External ID: 12hLO-f5-genesis-15-1hLO

Protocol: PROTOCOL TABLE
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION
1; Enzyme; 3 uL; 40 nM ALOX15 (15hLO-1) 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; 50 uM arachidonic acid (final concentration).
5; Incubate; 30 min; room temperature.
6; Reagent; 4 uL; Fe-XO solution was added to each well.
7; Centrifuge; 1000 rpm, 15 sec.
8; Incubation; 30 min; room temperature
9; Detector; absorbance; Read 1 at 405 nm absorbance using ViewLux (PerkinElmer) CCD imager.
10; Detector; absorbance; Read 2 at 573 nm absorbance using ViewLux (PerkinElmer) CCD imager.

NOTES (numbers refer to sequence above)
1; 40 nM ALOX15 (final concentration) in buffer solution (25 mM Hepes, 0.01% Triton X-100, pH 7.5) 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 10 mM in DMSO then 1:2 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; 50 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.
9, 10; 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: IP6K1-p1

Protocol: PROTOCOL TABLE
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE and DESCRIPTION.

1. Reagent, 3 uL of 1.3 mM ATP and 130 uM IP6 mixture in assay buffer was dispensed to a white, 1536-well assay plate.
2. Compound, 23 nL of compounds of the top two doses (57.5 uM and 19.1 uM final concentration) of the libraries were dispensed into the mixture using the Kalypsis pintool.
3. Reagent, 1 uL of 2.4 uM IP6K1 was dispensed to the assay plates.
4. Incubation, 2 hr incubation at room temperature.
5. Reagent, 2 uL of ADP-Glo reagent was added to the wells.
6. Incubation, 1 hr incubation at room temperature.
7. Reagent, 4 uL or ADP-Glo substate was added to the wells.
8. Incubation, 45 min incubation at room temperature.
9. Detection, luminescence signal was detected using the ViewLux microplate imager (PerkinElmer).

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 activity calls presented here are based on the NCATS analysis methods.

Compound Ranking:
1. Majority of the compounds were tested at 58uM and 11uM. Percent inhibition at 58uM (Max_Response) was obtained and used for compound ranking.
2. For all inactive compounds, with Max_Response >= -25.00, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds (Max_Response < -25.00, more than 25% inhibition) a score range was given between 25 and 100. The activity score is based on the absolute value of the Max_Response.

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: MTASE-p

Protocol: PROTOCOL TABLE (as described by Inglese J, Shamu CE and Guy RK. 2007)
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION.
1; Reagent; 2 uL; SMMTase Enzyme (2x) in reaction buffer, columns 1- 48.
2; Reagent; 1 uL; SAM (4x) in reaction buffer, columns 1-48.
3; Controls; 23 nL; DMSO in column 4; sinefungin in DMSO (0 uM - 40 uM) in 7-point 1:2 dilution series (n = 2) in column 2.
4; Compounds; 23 nL; Columns 5-48.
5; Reagent; 1 uL; Substrate (4x) in reaction buffer, columns 2-48.
6; Time; 20-30 min; Incubation.
7; Reagent; 1 uL; MTase-Glo reagent (5X), columns 1-48.
8; Time; 30 min; Incubation.
9; Reagent; 5 uL; MTase-Glo Detection reagent, columns 1-48.
10; Time; 30 min; Incubation, luminescence evolution.
11; Detection; Luminescence; ViewLux uHTS Microplate Imager (PerkinElmer).

NOTES (numbers refer to Sequence numbers above)
1, 2, 5. White Medium Binding Greiner 1536-well plates (Cat #789175-F, Greiner Bio-One, Monroe, NC); Reaction buffer: 50 mM Tris, pH 8.0, 3 mM MgCl2, 1 mM EDTA, 50 mM NaCl, 1 mM DTT, and 0.1 mg/mL BSA. Evaporation was prevented by covering assay plates with metal lids containing holes to allow gas diffusion.
1. Six SMMTase were profiled: HNMT, GNMT, PNMT, COMT, NNMT, GAMT
2. The corresponding SAM cofactor concentration was used for each SMMTase: HNMT - 9.3 uM SAM; GNMT - 12.1 uM SAM; PNMT - 3.6 uM SAM; COMT - 14.5 uM SAM; NNMT 11.9 uM SAM; GAMT - 5.6 uM SAM.
3, 4. Pintool transfer
5. The corresponding substrate was used for each enzyme: HNMT - 5.3 uM Histamine; GNMT - >500 uM Glycine; PNMT - 16.2 uM Norephinephrine; COMT - 13.7 uM Norephinephrine; NNMT - 3.8 uM Nicotinamide; GAMT - 2.5 uM Guanidinoacetate.6, 8, 10. Room temperature
6. Final reaction conditions: 10 nM COMT, 5 uM SAM, 15 uM norepinephrine, 50 mM Tris, pH 8.0, 3 mM MgCl2, 1 mM EDTA, 50 mM NaCl, 1 mM DTT, and 0.1 mg/mL BSA; refer to tables 2 and S1 for specific timing.
8. Conversion of SAH to ADP; MTase Glo Kit (Promega, Madison, WI).
10. Conversion of ADP to ATP and detection by UltraGlo luciferase; MTase Glo Kit (Promega, Madison, WI).
11. Settings: 20 s exposure, 1X binning, high gain, medium speed.

