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: CHEMBL750149

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Journal: J. Med. Chem.
Year: 1996
Volume: 39
Issue: 1
First Page: 183
Last Page: 190
DOI: 10.1021/jm950393q

Target ChEMBL ID: CHEMBL1907608
ChEMBL Target Name: Glutamate NMDA receptor
ChEMBL Target Type: PROTEIN COMPLEX GROUP - Target is a poorly defined protein complex, where subunit composition is unclear (e.g., GABA-A receptor)
Relationship Type: H - Homologous protein target assigned
Confidence: Multiple homologous protein targets may be assigned

External ID: CHEMBL699778

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Journal: Bioorg. Med. Chem. Lett.
Year: 1996
Volume: 6
Issue: 20
First Page: 2381
Last Page: 2386
DOI: 10.1016/0960-894X(96)00436-2

Target ChEMBL ID: CHEMBL2094119
ChEMBL Target Name: Glutamate receptor ionotropic, kainate
ChEMBL Target Type: PROTEIN COMPLEX GROUP - Target is a poorly defined protein complex, where subunit composition is unclear (e.g., GABA-A receptor)
Relationship Type: H - Homologous protein target assigned
Confidence: Multiple homologous protein targets may be assigned

External ID: TDP1100

Protocol: DT40-hTDP1 cells without 20 nM Camptothecin (add 20 microL of DMSO in 1 L of cell culture medium) were dispensed at 400 cells/5microL/well in tissue culture treated 1536-well white wall/solid bottom assay plates (Greiner Bio-One North America, NC, U.S.A.) using a Multidrop Combi 8 channel dispenser (Thermo Fisher, Waltham, MA, USA). 23 nL compounds and controls were transferred using the pin tool (Kalypsys, San Diego, CA, USA) to the assay plates. The assay plates were then incubated at 37 masculineC for a minimum 48 hr. Three microL of Cell Titer Glo solution was added to the plates and incubated at RT in dark for 30 min. Luminescence was read using ViewLux (Perkin Elmer) with 1 sec exposure slow speed, high gain and 2x binning.

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: TDP1101

Protocol: DT40-hTDP1 cells with 20 nM Camptothecin (add 20 microL of 1 mM CPT stock in 1 L of cell culture medium) were dispensed at 400 cells/5microL/well in tissue culture treated 1536-well white wall/solid bottom assay plates (Greiner Bio-One North America, NC, U.S.A.) using a Multidrop Combi 8 channel dispenser (Thermo Fisher, Waltham, MA, USA). 23 nL compounds and controls were transferred using the pin tool (Kalypsys, San Diego, CA, USA) to the assay plates. The assay plates were then incubated at 37 masculineC for a minimum 48 hr. Three microL of Cell Titer Glo solution was added to the plates and incubated at RT in dark for 30 min. Luminescence was read using ViewLux (Perkin Elmer) with 1 sec exposure slow speed, high gain and 2x binning.

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: CHEMBL633541

Protocol: N/A

Comment: Journal: J. Med. Chem.
Year: 1999
Volume: 42
Issue: 20
First Page: 4099
Last Page: 4107
DOI: 10.1021/jm991081g

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: CHEMBL633542

Protocol: N/A

Comment: Journal: J. Med. Chem.
Year: 1999
Volume: 42
Issue: 20
First Page: 4099
Last Page: 4107
DOI: 10.1021/jm991081g

