External ID: cmt-p4-fda-celltiter_regid

Protocol: Native S16 cells were seeded in white solid-bottom 1536 well plates at between 500-750 cells/6uL in DMEM medium containing 10% FBS, w/o phenol red. After overnight incubation at 37 degree Celsius/5% C02, 23 nL of compounds or DMSO were delivered to each well using a pin tool, followed by 24h-incubation at 37 degree Celsius/5% C02. Then one volume of CellTiter-Glo assay reagent (Promega) was added to each well for cell viability measured by a ViewLux plate reader (PerkinElmer). The % activity was determined by normalizing to the average readings from wells containing no cells relative to DMSO-treated cells (0% Activity). The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity.

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, toxic compounds are ranked higher than non-toxic compounds.
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. Toxic 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: adst-fluc-km-fda-o1_regid

Protocol: Three microliters/well of substrate- buffer solution (0.01 mM D-Luciferin, 0.01 mM ATP, 50 mM Tris Acetate, 10 mM Mg Acetate, 0.01% Tween-20 and 0.038% BSA final concentrations) were dispensed into white solid-bottom 1536 well plates. Compounds were transferred to the plates in 23nl, and one microliter/well luciferase enzyme-buffer solution (10 nM P. pyralis luciferase and 50 mM Tris Acetate final concentrations) was dispensed yielding a final reaction volume of 4 ul/well. Luciferase luminescence was measured by a ViewLux plate reader within 5 minutes of enzyme-buffer addition. The % activity was determined by normalizing to the average readings of PTC124 (a positive control) relative to DMSO (0% Activity). The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity.

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: cmt-p4-fluc-fda_regid

Protocol: S16 Schwann cell PMP22 intronic element firefly luciferase assay
The FLuc-expressing S16 cells were seeded in white solid 1536-well plates at between 500-750 cells/4uL in DMEM medium containing 10% FBS, w/o phenol red. After overnight incubation at 37 degree Celsius/5% C02, 23 nL of compounds or DMSO were delivered to each well using a pin tool, followed by 24h-incubation at 37 degree Celsius/5% C02. Then 4 uL ONE-Glo luminescent substrate mix (Promega) was added to each well. Luminescence was measured on a ViewLux plate reader (Perkin Elmer). The % activity was determined by normalizing to the average readings of PTC124 (a positive control) - treated cells relative to DMSO-treated cells (0% Activity). The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity.

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: PolK100

Protocol: Three microliters of reagents (buffer in column 3 and 4 as negative control and 10 nM Pol kappa in columns 1, 2, and 5-48) were dispensed into a 1536-well black solid-bottom plate. Compounds (23 nL) were transferred via Kalypsys pin tool equipped with 1536-pin array. The plates were then incubated for 15 min at room temperature, and 1 uL substrate (50 nM final concentration) were then 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.