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: CPF004

Protocol: Reagents: 50mM Tris acetate, pH 7.5; 10mM Mg acetate; 10uM D-luciferin (Sigma #L9504); 10uM ATP; 0.01% Tween-20; 0.05% BSA; 10nM P. pyralis luciferase (Sigma #L9506)
Control compounds used were two known firefly luciferase inhibitors (compounds (2) and (5) in Auld et al., 2010), and DMSO.

Assay Summary: Three microliters containing firefly luciferase substrates in buffer (final concentrations: 50mM Tris acetate, pH 7.5, 10mM Mg acetate, 0.01% Tween-20, 0.05% BSA, 10uM D-luciferin, and 10uM ATP) are dispensed into each well of a Greiner white, solid-bottom 1536-well format plate using a flying reagent dispenser (FRD). These assay plates were then treated with 23nL of compound or DMSO using a Kalypsys pin tool, which allows for delivery of a 6-point interplate titration of each compound to the assay plate (quantitative HTS), with a final compound concentrations ranging from approximately 60uM to 7pM. One microliter of firefly luciferase in 500mM Tris-acetate buffer was then delivered by FRD to each well for a final enzyme concentration of 10nM. Luciferase activity was then measured using a ViewLux CCD imager (PerkinElmer), with an average exposure time of 2-30 seconds (2X binning, medium/high gain).

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: hNprA-p1

Protocol: PROTOCOL TABLES (as described by Inglese J, Shamu CE and Guy RK. 2007)

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

1. Cells. Seed 1500 HEK293 cGMP-sensor cells expressing hNpr1 per well in 4uL into Greiner 1536-well white/solid bottom high base, tissue culture-treated plates.
2. Incubation. Incubate plated cells at 37C in 5% CO2, 95% RH overnight.
3. Reagent. Add 1uL per well GloSensor reagent from Promega (2% final concentration) or CO2 independent media vehicle control to respective columns of Greiner 1536-well plates.
4. Incubation. Incubate plated cells at room temperature for 2 h, protect from light.
5. Compounds. Library compounds and A-71915 control added at 23 nL per well, transferred by Pin Tool.
6. Incubation. Incubate plated cells for 30 min. at room temperature, protect from light.
7. Detector. GloSensor baseline pre-read luminescence measured using CCD-based camera (Viewlux).
8. Compound. Add 1uL of hNppA peptide to 0.1 nM final concentration to stimulate hNpr1 or CO2 independent media as vehicle control.
9. Incubation. Incubate treated cells at room temperature for 20 min., protect from light.
10. Detector. GloSensor post-hNppA luminescence measured using CCD-based camera (Viewlux).

NOTES (numbers refer to Sequence numbers above)

1. HEK293 cells expressing hNpr1 and GIP/pGS-40F grown to 85-95% confluence (48-72 h) in T175 culture flasks (CO2 independent media supplemented with 10% FBS, 1% Pen/Strep antibiotics and 1 ug/mL puromycin). Trypsinize and harvest cells from T175 culture flasks and filter through 40 um cell strainer. Cells counted with Invitrogen Countess (30,000,000 - 50,000,000 cells/flask). Cells diluted to a final concentration of 375 cells/uL in CO2 independent media and a Millipore multidrop used to dispense 1500 cells per well (4uL/well) into 403 x 1536-well Greiner White/Solid bottom, high base tissue-culture treated (TC) plates and one Aurora 1536-well Black/Clear bottom, low base, TC plate (for visual inspection of cell health throughout the duration of the assay).

2. Cells incubated overnight at ambient temperature, 24 h. at 37C in 95% RH incubator protected from light, covered with metal lid with gas-exchange holes.

3. Prepare GloSensor Reagent (Promega): Reconstitute GloSensor luciferase substrate by adding 8.2mL Hepes buffer to 250mg substrate, mix well, protect from light and stored at -80C. Prepare a fresh 12% GloSensor solution in CO2 independent supplemented plating media (see above). Mix well and store at ambient temperature, protected from light. 1uL 12% GloSensor Luciferase Reagent added (2% final concentration) to respective columns of 403 x 1536-well Greiner plate with BioRaptr, 1uL CO2 independent plating media added to respective columns as vehicle control with BioRaptr.

4. Cells incubated for 2 h. at ambient temperature, protected from light with metal lid with gas-exchange holes.

5. A-71915 stock solution prepared (10 mM in DMSO). Working dilution for dispense = 10 mM stock dilution (38.3 uM final concentration; 16-pt 1:2 titration in duplicate = final concentration range of 38.3 uM - 1.17 nM). 23nl of GENESIS library compounds, A-71915 antagonist control or DMSO vehicle control transferred to the 403 cell-containing Greiner white/solid bottom high base, tissue culture-treated plates in 5-, 6- or 7-pt. interplate titrations where concentrations range 10 mM to 640 uM stock concentrations (see Yasgar, A. et al. 2008) with Pintool. 27 of the 403 plates are DMSO-only in compound field interspersed approximately every 20 plates between 376 library-treated plates.

6. Cells incubated for 30-50 min. at ambient temperature protected from light with metal lid with gas-exchange holes.

7. Read cellular baseline GloSensor luminescence in presence of A-71915 antagonist or test library compounds on the ViewLux [Exposure = 10 s.; Gain = High; Speed = Slow; and Binning = 2X.]

8. hNppA peptide agonist solution prepared at 250 uM (in H2O) stock concentration. From this a working dilution was prepared at 0.6 nM in CO2 Independent media. 1uL of 0.6 nM hNppA agonist (0.1 nM final concentration) or CO2 Independent media vehicle control dispensed into respective columns of 403 x Greiner 1536-well white/solid bottom, tissue culture plates with BioRaptr.

9. Cells incubated with agonist for 20 min. at ambient temperature, protected from light, covered with metal lid with gas-exchange holes.

10. Read final Luminescence on the ViewLux. [Exposure = 10 s.; Gain = High; Speed = Slow; and Binning = 2X.]

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

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

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, with an 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 activity curve class = -1.1, score = 80+abs((log_ac50+4.5)*inf_activity/20). For activity.curve_class == -1.2 && abs(activity.max_response) > 6*10, score = 60+abs((log_ac50+4.5)*inf_activity/20). For activity.curve_class == -2.1 || ( activity.curve_class==-2.2 && abs(activity.max_response) > 6*10), score = 40+abs((log_ac50+4.5)*inf_activity/20). For activity.curve_class == -1.2 || activity.curve_class == -2.2, score = 20+abs((log_ac50+4.5)*inf_activity/20). Inconclusive compounds, with a preread curve class other than 4, have PUBCHEM_ACTIVITY_SCORE of 10.

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.