External ID: 12hLO-f-round2

Protocol: PROTOCOL TABLE
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION
1; Enzyme; 3 uL; 150 nM 12hLO enzyme solution dispensed in 1536-well Greiner black, clear bottom plates.
2; Compound; 23 nL; compounds and controls were added.
3; Incubate; 15 min; room temperature.
4; Substrate; 1 uL; 40 uM arachidonic acid (final concentration).
5; Incubate; 30 min; room temperature.
6; Reagent; 4 uL; Fe-XO solution was added to each well.
7; Incubation; 30 min; room temperature
8; Detector; absorbance; Read 1 at 405 nm absorbance using ViewLux (PerkinElmer) CCD imager.
9; Detector; absorbance; Read 2 at 573 nm absorbance using ViewLux (PerkinElmer) CCD imager.
NOTES (numbers refer to sequence above)
1; 150 nM 12hLO (final concentration) in buffer solution (25 mM Hepes, 0.01% Triton X-100, pH 7.0) was dispensed in columns 1, 2, 5-48. Column 1 is neutral (100% activity). Buffer only was dispensed in columns 3 and 4 as no enzyme negative controls. The plates were covered with metal lids with gas-exchange holes.
2; Compounds were pin transferred via a Kalypsys Pin Tool (Wako USA) in column 5 - 48. Control compound was transferred in column 2, titration of nordihydroguaiaretic acid (NDGA, Sigma-Aldrich, N5023) with top concentration of 20 mM in DMSO then 1:3 dilution in duplicate. Dilution factor of 23 nL into 4 uL final assay volume.
3; Covered assay plates were incubated for 15 min at room temperature.
4; Substrate was added to start the reaction; 40 uM arachidonic acid (Sigma-Aldrich, St. Louis, MO) was dispensed throughout the plate.
6; Chromogenic detection reagent solution (divalent iron/xylenol orange, Fe-XO) was added to each well: 200 uM xylenol orange, 300 uM ferrous ammonium sulfate prepared freshly in 50 mM sulfuric acid.
8, 9; Two absorbance readouts were obtained at 405 nm and at 573 nm using a ViewLux high-throughput CCD plate imager (PerkinElmer). The 573 to 405 (Read 2 / Read 1) absorbance ratio was used to compute reaction progress.

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: IucA Pilot Assay Tocris Library

Protocol: A solution containing 55.6 mM HEPES pH 7.5, 0.11% Tween 20, 16.7 mM MgCl2, 55.6 mM hydroxylamine, 55.6 microM ATP, 55.6 microM citrate, and 0.28 U/mL IPP was dispensed (45 microL) into clear polystyrene microplates (Corning, Inc.) using a BioTek MicroFlo dispenser.

Next, 40 nL of test compounds (10 mM in DMSO, 8 microM final concentration) were transferred from deep-well blocks to the reaction solution using a stainless-steel pin tool operated by a robotic workstation (JANUS, PerkinElmer, Waltham, MA). The IucA-catalyzed reaction was initiated by adding 5 microL of 3 microM IucA in 25 mM HEPES, 75 mM NaCl, and 0.1 mM TCEP at pH 7.5.

The reactions were allowed to proceed for 30 min at room temperature before being quenched by dispensing (microFill, BioTek) 13 microL of MG developing solution, containing 1.0 mg/mL MG oxalate, 1.5% (w/v) ammonium molybdate, 0.15% (v/v) Tween 20, and 4.7 N sulfuric acid. After allowing the assay color to develop/stabilize for 30 min, the absorbance at 620 nm was measured (EnVision 2103 Multilabel Microplate Reader, PerkinElmer).

Average positive controls from 24 wells with no test compound and average negative controls from 8 wells with no enzyme were calculated. Dynamic range was calculated by difference between Avg Pos Control and Avg Neg Control. Percent inhibition was calculated by the ratio of (Avg. Pos. Ctrl - Sample OD) to Dynamic Range.

Comment: Protein Target is
IucA

EMB09144
574 aa
G057_19877
Klebsiella pneumoniae hvKP1

Active compounds were defined by <80% activity at 8 microM screening concentration.

