ID: CHEMBL6036
ChEMBL Target Name: N(G),N(G)-dimethylarginine dimethylaminohydrolase 1
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL2343337

Protocol: N/A

Comment: Journal: J. Med. Chem.
Year: 2013
Volume: 56
Issue: 8
First Page: 3121
Last Page: 3147
DOI: 10.1021/jm3015926

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: CHEMBL2343334

Protocol: N/A

Comment: Journal: J. Med. Chem.
Year: 2013
Volume: 56
Issue: 8
First Page: 3121
Last Page: 3147
DOI: 10.1021/jm3015926

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: CHEMBL1056360

Protocol: N/A

Comment: Journal: Eur. J. Med. Chem.
Year: 2009
Volume: 44
Issue: 7
First Page: 2877
Last Page: 2887
DOI: 10.1016/j.ejmech.2008.12.007

Target ChEMBL ID: CHEMBL4803
ChEMBL Target Name: Nitric-oxide synthase, endothelial
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: epac1-activator-v

Protocol: Briefly, three uL of reagents (100 nM EPAC1, 250 nM RAP1B-BODIPY-GDP, 50 uM GDP) were dispensed into a 1536-well Greiner black solid-bottom medium binding assay plate. Controls and test compounds (23 nL) were transferred to the plate via a Kalypsys pin tool equipped with a 1536-pin array. The plates were centrifuged at 1,000 rpm for 15 seconds followed by 5 minute incubation at room temperature. The assay plates were read at 5 minute intervals for 30 minutes in the ViewLux plate reader using 480nm excitation and 540nm emission filters. The results were normalized to the agonist positive control of 6.5 mM cAMP.

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 activators are ranked higher than compounds that showed apparent inhibition.
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: CHEMBL1020501

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2008
Volume: 16
Issue: 24
First Page: 10205
Last Page: 10209
DOI: 10.1016/j.bmc.2008.10.058

Target ChEMBL ID: CHEMBL4803
ChEMBL Target Name: Nitric-oxide synthase, endothelial
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL1056358

Protocol: N/A

Comment: Journal: Eur. J. Med. Chem.
Year: 2009
Volume: 44
Issue: 7
First Page: 2877
Last Page: 2887
DOI: 10.1016/j.ejmech.2008.12.007

Target ChEMBL ID: CHEMBL4481
ChEMBL Target Name: Nitric oxide synthase, inducible
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: epac1-inhibitor-v

Protocol: Briefly, three uL of reagents (100 nM EPAC1, 250 nM RAP1B-BODIPY-GDP, 50 uM GDP) were dispensed into a 1536-well Greiner black solid-bottom medium binding assay plate. Controls and test compounds (23 nL) were transferred to the plate via a Kalypsys pin tool equipped with a 1536-pin array. The plates were centrifuged at 1,000 rpm for 15 seconds followed by 5 minute incubation at room temperature. The assay plates were read at 5 minute intervals for 30 minutes in the ViewLux plate reader using 480nm excitation and 540nm emission filters. The results were normalized to the agonist positive control ATA and DMSO.

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: CHEMBL1020502

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2008
Volume: 16
Issue: 24
First Page: 10205
Last Page: 10209
DOI: 10.1016/j.bmc.2008.10.058

Target ChEMBL ID: CHEMBL4481
ChEMBL Target Name: Nitric oxide synthase, inducible
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL1020499

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2008
Volume: 16
Issue: 24
First Page: 10205
Last Page: 10209
DOI: 10.1016/j.bmc.2008.10.058

Target ChEMBL ID: CHEMBL4481
ChEMBL Target Name: Nitric oxide synthase, inducible
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL1056356

Protocol: N/A

Comment: Journal: Eur. J. Med. Chem.
Year: 2009
Volume: 44
Issue: 7
First Page: 2877
Last Page: 2887
DOI: 10.1016/j.ejmech.2008.12.007

Target ChEMBL ID: CHEMBL3568
ChEMBL Target Name: Nitric-oxide synthase, brain
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL1020500

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2008
Volume: 16
Issue: 24
First Page: 10205
Last Page: 10209
DOI: 10.1016/j.bmc.2008.10.058

Target ChEMBL ID: CHEMBL3568
ChEMBL Target Name: Nitric-oxide synthase, brain
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: PolI100

Protocol: Three microliters of reagents (buffer in column 3 and 4 as negative control and 10 nM Pol iota in columns 1, 2, and 5-48) will be dispensed into 1,536-well black solid-bottomed plate. Compounds (23 nL) will be transferred via Kalypsys pin tool equipped with 1536-pin array. The plates will then be incubated for 15 min at room temperature, and 1 muL substrate (50 nM final concentration) will be added to start the reaction and kinetically read twice at 0 min and 10 min on the Viewlux reader.

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: epac2-activator-v2

Protocol: Briefly, three uL of reagents (100 nM EPAC2, 250 nM RAP1B-BODIPY-GDP, 50 uM GDP) were dispensed into a 1536-well Greiner black solid-bottom medium binding assay plate. Controls and test compounds (23 nL) were transferred to the plate via a Kalypsys pin tool equipped with a 1536-pin array. The plates were centrifuged at 1,000 rpm for 15 seconds followed by 5 minute incubation at room temperature. The assay plates were read at 5 minute intervals for 30 minutes in the ViewLux plate reader using 480nm excitation and 540nm emission filters. The results were normalized to the agonist positive control of 6.5 mM cAMP.

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 activators are ranked higher than compounds that showed apparent inhibition.
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.