REFERENCES:
Inglese J, Shamu CE and Guy RK, Reporting data from high throughput screening of small molecule libraries, Nature Chemical Biology, 2007, 3(8): 438-441. doi.org/10.1038/nchembio0807-438.

Comment: Disclaimer:
Although all reasonable efforts have been made to ensure the accuracy and reliability of the data, caution should be exercised when interpreting the results as artifacts are possible from nonspecific effects such as assay signal interference. The curve fitting and activity calls presented here are based on the NCATS analysis methods.

Compound Ranking:
1. Compounds are assayed at single point activity (40 uM).
2. Active / Inactive compound calling is based on the results of the enzyme assay panel. ACTIVE compounds have PUBCHEM_ACTIVITY_SCORE = 50 and are compounds that have <= -50% max response in ANY ONE of the six enzymes. INACTIVE compounds have PUBCHEM_ACTIVITY_SCORE = 0 and are those that have >= -30% max response in ALL SIX enzymes. The remaining compounds are INCONCLUSIVE and have PUBCHEM_ACITIVITY_SCORE = 30.

External ID: MScreen:TargetID_600

Protocol: C-terminally 6xHis tagged CDK2/Cyclin A complex and N-terminally Flag tagged CDC25B C473S (372-566) were expressed and purified. Proteins were incubated together at a final concentration of 125 nM each for 1 hr prior to incubation with compound for 1 hr, followed by addition of anti-6xHis europium cryptate donor beads (Cisbio) and anti-Flag XL-665 acceptor beads (Cisbio) at a final dilution of 1:350 for 1 hr. 20mM potassium fluoride was added 10 minute prior to plate reading. Assay reagents were dispensed using a multidrop liquid dispenser (Thermo Scientific) onto uncoated, black, low-volume, 384-well plates (Corning). Assay plates were quantified using an Envision plate reader (Perkin-Elmer) with excitation of the europium crytate donor at 337 nm wavelength and emission of the donor at 620 nm and emission of the XL-665 acceptor at 665 nm in 18 uL volumes. Assays were performed in a buffer containing 50 mM Tris (pH 7.5), 50 mM NaCl, 10mM MgCl2, 1mM TCEP, with addition of and 1mM ATP, 0.05% BSA, and 0.05% Tween-20 immediately prior to the start of the assay.

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.

This screen was funded by NIH grant number: R01CA181185

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: CPF003

Protocol: NIH 3T3 cells were seeded into white 1536-well plates using a Multidrop Combi peristaltic dispenser (ThermoFisher, Waltham, MA) at a density of 400 cells/well in 5 uL of medium respectively. A pintool (Kalypsys) was used to transfer 23 nL of compound solution to the 1536-well assay plates. After 48 or 72 hr incubation at 37 degree celcius, 5% CO and 95% humidity, 2.5 uL of CellTiter-Glo (Promega) was dispensed into each well using a dispenser (Aspect Automation, St. Paul, MN) with solenoid valves (Lee Valves, Westbrook CT). Plates were left at room temperature for 10 min before imaging the ATP-coupled luminescence using a ViewLux microplate imager (PerkinElmer, Waltham, MA).

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: CPF002

Protocol: HEK 293 cells were seeded into white 1536-well plates using a Multidrop Combi peristaltic dispenser (ThermoFisher, Waltham, MA) at a density of 250 cells/well in 5 uL of medium respectively. A pintool (Kalypsys) was used to transfer 23 nL of compound solution to the 1536-well assay plates. After 48 or 72 hr incubation at 37 degree celcius, 5% CO and 95% humidity, 2.5 uL of CellTiter-Glo (Promega) was dispensed into each well using a dispenser (Aspect Automation, St. Paul, MN) with solenoid valves (Lee Valves, Westbrook CT). Plates were left at room temperature for 10 min before imaging the ATP-coupled luminescence using a ViewLux microplate imager (PerkinElmer, Waltham, MA).

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.