External ID: CHEMBL788866

Protocol: N/A

Comment: Journal: J. Med. Chem.
Year: 1985
Volume: 28
Issue: 5
First Page: 668
Last Page: 672
DOI: 10.1021/jm50001a022

Target ChEMBL ID: CHEMBL376
ChEMBL Target Name: Rattus norvegicus
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL696376

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Journal: J. Med. Chem.
Year: 1997
Volume: 40
Issue: 20
First Page: 3182
Last Page: 3191
DOI: 10.1021/jm950028z

Target ChEMBL ID: CHEMBL2094119
ChEMBL Target Name: Glutamate receptor ionotropic, kainate
ChEMBL Target Type: PROTEIN COMPLEX GROUP - Target is a poorly defined protein complex, where subunit composition is unclear (e.g., GABA-A receptor)
Relationship Type: H - Homologous protein target assigned
Confidence: Multiple homologous protein targets may be assigned

External ID: CHEMBL878883

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Journal: J. Med. Chem.
Year: 1997
Volume: 40
Issue: 22
First Page: 3645
Last Page: 3650
DOI: 10.1021/jm9702387

Target ChEMBL ID: CHEMBL2009
ChEMBL Target Name: Glutamate receptor ionotropic, AMPA 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: CHEMBL696377

Protocol: N/A

Comment: Journal: J. Med. Chem.
Year: 1996
Volume: 39
Issue: 1
First Page: 183
Last Page: 190
DOI: 10.1021/jm950393q

Target ChEMBL ID: CHEMBL2094119
ChEMBL Target Name: Glutamate receptor ionotropic, kainate
ChEMBL Target Type: PROTEIN COMPLEX GROUP - Target is a poorly defined protein complex, where subunit composition is unclear (e.g., GABA-A receptor)
Relationship Type: H - Homologous protein target assigned
Confidence: Multiple homologous protein targets may be assigned

External ID: CHEMBL1072823

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2010
Volume: 18
Issue: 1
First Page: 202
Last Page: 213
DOI: 10.1016/j.bmc.2009.11.001

External ID: CHEMBL828867

Protocol: N/A

Comment: Journal: J. Med. Chem.
Year: 2005
Volume: 48
Issue: 9
First Page: 3438
Last Page: 3442
DOI: 10.1021/jm050014l

Target ChEMBL ID: CHEMBL1907608
ChEMBL Target Name: Glutamate NMDA receptor
ChEMBL Target Type: PROTEIN COMPLEX GROUP - Target is a poorly defined protein complex, where subunit composition is unclear (e.g., GABA-A receptor)
Relationship Type: D - Direct protein target assigned
Confidence: Multiple direct protein targets may be assigned

External ID: VSVM-OFFLINE

Protocol: Two uL of HEK293 cell suspension are dispensed at 1000 cells/well into solid white 1536-well plates (Grenier) using a Multidrop Combi (Thermo Scientific). After addition of 23 nL compound by a pin tool (Kalypsys), the plate is incubated 1 h at 37 degrees C and then 3 uL of virus 1:100 dilution VSV-MARV is added. After 28 hr, 4 uL of assay reagent is added and the plates are read using a ViewLux (Perkin Elmer). Assays are performed in sub-saturating amounts of virus (MOI <0.5), therefore luciferase signals reflect the amount (titer) of virus able to infect the cells in presence of the compound.

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: VSVL-OFFLINE

Protocol: For screening, 1000 cells in 2 ul/well were dispensed into white solid 1536-well plates (Greiner) using a solenoid-based dispenser. Following transfer of 23nl compound or DMSO vehicle by a pin tool, 3 ul/well of VSV-LV was added. The plates were centrifuged 1 min at 1000 RPM and then incubated 16 hr at 37 C and 5% CO2. After addition of 4 ul/well SteadyLite (PerkinElmer) detection reagent, the plates were incubated 10 min at ambient temperature and luminescence was measured on a ViewLux (Perkin Elmer) plate reader.

Keywords: NIH Roadmap, MLPCN, MLI, MLSMR, qHTS, NCGC, Lassa virus, luciferase, cell assay, infection

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: 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: 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.